Mechanisms of DNA replication termination.

PubMed

Dewar, James M; Walter, Johannes C

2017-08-01

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach.

PubMed

Frimodt-Møller, Jakob; Charbon, Godefroid; Krogfelt, Karen A; Løbner-Olesen, Anders

2017-09-11

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Origin recognition is the predominant role for DnaA-ATP in initiation of chromosome replication.

PubMed

Grimwade, Julia E; Rozgaja, Tania A; Gupta, Rajat; Dyson, Kyle; Rao, Prassanna; Leonard, Alan C

2018-05-25

In all cells, initiation of chromosome replication depends on the activity of AAA+ initiator proteins that form complexes with replication origin DNA. In bacteria, the conserved, adenosine triphosphate (ATP)-regulated initiator protein, DnaA, forms a complex with the origin, oriC, that mediates DNA strand separation and recruitment of replication machinery. Complex assembly and origin activation requires DnaA-ATP, which differs from DnaA-ADP in its ability to cooperatively bind specific low affinity sites and also to oligomerize into helical filaments. The degree to which each of these activities contributes to the DnaA-ATP requirement for initiation is not known. In this study, we compared the DnaA-ATP dependence of initiation from wild-type Escherichia coli oriC and a synthetic origin (oriCallADP), whose multiple low affinity DnaA sites bind DnaA-ATP and DnaA-ADP similarly. OriCallADP was fully occupied and unwound by DnaA-ADP in vitro, and, in vivo, oriCallADP suppressed lethality of DnaA mutants defective in ATP binding and ATP-specific oligomerization. However, loss of preferential DnaA-ATP binding caused over-initiation and increased sensitivity to replicative stress. The findings indicate both DnaA-ATP and DnaA-ADP can perform most of the mechanical functions needed for origin activation, and suggest that a key reason for ATP-regulation of DnaA is to control replication initiation frequency.

Analysis of the temporal program of replication initiation in yeast chromosomes.

PubMed

Friedman, K L; Raghuraman, M K; Fangman, W L; Brewer, B J

1995-01-01

The multiple origins of eukaryotic chromosomes vary in the time of their initiation during S phase. In the chromosomes of Saccharomyces cerevisiae the presence of a functional telomere causes nearby origins to delay initiation until the second half of S phase. The key feature of telomeres that causes the replication delay is the telomeric sequence (C(1-3)A/G(1-3)T) itself and not the proximity of the origin to a DNA end. A second group of late replicating origins has been found at an internal position on chromosome XIV. Four origins, spanning approximately 140 kb, initiate replication in the second half of S phase. At least two of these internal origins maintain their late replication time on circular plasmids. Each of these origins can be separated into two functional elements: those sequences that provide origin function and those that impose late activation. Because the assay for determining replication time is costly and laborious, it has not been possible to analyze in detail these 'late' elements. We report here the development of two new assays for determining replication time. The first exploits the expression of the Escherichia coli dam methylase in yeast and the characteristic period of hemimethylation that transiently follows the passage of a replication fork. The second uses quantitative hybridization to detect two-fold differences in the amount of specific restriction fragments as a function of progress through S phase. The novel aspect of this assay is the creation in vivo of a non-replicating DNA sequence by site-specific pop-out recombination. This non-replicating fragment acts as an internal control for copy number within and between samples. Both of these techniques are rapid and much less costly than the more conventional density transfer experiments that require CsCl gradients to detect replicated DNA. With these techniques it should be possible to identify the sequences responsible for late initiation, to search for other late replicating regions in the genome, and to begin to analyze the effect that altering the temporal program has on chromosome function.

Functional characterization of replication and stability factors of an incompatibility group P-1 plasmid from Xylella fastidiosa.

PubMed

Lee, Min Woo; Rogers, Elizabeth E; Stenger, Drake C

2010-12-01

Xylella fastidiosa strain riv11 harbors a 25-kbp plasmid (pXF-RIV11) belonging to the IncP-1 incompatibility group. Replication and stability factors of pXF-RIV11 were identified and used to construct plasmids able to replicate in X. fastidiosa and Escherichia coli. Replication in X. fastidiosa required a 1.4-kbp region from pXF-RIV11 containing a replication initiation gene (trfA) and the adjacent origin of DNA replication (oriV). Constructs containing trfA and oriV from pVEIS01, a related IncP-1 plasmid of the earthworm symbiont Verminephrobacter eiseniae, also were competent for replication in X. fastidiosa. Constructs derived from pXF-RIV11 but not pVEIS01 replicated in Agrobacterium tumefaciens, Xanthomonas campestris, and Pseudomonas syringae. Although plasmids bearing replication elements from pXF-RIV11 or pVEIS01 could be maintained in X. fastidiosa under antibiotic selection, removal of selection resulted in plasmid extinction after 3 weekly passages. Addition of a toxin-antitoxin addiction system (pemI/pemK) from pXF-RIV11 improved plasmid stability such that >80 to 90% of X. fastidiosa cells retained plasmid after 5 weekly passages in the absence of antibiotic selection. Expression of PemK in E. coli was toxic for cell growth, but toxicity was nullified by coexpression of PemI antitoxin. Deletion of N-terminal sequences of PemK containing the conserved motif RGD abolished toxicity. In vitro assays revealed a direct interaction of PemI with PemK, suggesting that antitoxin activity of PemI is mediated by toxin sequestration. IncP-1 plasmid replication and stability factors were added to an E. coli cloning vector to constitute a stable 6.0-kbp shuttle vector (pXF20-PEMIK) suitable for use in X. fastidiosa.

Replication of the Escherichia coli chromosome in RNase HI-deficient cells: multiple initiation regions and fork dynamics.

PubMed

Maduike, Nkabuije Z; Tehranchi, Ashley K; Wang, Jue D; Kreuzer, Kenneth N

2014-01-01

DNA replication in Escherichia coli is normally initiated at a single origin, oriC, dependent on initiation protein DnaA. However, replication can be initiated elsewhere on the chromosome at multiple ectopic oriK sites. Genetic evidence indicates that initiation from oriK depends on RNA-DNA hybrids (R-loops), which are normally removed by enzymes such as RNase HI to prevent oriK from misfiring during normal growth. Initiation from oriK sites occurs in RNase HI-deficient mutants, and possibly in wild-type cells under certain unusual conditions. Despite previous work, the locations of oriK and their impact on genome stability remain unclear. We combined 2D gel electrophoresis and whole genome approaches to map genome-wide oriK locations. The DNA copy number profiles of various RNase HI-deficient strains contained multiple peaks, often in consistent locations, identifying candidate oriK sites. Removal of RNase HI protein also leads to global alterations of replication fork migration patterns, often opposite to normal replication directions, and presumably eukaryote-like replication fork merging. Our results have implications for genome stability, offering a new understanding of how RNase HI deficiency results in R-loop-mediated transcription-replication conflict, as well as inappropriate replication stalling or blockage at Ter sites outside of the terminus trap region and at ribosomal operons. © 2013 John Wiley & Sons Ltd.

Stable DNA replication: interplay between DNA replication, homologous recombination, and transcription.

PubMed

Kogoma, T

1997-06-01

Chromosome replication in Escherichia coli is normally initiated at oriC, the origin of chromosome replication. E. coli cells possess at least three additional initiation systems for chromosome replication that are normally repressed but can be activated under certain specific conditions. These are termed the stable DNA replication systems. Inducible stable DNA replication (iSDR), which is activated by SOS induction, is proposed to be initiated from a D-loop, an early intermediate in homologous recombination. Thus, iSDR is a form of recombination-dependent DNA replication (RDR). Analysis of iSDR and RDR has led to the proposal that homologous recombination and double-strand break repair involve extensive semiconservative DNA replication. RDR is proposed to play crucial roles in homologous recombination, double-strand break repair, restoration of collapsed replication forks, and adaptive mutation. Constitutive stable DNA replication (cSDR) is activated in mhA mutants deficient in RNase HI or in recG mutants deficient in RecG helicase. cSDR is proposed to be initiated from an R-loop that can be formed by the invasion of duplex DNA by an RNA transcript, which most probably is catalyzed by RecA protein. The third form of SDR is nSDR, which can be transiently activated in wild-type cells when rapidly growing cells enter the stationary phase. This article describes the characteristics of these alternative DNA replication forms and reviews evidence that has led to the formulation of the proposed models for SDR initiation mechanisms. The possible interplay between DNA replication, homologous recombination, DNA repair, and transcription is explored.

Evidence for roles of the Escherichia coli Hda protein beyond regulatory inactivation of DnaA.

PubMed

Baxter, Jamie C; Sutton, Mark D

2012-08-01

The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication (oriC) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. Here, we used the mutant dnaN159 strain, which expresses the β159 clamp protein, to gain insight into how the actions of Hda are co-ordinated with replication. Elevated expression of Hda impeded growth of the dnaN159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of -1 frameshift mutations characteristic of Pol IV, while loss of hda conferred cold sensitivity. Using the dnaN159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Taken together, these findings suggest that although one or more Hda functions are essential for cell viability, RIDA may be dispensable. © 2012 Blackwell Publishing Ltd.

Interrogating the Escherichia coli cell cycle by cell dimension perturbations.

PubMed

Zheng, Hai; Ho, Po-Yi; Jiang, Meiling; Tang, Bin; Liu, Weirong; Li, Dengjin; Yu, Xuefeng; Kleckner, Nancy E; Amir, Ariel; Liu, Chenli

2016-12-27

Bacteria tightly regulate and coordinate the various events in their cell cycles to duplicate themselves accurately and to control their cell sizes. Growth of Escherichia coli, in particular, follows a relation known as Schaechter's growth law. This law says that the average cell volume scales exponentially with growth rate, with a scaling exponent equal to the time from initiation of a round of DNA replication to the cell division at which the corresponding sister chromosomes segregate. Here, we sought to test the robustness of the growth law to systematic perturbations in cell dimensions achieved by varying the expression levels of mreB and ftsZ We found that decreasing the mreB level resulted in increased cell width, with little change in cell length, whereas decreasing the ftsZ level resulted in increased cell length. Furthermore, the time from replication termination to cell division increased with the perturbed dimension in both cases. Moreover, the growth law remained valid over a range of growth conditions and dimension perturbations. The growth law can be quantitatively interpreted as a consequence of a tight coupling of cell division to replication initiation. Thus, its robustness to perturbations in cell dimensions strongly supports models in which the timing of replication initiation governs that of cell division, and cell volume is the key phenomenological variable governing the timing of replication initiation. These conclusions are discussed in the context of our recently proposed "adder-per-origin" model, in which cells add a constant volume per origin between initiations and divide a constant time after initiation.

Sequencing a piece of history: complete genome sequence of the original Escherichia coli strain

PubMed Central

Dunne, Karl A; Chaudhuri, Roy R; Rossiter, Amanda E; Beriotto, Irene; Browning, Douglas F; Squire, Derrick; Cunningham, Adam F; Cole, Jeffrey A; Loman, Nicholas

2017-01-01

In 1885, Theodor Escherich first described the Bacillus coli commune, which was subsequently renamed Escherichia coli. We report the complete genome sequence of this original strain (NCTC 86). The 5 144 392 bp circular chromosome encodes the genes for 4805 proteins, which include antigens, virulence factors, antimicrobial-resistance factors and secretion systems, of a commensal organism from the pre-antibiotic era. It is located in the E. coli A subgroup and is closely related to E. coli K-12 MG1655. E. coli strain NCTC 86 and the non-pathogenic K-12, C, B and HS strains share a common backbone that is largely co-linear. The exception is a large 2 803 932 bp inversion that spans the replication terminus from gmhB to clpB. Comparison with E. coli K-12 reveals 41 regions of difference (577 351 bp) distributed across the chromosome. For example, and contrary to current dogma, E. coli NCTC 86 includes a nine gene sil locus that encodes a silver-resistance efflux pump acquired before the current widespread use of silver nanoparticles as an antibacterial agent, possibly resulting from the widespread use of silver utensils and currency in Germany in the 1800s. In summary, phylogenetic comparisons with other E. coli strains confirmed that the original strain isolated by Escherich is most closely related to the non-pathogenic commensal strains. It is more distant from the root than the pathogenic organisms E. coli 042 and O157 : H7; therefore, it is not an ancestral state for the species. PMID:28663823

Back to basics: pBR322 and protein expression systems in E. coli.

PubMed

Balbás, Paulina; Bolívar, Francisco

2004-01-01

The extensive variety of plasmid-based expression systems in E. coli resulted from the fact that there is no single strategy for achieving maximal expression of every cloned gene. Although a number of strategies have been implemented to deal with problems associated to gene transcription and translation, protein folding, secretion, location, posttranslational modifications, particularities of different strains, and the like and more integrated processes have been developed, the basic plasmid-borne elements and their interaction with the particular host strain will influence the overall expression system and final productivity. Plasmid vector pBR322 is a well-established multipurpose cloning vector in laboratories worldwide, and a large number of derivatives have been created for specific applications and research purposes, including gene expression in its natural host, E. coli, and few other bacteria. The early characterization of the molecule, including its nucleotide sequence, replication and maintenance mechanisms, and determination of its coding regions, accounted for its success, not only as a universal cloning vector, but also as a provider of genes and an origin of replication for other intraspecies vectors. Since the publication of the aforementioned reviews, novel discoveries pertaining to these issues have appeared in the literature that deepen the understanding of the plasmid's features, behavior, and impact in gene expression systems, as well as some important strain characteristics that affect plasmid replication and stability. The objectives of this review include updating and discussing the new information about (1) the replication and maintenance of pBR322; (2) the host-related modulation mechanisms of plasmid replication; (3) the effects of growth rate on replication control, stability, and recombinant gene expression; (4) ways for plasmid amplification and elimination. Finally, (5) a summary of novel ancillary studies about pBR322 is presented.

Molecular flip–flops formed by overlapping Fis sites

PubMed Central

Hengen, Paul N.; Lyakhov, Ilya G.; Stewart, Lisa E.; Schneider, Thomas D.

2003-01-01

The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart. Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication. Only one of these sites is bound by Fis at a time, so the structure is a molecular flip–flop that could direct alternative firing of replication complexes in opposite directions. Alternatively, the flip–flop could represent part of an on–off switch for replication. Because they can be used to create precise switched states, molecular flip–flops could be used as the basis of a novel molecular computer. PMID:14602927

Molecular flip-flops formed by overlapping Fis sites.

PubMed

Hengen, Paul N; Lyakhov, Ilya G; Stewart, Lisa E; Schneider, Thomas D

2003-11-15

The DNA-binding protein Fis frequently uses pairs of sites 7 or 11 base pairs (bp) apart. Two overlapping Fis sites separated by 11 bp are found in the Escherichia coli origin of chromosomal replication. Only one of these sites is bound by Fis at a time, so the structure is a molecular flip-flop that could direct alternative firing of replication complexes in opposite directions. Alternatively, the flip-flop could represent part of an on-off switch for replication. Because they can be used to create precise switched states, molecular flip-flops could be used as the basis of a novel molecular computer.

Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis.

PubMed

Salazar, L; Fsihi, H; de Rossi, E; Riccardi, G; Rios, C; Cole, S T; Takiff, H E

1996-04-01

The genus Mycobacterium is composed of species with widely differing growth rates ranging from approximately three hours in Mycobacterium smegmatis to two weeks in Mycobacterium leprae. As DNA replication is coupled to cell duplication, it may be regulated by common mechanisms. The chromosomal regions surrounding the origins of DNA replication from M. smegmatis, M. tuberculosis, and M. leprae have been sequenced, and show very few differences. The gene order, rnpA-rpmH-dnaA-dnaN-recF-orf-gyrB-gyrA, is the same as in other Gram-positive organisms. Although the general organization in M. smegmatis is very similar to that of Streptomyces spp., a closely related genus, M. tuberculosis and M. leprae differ as they lack an open reading frame, between dnaN and recF, which is similar to the gnd gene of Escherichia coli. Within the three mycobacterial species, there is extensive sequence conservation in the intergenic regions flanking dnaA, but more variation from the consensus DnaA box sequence was seen than in other bacteria. By means of subcloning experiments, the putative chromosomal origin of replication of M. smegmatis, containing the dnaA-dnaN region, was shown to promote autonomous replication in M. smegmatis, unlike the corresponding regions from M. tuberculosis or M. leprae.

High carriage of adherent invasive E. coli in wildlife and healthy individuals.

PubMed

Rahmouni, Oumaïra; Vignal, Cécile; Titécat, Marie; Foligné, Benoît; Pariente, Benjamin; Dubuquoy, Laurent; Desreumaux, Pierre; Neut, Christel

2018-01-01

Adherent invasive Escherichia coli (AIEC) are suspected to be involved in the pathogenesis of inflammatory bowel diseases. Since AIEC was first described in 1999, despite important progress on its genomic and immune characterizations, some crucial questions remain unanswered, such as whether there exists a natural reservoir, or whether there is asymptomatic carriage. The ECOR collection, including E. coli strains isolated mainly from the gut of healthy humans and animals, constitutes an ideal tool to investigate AIEC prevalence in healthy condition. A total of 61 E. coli strains were examined for characteristics of AIEC. The adhesion, invasion and intramacrophage replication capabilities (AIEC phenotype) of 61 intestinal E. coli strains were determined. The absence of virulence-associated diarrheagenic E. coli pathotypes (EPEC, ETEC, EIEC, EHEC, DAEC, EAEC), and uropathogenic E. coli was checked. Out of 61 intestinal strains, 13 (21%) exhibit the AIEC phenotype, 7 are from human origin and 6 are from animal origin. Prevalence of AIEC strains is about 24 and 19% in healthy humans and animals respectively. These strains are highly genetically diverse as they are distributed among the main described phylogroups. Among E. coli strains from the ECOR collection, we also detected strains able to detach I-407 cells. Our study described for the first time AIEC strains isolated from the feces of healthy humans and animals.

Construction of an easy-to-use CRISPR-Cas9 system by patching a newly designed EXIT circuit.

PubMed

Tang, Qiang; Lou, Chunbo; Liu, Shuang-Jiang

2017-01-01

Plasmid-borne genetic editing tools, including the widely used CRISPR-Cas9 system, have greatly facilitated bacterial programming to obtain novel functionalities. However, the lack of effective post-editing plasmid elimination methods impedes follow-up genetic manipulation or application. Conventional strategies including exposure to physical and chemical treatments, or exploiting temperature-sensitive replication origins have several drawbacks (e.g., they are limited for efficiency and are time-consuming). Therefore, the demand is apparent for easy and rapid elimination of the tool plasmids from their bacterial hosts after genetic manipulation. To bridge this gap, we designed a novel EXIT circuit with the homing endonuclease, which can be exploited for rapid and efficient elimination of various plasmids with diverse replication origins. As a proof of concept, we validated the EXIT circuit in Escherichia coli by harnessing homing endonuclease I- Sce I and its cleavage site. When integrated into multiple plasmids with different origins, the EXIT circuit allowed them to be eliminated from the host cells, simultaneously. By combining the widely used plasmid-borne CRISPR-Cas9 system and the EXIT circuit, we constructed an easy-to-use CRISPR-Cas9 system that eliminated the Cas9- and the single-guide RNA (sgRNA)-encoding plasmids in one-step. Within 3 days, we successfully constructed an atrazine-degrading E. coli strain, thus further demonstrating the advantage of this new CRISPR-Cas9 system for bacterial genome editing. Our novel EXIT circuit, which exploits the homing endonuclease I- Sce I, enables plasmid(s) with different replication origins to be eliminated from their host cells rapidly and efficiently. We also developed an easy-to-use CRISPR-Cas9 system with the EXIT circuit, and this new system can be widely applied to bacterial genome editing.

[The effects of TorR protein on initiation of DNA replication in Escherichia coli].

PubMed

Yuan, Yao; Jiaxin, Qiao; Jing, Li; Hui, Li; Morigen, Morigen

2015-03-01

The two-component systems, which could sense and respond to environmental changes, widely exist in bacteria as a signal transduction pathway. The bacterial CckA/CtrA, ArcA/ArcB and PhoP/PhoQ two-component systems are associated with initiation of DNA replication and cell division, however, the effects of the TorS/TorR system on cell cycle and DNA replication remains unknown. The TorS/TorR system in Escherichia coli can sense changes in trimethylamine oxide (TMAO) concentration around the cells. However, it is unknown if it also affects initiation of DNA replication. We detected DNA replication patterns in ΔtorS and ΔtorR mutant strains by flow cytometry. We found that the average number of replication origins (oriCs) per cell and doubling time in ΔtorS mutants were the same while the average number of oriCs in ΔtorR mutants was increased compared with that in wild-type cells. These results indicated that absence of TorR led to an earlier initiation of DNA replication than that in wild-type cells. Strangely, neither overexpression of TorR nor co-expression of TorR and TorS could restore ΔtorR mutant phenotype to the wild type. However, overexpression of SufD in both wild type and ΔtorR mutants promoted initiation of DNA replication, while mutation of SufD delayed it in ΔtorR mutants. Thus, TorR may affect initiation of DNA replication indirectly through regulating gene expression of sufD.

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed Central

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-01-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus. PMID:9097439

Genetic manipulation of Bacillus methanolicus, a gram-positive, thermotolerant methylotroph.

PubMed

Cue, D; Lam, H; Dillingham, R L; Hanson, R S; Flickinger, M C

1997-04-01

We report the fist genetic transformation system, shuttle vectors, and integrative vectors for the thermotolerant, methylotrophic bacterium Bacillus methanolicus. By using a polyethylene glycol-mediated transformation procedure, we have successfully transformed B. methanolicus with both integrative and multicopy plasmids. For plasmids with a single BmeTI recognition site, dam methylation of plasmid DNA (in vivo or in vitro) was found to enhance transformation efficiency from 7- to 11-fold. Two low-copy-number Escherichia coli-B, methanolicus shuttle plasmids, pDQ507 and pDQ508, are described. pDQ508 caries the replication origin cloned from a 17-kb endogenous B. methanolicus plasmid, pBM1. pDQ507 carries a cloned B. methanolicus DNA fragment, pmr-1, possibly of chromosomal origin, that supports maintenance of pDQ507 as a circular, extrachromosomal DNA molecule. Deletion analysis of pDQ507 indicated two regions required for replication, i.e., a 90-bp AT-rich segment containing a 46-bp imperfect, inverted repeat sequence and a second region 65% homologous to the B. subtilis dpp operon. We also evaluated two E. coli-B. subtilis vectors, pEN1 and pHP13, for use as E. coli-B. methanolicus shuttle vectors. The plasmids pHP13, pDQ507, and pDQ508 were segregationally and structurally stable in B. methanolicus for greater than 60 generations of growth under nonselective conditions; pEN1 was segregationally unstable. Single-stranded plasmid DNA was detected in B. methanolicus transformants carrying either pEN1, pHP13, or pDQ508, suggesting that pDQ508, like the B. subtilis plasmids, is replicated by a rolling-circle mechanism. These studies provide the basic tools for the genetic manipulation of B. methanolicus.

Killing of Escherichia coli by Crohn's Disease Monocyte-derived Macrophages and Its Enhancement by Hydroxychloroquine and Vitamin D

PubMed Central

Flanagan, Paul K.; Chiewchengchol, Direkrit; Wright, Helen L.; Edwards, Steven W.; Alswied, Abdullah; Satsangi, Jack; Subramanian, Sreedhar; Rhodes, Jonathan M.

2015-01-01

Background: Crohn's disease (CD) is associated with defective innate immunity, including impaired neutrophil chemotaxis, and mucosal invasion by bacteria, particularly adherent and invasive Escherichia coli that replicate inside macrophage phagolysosomes. We compared CD and healthy control (HC) macrophages for their abilities to kill E. coli and generate neutrophil chemoattractants and also assessed the effects of hydroxychloroquine (HCQ) and vitamin D on killing of phagocytosed E. coli. Methods: Peripheral blood monocyte-derived macrophages from CD and HC were compared for bacterial killing and generation of neutrophil chemoattractants in response to CD-derived E. coli. Escherichia coli replication was also assessed in the presence and absence of HCQ, alone and with antibiotics, and vitamin D. Results: Monocyte-derived macrophages from patients with CD were similar to HC in allowing replication of phagocytosed CD-derived E. coli: HM605 {CD: N = 10, mean fold replication in 3 hr = 1.08 (95% confidence interval [CI], 0.39–1.78); HC: N = 9, 1.50 (95% CI, 1.02–1.97); P = 0.15} and also in generation of neutrophil chemoattractants in response to E. coli (mean fold chemotaxis relative to control: CD = 2.55 [95% CI, 2.31–2.80]; HC = 2.65 [95% CI, 2.46–2.85], P = 0.42). HCQ and 1,25 OH2-vitamin D3 both caused dose-dependent inhibition of intramacrophage E. coli replication 3-hour postinfection; HCQ: 73.9% inhibition (P < 0.001) at 1 μg/mL, accompanied by raised intraphagosomal pH, and 1,25 OH2-vitamin D3: 80.7% inhibition (P < 0.05) at 80 nM. HCQ had synergistic effects with doxycycline and ciprofloxacin. Conclusions: CD and HC macrophages perform similarly in allowing replication of phagocytosed E. coli and generating neutrophil chemoattractants. Replication of phagocytosed E. coli was substantially decreased by HCQ and vitamin D. These warrant further therapeutic trials in CD in combination with relevant antibiotics. PMID:25839777

In vivo replication of T4 and T7 bacteriophages in germ-free mice colonized with Escherichia coli.

PubMed

Weiss, Marietta; Denou, Emmanuel; Bruttin, Anne; Serra-Moreno, Ruth; Dillmann, Marie-Lise; Brüssow, Harald

2009-10-10

The gut transit of T4 phages was studied in axenic mice mono-colonized with the non-pathogenic Escherichia coli strain K-12. Thirty minutes, 1 and 2 h after phage feeding, T4 phage had reached the jejunum, ileum and cecum, respectively. Phage was found in the lumen and was also associated with the mucosa. One day later no phage was detected in the feces. Compared to germ-free control animals, oral T4 phage led to a 300-fold higher fecal phage titer in mice mono-colonized with E. coli strain WG-5. The in vivo T4 phage replication was transient and reached peak fecal titers about 8 h after oral phage application followed by a rapid titer decrease over two days. Similar data were obtained in mice colonized with E. coli strain Nissle. In contrast, orally applied T7 phage experienced a massive and sustained in vivo replication in mice mono-colonized with E. coli strain WG-5 irrespective whether phage or E. coli host was applied first. T7 phage replication occurred mainly in the large intestine. High titers of T7 phage and high E. coli cell counts coexisted in the feces. The observation of only 20% T7 phage-resistant fecal E. coli colonies suggests a refuge model where phage-sensitive E. coli cells are physically or physiologically protected from phage infection in the gut. The difference between T7 and T4 with respect to gut replication might partly reflect their distinct in vitro capacity to replicate on slowly growing cells.

Survival of Escherichia coli, enterococci and Campylobacter jejuni in Canada goose faeces on pasture.

PubMed

Moriarty, E M; Weaver, L; Sinton, L W; Gilpin, B

2012-11-01

Freshly excreted Canada goose faeces pose a public health risk as they contain pathogenic microorganisms. Accordingly, a study was carried out on the growth and survival of resident indicator bacteria (enterococci and Escherichia coli) and inoculated Campylobacter jejuni in freshly excreted faeces over summer and winter. Canada goose faeces were collected, mixed thoroughly and inoculated with 10⁸ g⁻¹ C. jejuni. The faeces were mixed again before making the Canada goose dropping. The simulated goose droppings (N = 70) were placed on pasture, and the concentrations of E. coli, enterococci and the pathogen, C. jejuni, were monitored. In summer only, the molecular marker of E. coli LacZ and the avian-associated bacteria E2 was also monitored. Results of the survival study indicated that significant growth of enterococci and E. coli occurred in summer, before concentrations decreased to less than 15% of the original concentration (day 77) for enterococci and 0.01% for E. coli. LacZ followed a similar pattern to E. coli, while the E2 marker dropped to below 0.1% of the original concentration within 4 days. In winter, enterococci grew slightly, while no growth of E. coli occurred. In both summer and winter, C. jejuni was rapidly inactivated. This research highlights the ability of bacterial indicators to replicate and survive in the environment when harboured by avian faeces, and the limited risk aged Canada goose faeces pose as an environmental source of Campylobacter spp. © 2012 Blackwell Verlag GmbH.

A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation.

PubMed

Heuermann, D; Haas, R

1998-03-01

A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHel2 carries a chloramphenicol resistance cassette (catGC) and pHel3 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 x 10(-7) and 4.7 x 10(-7) transformants per viable recipient was achieved with pHel2 and pHel3, respectively, and both vectors showed stable, autonomous replication in H. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H. pylori recipients, with pHel2 showing an efficiency of 2.0 x 10(-5) transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a recA-deficient H. pylori mutant by the cloned H. pylori recA+ gene, and the expression of the heterologous green fluorescent protein (GFP) in H. pylori demonstrate the general usefulness of this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.

Long range chromosome organization in Escherichia coli: The position of the replication origin defines the non-structured regions and the Right and Left macrodomains

PubMed Central

2017-01-01

The Escherichia coli chromosome is organized into four macrodomains (Ori, Ter, Right and Left) and two non-structured regions. This organization influences the segregation of sister chromatids, the mobility of chromosomal DNA, and the cellular localization of the chromosome. The organization of the Ter and Ori macrodomains relies on two specific systems, MatP/matS for the Ter domain and MaoP/maoS for the Ori domain, respectively. Here by constructing strains with chromosome rearrangements to reshuffle the distribution of chromosomal segments, we reveal that the difference between the non-structured regions and the Right and Left lateral macrodomains relies on their position on the chromosome. A change in the genetic location of oriC generated either by an inversion within the Ori macrodomain or by the insertion of a second oriC modifies the position of Right and Left macrodomains, as the chromosome region the closest to oriC are always non-structured while the regions further away behave as macrodomain regardless of their DNA sequence. Using fluorescent microscopy we estimated that loci belonging to a non-structured region are significantly closer to the Ori MD than loci belonging to a lateral MD. Altogether, our results suggest that the origin of replication plays a prominent role in chromosome organization in E. coli, as it determines structuring and localization of macrodomains in growing cell. PMID:28486476

Escherichia coli DNA polymerase I can disrupt G-quadruplex structures during DNA replication.

PubMed

Teng, Fang-Yuan; Hou, Xi-Miao; Fan, San-Hong; Rety, Stephane; Dou, Shuo-Xing; Xi, Xu-Guang

2017-12-01

Non-canonical four-stranded G-quadruplex (G4) DNA structures can form in G-rich sequences that are widely distributed throughout the genome. The presence of G4 structures can impair DNA replication by hindering the progress of replicative polymerases (Pols), and failure to resolve these structures can lead to genetic instability. In the present study, we combined different approaches to address the question of whether and how Escherichia coli Pol I resolves G4 obstacles during DNA replication and/or repair. We found that E. coli Pol I-catalyzed DNA synthesis could be arrested by G4 structures at low protein concentrations and the degree of inhibition was strongly dependent on the stability of the G4 structures. Interestingly, at high protein concentrations, E. coli Pol I was able to overcome some kinds of G4 obstacles without the involvement of other molecules and could achieve complete replication of G4 DNA. Mechanistic studies suggested that multiple Pol I proteins might be implicated in G4 unfolding, and the disruption of G4 structures requires energy derived from dNTP hydrolysis. The present work not only reveals an unrealized function of E. coli Pol I, but also presents a possible mechanism by which G4 structures can be resolved during DNA replication and/or repair in E. coli. © 2017 Federation of European Biochemical Societies.

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme, Sphingobium fuliginis ATCC 27551.

PubMed

Nakayama, Kosuke; Ohmori, Takeshi; Ishikawa, Satoshi; Iwata, Natsumi; Seto, Yasuo; Kawahara, Kazuyoshi

2016-05-01

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.

Characterization of the Basic Replicon of Rhodococcus Plasmid pSOX and Development of a Rhodococcus-Escherichia coli Shuttle Vector†

PubMed Central

Denis-Larose, Claude; Bergeron, Hélène; Labbé, Diane; Greer, Charles W.; Hawari, Jalal; Grossman, Matthew J.; Sankey, Bruce M.; Lau, Peter C. K.

1998-01-01

The replication region of a 100-kb desulfurization plasmid (pSOX) from Rhodococcus sp. strain X309 was localized to a 4-kb KpnI fragment, and its sequence was determined. The amino acid sequence of one of the predicted open reading frames (ORFs) was related to the putative replication (Rep) protein sequences of the mycobacterial pLR7 family of plasmids. Three of the five predicted ORF products were identified by radiolabelling with the Escherichia coli T7 polymerase/promoter system. In E. coli, the Rep protein of pSOX was apparently synthesized in a shortened form, 21.3 kDa instead of the predicted 41.3 kDa, as a result of an internal initiation. This situation is reminescent of that for some bacterial Rep proteins. A shuttle plasmid was constructed with the pSOX origin, pBluescript II KS−, and the chloramphenicol resistance (Cmr) gene from pRF29. This new shuttle plasmid was used to demonstrate expression of the Bacillus subtilis sacB gene in a strain of Rhodococcus, rendering it sensitive to the presence of sucrose. PMID:9797291

Characterization of the cryptic plasmid pOfk55 from Legionella pneumophila and construction of a pOfk55-derived shuttle vector.

PubMed

Nishida, Takashi; Watanabe, Kenta; Tachibana, Masato; Shimizu, Takashi; Watarai, Masahisa

2017-03-01

In this study, a cryptic plasmid pOfk55 from Legionella pneumophila was isolated and characterized. pOfk55 comprised 2584bp with a GC content of 37.3% and contained three putative open reading frames (ORFs). orf1 encoded a protein of 195 amino acids and the putative protein shared 39% sequence identity with a putative plasmid replication protein RepL. ORF1 was needed for replication in L. pneumophila but pOfk55 did not replicate in Escherichia coli. orf2 and orf3 encoded putative hypothetical proteins of 114 amino acids and 78 amino acids, respectively, but the functions of the putative proteins ORF2 and OFR3 are not clear. The transfer mechanism for pOfk55 was independent on the type IVB secretion system in the original host. A L. pneumophila-E. coli shuttle vector, pNT562 (5058bp, Km R ), was constructed by In-Fusion Cloning of pOfk55 with a kanamycin-resistance gene from pUTmini-Tn5Km and the origin of replication from pBluescript SK(+) (pNT561). Multiple cloning sites from pBluescript SK(+) as well as the tac promoter region and lacI gene from pAM239-GFP were inserted into pNT561 to construct pNT562. The transformation efficiency of pNT562 in L. pneumophila strains ranged from 1.6×10 1 to 1.0×10 5 CFU/ng. The relative number of pNT562 was estimated at 5.7±1.0 copies and 73.6% of cells maintained the plasmid after 1week in liquid culture without kanamycin. A green fluorescent protein (GFP) expression vector, pNT563, was constructed by ligating pNT562 with the gfpmut3 gene from pAM239-GFP. pNT563 was introduced into L. pneumophila Lp02 and E. coli DH5α, and both strains expressed GFP successfully. These results suggest that the shuttle vector is useful for genetic studies in L. pneumophila. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutations reducing replication from R-loops suppress the defects of growth, chromosome segregation and DNA supercoiling in cells lacking topoisomerase I and RNase HI activity.

PubMed

Usongo, Valentine; Martel, Makisha; Balleydier, Aurélien; Drolet, Marc

2016-04-01

R-loop formation occurs when the nascent RNA hybridizes with the template DNA strand behind the RNA polymerase. R-loops affect a wide range of cellular processes and their use as origins of replication was the first function attributed to them. In Escherichia coli, R-loop formation is promoted by the ATP-dependent negative supercoiling activity of gyrase (gyrA and gyrB) and is inhibited by topoisomerase (topo) I (topA) relaxing transcription-induced negative supercoiling. RNase HI (rnhA) degrades the RNA moiety of R-loops. The depletion of RNase HI activity in topA null mutants was previously shown to lead to extensive DNA relaxation, due to DNA gyrase inhibition, and to severe growth and chromosome segregation defects that were partially corrected by overproducing topo III (topB). Here, DNA gyrase assays in crude cell extracts showed that the ATP-dependent activity (supercoiling) of gyrase but not its ATP-independent activity (relaxation) was inhibited in topA null cells lacking RNase HI. To characterize the cellular event(s) triggered by the absence of RNase HI, we performed a genetic screen for suppressors of the growth defect of topA rnhA null cells. Suppressors affecting genes in replication (holC2::aph and dnaT18::aph) nucleotide metabolism (dcd49::aph), RNA degradation (rne59::aph) and fimbriae synthesis (fimD22::aph) were found to reduce replication from R-loops and to restore supercoiling, thus pointing to a correlation between R-loop-dependent replication in topA rnhA mutants and the inhibition of gyrase activity and growth. Interestingly, the position of fimD on the E. coli chromosome corresponds to the site of one of the five main putative origins of replication from R-loops in rnhA null cells recently identified by next-generation sequencing, thus suggesting that the fimD22::aph mutation inactivated one of these origins. Furthermore, we show that topo III overproduction is unable to complement the growth defect of topA rnhA null mutants at low temperatures that stabilizes hyper-negatively supercoiled DNA. Copyright © 2016 Elsevier B.V. All rights reserved.

Requirement of RecBC enzyme and an elevated level of activated RecA for induced stable DNA replication in Escherichia coli.

PubMed Central

Magee, T R; Kogoma, T

1990-01-01

During SOS induction, Escherichia coli cells acquire the ability to replicate DNA in the absence of protein synthesis, i.e., induced stable DNA replication (iSDR). Initiation of iSDR can occur in the absence of transcription and DnaA protein activity, which are both required for initiation of normal DNA replication at the origin of replication, oriC. In this study we examined the requirement of recB, recC, and recA for the induction and maintenance of iSDR. We found that recB and recC mutations blocked the induction of iSDR by UV irradiation and nalidixic acid treatment. In recB(Ts) strains, iSDR activity induced at 30 degrees C was inhibited by subsequent incubation at 42 degrees C. In addition, iSDR that was induced after heat activation of the RecA441 protein was abolished by the recB21 mutation. These results indicated that the RecBC enzyme was essential not only for SOS signal generation but also for the reinitiation of DNA synthesis following DNA damage. recAo(Con) lexA3(Ind-) strains were found to be capable of iSDR after nalidixic acid treatment, indicating that the derepression of the recA gene and the activation of the elevated level of RecA protein were the necessary and sufficient conditions for the induction of iSDR. PMID:2180906

Electron microscopic studies of bacteriophage M13 DNA replication. [Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allison, D.P.; Ganesan, A.T.; Olson, A.C.

Intracellular forms of M13 phage DNA isolated after infection of Escherichia coli with wild-type phage have been studied by electron microscopy and ultracentrifugation. The data indicate the involvement of rolling-circle intermediates in single-stranded DNA synthesis. In addition to single-stranded, circular DNA, we observed covalently closed and nicked replicative-form (RF) DNAs, dimer RF DNAs, concatenated RF DNAs, RF DNAs with single-stranded tails (sigma, rolling circles), and, occasionally, RF DNAs with theta structures. The tails in sigma molecules are always single stranded and are never longer than the DNA from mature phage; the proportion of sigma to other RF molecules does notmore » change significantly with time after infection. The origin of single-stranded DNA synthesis has been mapped by electron microscopy at a unique location on RF DNA by use of partial denaturation mapping and restriction endonuclease digestion. This location is between gene IV and gene II, and synthesis proceeds in a counterclockwise direction on the conventional genetic map.« less

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

PubMed

Rangarajan, Savithri; Woodgate, Roger; Goodman, Myron F

2002-02-01

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

DNA-directed mutations. Leading and lagging strand specificity

NASA Technical Reports Server (NTRS)

Sinden, R. R.; Hashem, V. I.; Rosche, W. A.

1999-01-01

The fidelity of replication has evolved to reproduce B-form DNA accurately, while allowing a low frequency of mutation. The fidelity of replication can be compromised, however, by defined order sequence DNA (dosDNA) that can adopt unusual or non B-DNA conformations. These alternative DNA conformations, including hairpins, cruciforms, triplex DNAs, and slipped-strand structures, may affect enzyme-template interactions that potentially lead to mutations. To analyze the effect of dosDNA elements on spontaneous mutagenesis, various mutational inserts containing inverted repeats or direct repeats were cloned in a plasmid containing a unidirectional origin of replication and a selectable marker for the mutation. This system allows for analysis of mutational events that are specific for the leading or lagging strands during DNA replication in Escherichia coli. Deletions between direct repeats, involving misalignment stabilized by DNA secondary structure, occurred preferentially on the lagging strand. Intermolecular strand switch events, correcting quasipalindromes to perfect inverted repeats, occurred preferentially during replication of the leading strand.

Characterization of the Campylobacter jejuni cryptic plasmid pTIW94 recovered from wild birds in the southeastern United States.

PubMed

Hiett, Kelli L; Rothrock, Michael J; Seal, Bruce S

2013-09-01

The complete nucleotide sequence was determined for a cryptic plasmid, pTIW94, recovered from several Campylobacter jejuni isolates from wild birds in the southeastern United States. pTIW94 is a circular molecule of 3860 nucleotides, with a G+C content (31.0%) similar to that of many Campylobacter spp. genomes. A typical origin of replication, with iteron sequences, was identified upstream of DNA sequences that demonstrated similarity to replication initiation proteins. A total of five open reading frames (ORFs) were identified; two of the five ORFs demonstrated significant similarity to plasmid pCC2228-2 found within Campylobacter coli. These two ORFs were similar to essential replication proteins RepA (100%; 26/26 aa identity) and RepB (95%; 327/346 aa identity). A third identified ORF demonstrated significant similarity (99%; 421/424 aa identity) to the MOB protein from C. coli 67-8, originally recovered from swine. The other two identified ORFs were either similar to hypothetical proteins from other Campylobacter spp., or exhibited no significant similarity to any DNA or protein sequence in the GenBank database. Promoter regions (-35 and -10 signal sites), ribosomal binding sites upstream of ORFs, and stem-loop structures were also identified within the plasmid. These results demonstrate that pTIW94 represents a previously un-reported small cryptic plasmid with unique sequences as well as highly similar sequences to other small plasmids found within Campylobacter spp., and that this cryptic plasmid is present among Campylobacter spp. recovered from different genera of wild birds. Copyright © 2013. Published by Elsevier Inc.

Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

PubMed Central

Possoz, Christophe; Durand, Adeline; Desfontaines, Jean-Michel; Barre, François-Xavier; Leach, David R. F.

2018-01-01

It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this “σ-replicating chromosome” causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations. PMID:29522563

Replication and Transcription of Eukaryotic DNA in Esherichia coli

PubMed Central

Morrow, John F.; Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Goodman, Howard M.; Helling, Robert B.

1974-01-01

Fragments of amplified Xenopus laevis DNA, coding for 18S and 28S ribosomal RNA and generated by EcoRI restriction endonuclease, have been linked in vitro to the bacterial plasmid pSC101; and the recombinant molecular species have been introduced into E. coli by transformation. These recombinant plasmids, containing both eukaryotic and prokaryotic DNA, replicate stably in E. coli. RNA isolated from E. coli minicells harboring the plasmids hybridizes to amplified X. laevis rDNA. Images PMID:4600264

Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo.

PubMed

Tal, Asaf; Arbel-Goren, Rinat; Costantino, Nina; Court, Donald L; Stavans, Joel

2014-05-20

The search for specific sequences on long genomes is a key process in many biological contexts. How can specific target sequences be located with high efficiency, within physiologically relevant times? We addressed this question for viral integration, a fundamental mechanism of horizontal gene transfer driving prokaryotic evolution, using the infection of Escherichia coli bacteria with bacteriophage λ and following the establishment of a lysogenic state. Following the targeting process in individual live E. coli cells in real time revealed that λ DNA remains confined near the entry point of a cell following infection. The encounter between the 15-bp-long target sequence on the chromosome and the recombination site on the viral genome is facilitated by the directed motion of bacterial DNA generated during chromosome replication, in conjunction with constrained diffusion of phage DNA. Moving the native bacterial integration site to different locations on the genome and measuring the integration frequency in these strains reveals that the frequencies of the native site and a site symmetric to it relative to the origin are similar, whereas both are significantly higher than when the integration site is moved near the terminus, consistent with the replication-driven mechanism we propose. This novel search mechanism is yet another example of the exquisite coevolution of λ with its host.

Phage transposon mutagenesis.

PubMed

Siegrist, M Sloan; Rubin, Eric J

2009-01-01

Phage transduction is an attractive method of genetic manipulation in mycobacteria. PhiMycoMarT7 is well suited for transposon mutagenesis as it is temperature sensitive for replication and contains T7 promoters that promote transcription, a highly active transposase gene, and an Escherichia coli oriR6 K origin of replication. Mycobacterial transposon mutant libraries produced by PhiMycoMarT7 transduction are amenable to both forward and reverse genetic studies. In this protocol, we detail the preparation of PhiMycoMarT7, including a description of the phage, reconstitution of the phage, purification of plaques, preparation of phage stock, and titering of phage stock. We then describe the transduction procedure and finally outline the isolation of individual transposon mutants.

Interrogating the Escherichia coli cell cycle by cell dimension perturbations

PubMed Central

Zheng, Hai; Ho, Po-Yi; Jiang, Meiling; Tang, Bin; Liu, Weirong; Li, Dengjin; Yu, Xuefeng; Kleckner, Nancy E.; Amir, Ariel; Liu, Chenli

2016-01-01

Bacteria tightly regulate and coordinate the various events in their cell cycles to duplicate themselves accurately and to control their cell sizes. Growth of Escherichia coli, in particular, follows a relation known as Schaechter’s growth law. This law says that the average cell volume scales exponentially with growth rate, with a scaling exponent equal to the time from initiation of a round of DNA replication to the cell division at which the corresponding sister chromosomes segregate. Here, we sought to test the robustness of the growth law to systematic perturbations in cell dimensions achieved by varying the expression levels of mreB and ftsZ. We found that decreasing the mreB level resulted in increased cell width, with little change in cell length, whereas decreasing the ftsZ level resulted in increased cell length. Furthermore, the time from replication termination to cell division increased with the perturbed dimension in both cases. Moreover, the growth law remained valid over a range of growth conditions and dimension perturbations. The growth law can be quantitatively interpreted as a consequence of a tight coupling of cell division to replication initiation. Thus, its robustness to perturbations in cell dimensions strongly supports models in which the timing of replication initiation governs that of cell division, and cell volume is the key phenomenological variable governing the timing of replication initiation. These conclusions are discussed in the context of our recently proposed “adder-per-origin” model, in which cells add a constant volume per origin between initiations and divide a constant time after initiation. PMID:27956612

Utilization of Virus ϕCh1 Elements To Establish a Shuttle Vector System for Halo(alkali)philic Archaea via Transformation of Natrialba magadii

PubMed Central

Mayrhofer-Iro, M.; Ladurner, A.; Meissner, C.; Derntl, C.; Reiter, M.; Haider, F.; Dimmel, K.; Rössler, N.; Klein, R.; Baranyi, U.; Scholz, H.

2013-01-01

In the study described here, we successfully developed a transformation system for halo(alkali)philic members of the Archaea. This transformation system comprises a series of Natrialba magadii/Escherichia coli shuttle vectors based on a modified method to transform halophilic members of the Archaea and genomic elements of the N. magadii virus ϕCh1. The shuttle vector pRo-5, based on the repH-containing region of ϕCh1, stably replicated in E. coli and N. magadii and in several halophilic and haloalkaliphilic members of the Archaea not transformable so far. The ϕCh1 operon ORF53/ORF54 (repH) was essential for pRo-5 replication and was thus identified as the minimal replication origin. The plasmid allowed homologous and heterologous gene expression, as exemplified by the expression of ϕCh1 ORF3452, which encodes a structural protein, and the reporter gene bgaH of Haloferax lucentense in N. magadii. The new transformation/vector system will facilitate genetic studies within N. magadii and other haloalkaliphilic archaea and will allow the detailed characterization of the gene functions of N. magadii virus ϕCh1 in their extreme environments. PMID:23416999

An 'instant gene bank' method for gene cloning by mutant complementation.

PubMed

Gems, D; Aleksenko, A; Belenky, L; Robertson, S; Ramsden, M; Vinetski, Y; Clutterbuck, A J

1994-02-01

We describe a new method of gene cloning by complementation of mutant alleles which obviates the need for construction of a gene library in a plasmid vector in vitro and its amplification in Escherichia coli. The method involves simultaneous transformation of mutant strains of the fungus Aspergillus nidulans with (i) fragmented chromosomal DNA from a donor species and (ii) DNA of a plasmid without a selectable marker gene, but with a fungal origin of DNA replication ('helper plasmid'). Transformant colonies appear as the result of the joining of chromosomal DNA fragments carrying the wild-type copies of the mutant allele with the helper plasmid. Joining may occur either by ligation (if the helper plasmid is in linear form) or recombination (if it is cccDNA). This event occurs with high efficiency in vivo, and generates an autonomously replicating plasmid cointegrate. Transformants containing Penicillium chrysogenum genomic DNA complementing A. nidulans niaD, nirA and argB mutations have been obtained. While some of these cointegrates were evidently rearranged or consisted only of unaltered replicating plasmid, in other cases plasmids could be recovered into E. coli and were subsequently shown to contain the selected gene. The utility of this "instant gene bank" technique is demonstrated here by the molecular cloning of the P. canescens trpC gene.

Controlled initiation of chromosomal replication in Escherichia coli requires functional Hda protein.

PubMed

Camara, Johanna Eltz; Skarstad, Kirsten; Crooke, Elliott

2003-05-01

Regulatory inactivation of DnaA helps ensure that the Escherichia coli chromosome is replicated only once per cell cycle, through accelerated hydrolysis of active replication initiator ATP-DnaA to inactive ADP-DnaA. Analysis of deltahda strains revealed that the regulatory inactivation of DnaA component Hda is necessary for maintaining controlled initiation but not for cell growth or viability.

Entry and intracellular replication of Escherichia coli K1 in macrophages require expression of outer membrane protein A.

PubMed

Sukumaran, Sunil K; Shimada, Hiroyuki; Prasadarao, Nemani V

2003-10-01

Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA (+) E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA(+) E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA(+) E. coli by RAW 264.7 cells occurred by both actin- and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.

Suppression of the Escherichia coli dnaA46 mutation by changes in the activities of the pyruvate-acetate node links DNA replication regulation to central carbon metabolism.

PubMed

Tymecka-Mulik, Joanna; Boss, Lidia; Maciąg-Dorszyńska, Monika; Matias Rodrigues, João F; Gaffke, Lidia; Wosinski, Anna; Cech, Grzegorz M; Szalewska-Pałasz, Agnieszka; Węgrzyn, Grzegorz; Glinkowska, Monika

2017-01-01

To ensure faithful transmission of genetic material to progeny cells, DNA replication is tightly regulated, mainly at the initiation step. Escherichia coli cells regulate the frequency of initiation according to growth conditions. Results of the classical, as well as the latest studies, suggest that the DNA replication in E. coli starts at a predefined, constant cell volume per chromosome but the mechanisms coordinating DNA replication with cell growth are still not fully understood. Results of recent investigations have revealed a role of metabolic pathway proteins in the control of cell division and a direct link between metabolism and DNA replication has also been suggested both in Bacillus subtilis and E. coli cells. In this work we show that defects in the acetate overflow pathway suppress the temperature-sensitivity of a defective replication initiator-DnaA under acetogenic growth conditions. Transcriptomic and metabolic analyses imply that this suppression is correlated with pyruvate accumulation, resulting from alterations in the pyruvate dehydrogenase (PDH) activity. Consequently, deletion of genes encoding the pyruvate dehydrogenase subunits likewise resulted in suppression of the thermal-sensitive growth of the dnaA46 strain. We propose that the suppressor effect may be directly related to the PDH complex activity, providing a link between an enzyme of the central carbon metabolism and DNA replication.

Cloning of an origin of DNA replication of Xenopus laevis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, S.; Taylor, J.H.

1980-09-01

DNA fragments of Xenopus laevis, the African frog, were cloned in the EcoRI site of the Eschrichia coli plasmid pACYC189 and tested for ability to initiate and complete replication of the recombinant plasmid when injected into unfertilized eggs of X. laevis. After measurement of the (/sup 3/H)-thymidine incorporation per egg for a number of recombinant plasmids, pSW14 and pSW9, which respectively contain a small segment (550 base pairs) and several kilobases of frog DNA, were selected for more extensive analysis. In spite of the small size of th segment in pSW14, it incorporates in 2 hr at least 3 timesmore » as much labeled thymidine as either pSW9 or the vector alone. To determine the number of replications of pSW14, a novel method was employed. The results showed that about 50% of the labeled, supercoiled DNA recovered from eggs after 4 hr was sensitive to EcoRI digestion, which indicates that most of the DNA that incorporated (/sup 3/H)thymidine had replicated twice during the 4 hr in the unfertilized eggs of X. laevis. We conclude the pSW14 has a functional origin in the Xenopus DNA segment.« less

The positioning logic and copy number control of genes in bacteria under stress

NASA Astrophysics Data System (ADS)

Zhang, Qiucen; Austin, Robert; Vyawahare, Saurabh; Lau, Alexandra

2013-03-01

Escherichia coli (E. coli) cells when challenged with sublethal concentrations of the genotoxic antibiotic ciprofloxacin cease to divide and form long filaments which contain multiple bacterial chromosomes. These filaments are individual mesoscopic environmental niches which provide protection for a community of chromosomes (as opposed to cells) under mutagenic stress and can provide an evolutionary fitness advantage within the niche. We use comparative genomic hybridization to show that the mesoscopic niche evolves within 20 minutes of ciprofloxacin exposure via replication of multiple copies of genes expressing ATP dependent transporters. We show that this rapid genomic amplification is done in a time efficient manner via placement of the genes encoding the pumps near the origin of replication on the bacterial chromosome. The de-amplification of multiple copies back to the wild type number is a function of the duration is a function of the ciprofloxacin exposure duration: the longer the exposure, the slower the removal of the multiple copies. The project described was supported by the National Science Foundation and the National Cancer Institute

Physical interactions between bacteriophage and Escherichia coli proteins required for initiation of lambda DNA replication.

PubMed

Liberek, K; Osipiuk, J; Zylicz, M; Ang, D; Skorko, J; Georgopoulos, C

1990-02-25

The process of initiation of lambda DNA replication requires the assembly of the proper nucleoprotein complex at the origin of replication, ori lambda. The complex is composed of both phage and host-coded proteins. The lambda O initiator protein binds specifically to ori lambda. The lambda P initiator protein binds to both lambda O and the host-coded dnaB helicase, giving rise to an ori lambda DNA.lambda O.lambda P.dnaB structure. The dnaK and dnaJ heat shock proteins have been shown capable of dissociating this complex. The thus freed dnaB helicase unwinds the duplex DNA template at the replication fork. In this report, through cross-linking, size chromatography, and protein affinity chromatography, we document some of the protein-protein interactions occurring at ori lambda. Our results show that the dnaK protein specifically interacts with both lambda O and lambda P, and that the dnaJ protein specifically interacts with the dnaB helicase.

Recombinant Clone Heterogeneity in ESCHERICHIA COLI Conjunction: Effect of Ph and Partially Replicated Recipient Deoxyribonucleic Acid

PubMed Central

Ou, Jonathan T.

1975-01-01

At pH 6.8, a substantial fraction of recombinant colonies obtained from conjugation with an HfrH donor contained multiple recombinant classes in a single colony (polygenotypic colony). In contrast, when the conjugation was performed at pH 7.6, the number of polygenotypic colonies was drastically reduced, and the recombinant colonies were predominantly monogenotypic or digenotypic. Genetic analysis revealed that the digenotypic recombinants differ in those donor markers near the origin of DNA replication but share those donor markers near the terminus. This integration pattern suggests that the formation of digenotypic recombinants involves recombination of a single copy of the exogenome with a partially replicated recipient DNA molecule. This suggestion was supported by examination of the genotype of recombinant colonies recovered from crosses with an HfrKL96 donor which was derived from HfrH but transfers its chromosome in the reverse direction. PMID:8360

Slow Joining of Newly Replicated DNA Chains in DNA Polymerase I-Deficient Escherichia coli Mutants*

PubMed Central

Okazaki, Reiji; Arisawa, Mikio; Sugino, Akio

1971-01-01

In Escherichia coli mutants deficient in DNA polymerase I, newly replicated short DNA is joined at about 10% of the rate in the wild-type strains. It is postulated that DNA polymerase I normally functions in filling gaps between the nascent short segments synthesized by the replication complex. Possible implications of the finding are discussed in relation to other abnormal properties of these mutants. PMID:4943548

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

PubMed Central

Alfano, C; McMacken, R

1988-01-01

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands. Images PMID:2847118

Roles of Long and Short Replication Initiation Proteins in the Fate of IncP-1 Plasmids

PubMed Central

Yano, Hirokazu; Deckert, Gail E.; Rogers, Linda M.

2012-01-01

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts. PMID:22228734

Alteration in levels of unsaturated fatty acids in mutants of Escherichia coli defective in DNA replication.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-07-01

We previously reported that mutations in the dnaA gene which encodes the initiator of chromosomal DNA replication in Escherichia coli caused an alteration in the levels of unsaturated fatty acids of phospholipids in membranes. In this study, we examined fatty acid compositions in other mutants which are defective in DNA replication. As in the case of temperature-sensitive dnaA mutants, temperature-sensitive dnaC and dnaE mutants, which have defects in initiation and elongation, respectively, of DNA replication showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) compared with the wild-type strain, especially at high temperatures. On the other hand, temperature-sensitive mutants defective in cellular processes other than DNA replication, such as RNA synthesis and cell division, did not show a lower level of unsaturation of fatty acids compared with the wild-type strain. These results suggest that the inhibition of DNA replication causes a lower level of unsaturation of fatty acids in Escherichia coli cells.

Electrotransformation of highly DNA-restrictive corynebacteria with synthetic DNA.

PubMed

Ankri, S; Reyes, O; Leblon, G

1996-01-01

Highly DNA-restrictive Corynebacteria can be transformed with DNA made in vitro by PCR amplification of a sequence that contains the replication origin of pBL1, a plasmid common to many Corynebacteria. In all strains examined, the transformation efficiencies of PCR-synthetized DNA equal or improve the performances of heterologous DNA extracted from wild-type and dam(-)-dcm-strains of Escherichia coli. The transformation efficiencies obtained with PCR-made DNA may be high enough to permit its general application to experiments of gene integration.

Properties of new Escherichia coli Hfr strains constructed by integration of pSC101-derived conjugative plasmids.

PubMed Central

François, V; Conter, A; Louarn, J M

1990-01-01

Conjugative temperature-sensitive plasmids were derived from pSC101. These plasmids are useful in genetic analysis for two reasons: (i) they render possible the construction of new Hfr lines by plasmid integration at predetermined chromosomal loci via Tn10 inverse transposition, and (ii) the Hfr characters are transducible via bacteriophage P1. We also showed that replication from pSC101 origin is deleterious for the plasmid-chromosome fusion. PMID:2155201

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage

PubMed Central

Zygiel, Emily M.; Noren, Karen A.; Adamkiewicz, Marta A.; Aprile, Richard J.; Bowditch, Heather K.; Carroll, Christine L.; Cerezo, Maria Abigail S.; Dagher, Adelle M.; Hebert, Courtney R.; Hebert, Lauren E.; Mahame, Gloria M.; Milne, Stephanie C.; Silvestri, Kelly M.; Sutherland, Sara E.; Sylvia, Alexandria M.; Taveira, Caitlyn N.; VanValkenburgh, David J.; Noren, Christopher J.

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5’-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5’-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries. PMID:28445507

Various mutations compensate for a deleterious lacZα insert in the replication enhancer of M13 bacteriophage.

PubMed

Zygiel, Emily M; Noren, Karen A; Adamkiewicz, Marta A; Aprile, Richard J; Bowditch, Heather K; Carroll, Christine L; Cerezo, Maria Abigail S; Dagher, Adelle M; Hebert, Courtney R; Hebert, Lauren E; Mahame, Gloria M; Milne, Stephanie C; Silvestri, Kelly M; Sutherland, Sara E; Sylvia, Alexandria M; Taveira, Caitlyn N; VanValkenburgh, David J; Noren, Christopher J; Hall, Marilena Fitzsimons

2017-01-01

M13 and other members of the Ff class of filamentous bacteriophages have been extensively employed in myriad applications. The Ph.D. series of phage-displayed peptide libraries were constructed from the M13-based vector M13KE. As a direct descendent of M13mp19, M13KE contains the lacZα insert in the intergenic region between genes IV and II, where it interrupts the replication enhancer of the (+) strand origin. Phage carrying this 816-nucleotide insert are viable, but propagate in E. coli at a reduced rate compared to wild-type M13 phage, presumably due to a replication defect caused by the insert. We have previously reported thirteen compensatory mutations in the 5'-untranslated region of gene II, which encodes the replication initiator protein gIIp. Here we report several additional mutations in M13KE that restore a wild-type propagation rate. Several clones from constrained-loop variable peptide libraries were found to have ejected the majority of lacZα gene in order to reconstruct the replication enhancer, albeit with a small scar. In addition, new point mutations in the gene II 5'-untranslated region or the gene IV coding sequence have been spontaneously observed or synthetically engineered. Through phage propagation assays, we demonstrate that all these genetic modifications compensate for the replication defect in M13KE and restore the wild-type propagation rate. We discuss the mechanisms by which the insertion and ejection of the lacZα gene, as well as the mutations in the regulatory region of gene II, influence the efficiency of replication initiation at the (+) strand origin. We also examine the presence and relevance of fast-propagating mutants in phage-displayed peptide libraries.

Control of Initiation of DNA Replication in Bacillus subtilis and Escherichia coli

PubMed Central

Jameson, Katie H.; Wilkinson, Anthony J.

2017-01-01

Initiation of DNA Replication is tightly regulated in all cells since imbalances in chromosomal copy number are deleterious and often lethal. In bacteria such as Bacillus subtilis and Escherichia coli, at the point of cytokinesis, there must be two complete copies of the chromosome to partition into the daughter cells following division at mid-cell during vegetative growth. Under conditions of rapid growth, when the time taken to replicate the chromosome exceeds the doubling time of the cells, there will be multiple initiations per cell cycle and daughter cells will inherit chromosomes that are already undergoing replication. In contrast, cells entering the sporulation pathway in B. subtilis can do so only during a short interval in the cell cycle when there are two, and only two, chromosomes per cell, one destined for the spore and one for the mother cell. Here, we briefly describe the overall process of DNA replication in bacteria before reviewing initiation of DNA replication in detail. The review covers DnaA-directed assembly of the replisome at oriC and the multitude of mechanisms of regulation of initiation, with a focus on the similarities and differences between E. coli and B. subtilis. PMID:28075389

Agrobacterium tumefaciens supports DNA replication of diverse geminivirus types.

PubMed

Selth, Luke A; Randles, John W; Rezaian, M Ali

2002-04-10

We have previously shown that the soil-borne plant pathogen Agrobacterium tumefaciens supports the replication of tomato leaf curl geminivirus (Australian isolate) (TLCV) DNA. However, the reproducibility of this observation with other geminiviruses has been questioned. Here, we show that replicative DNA forms of three other geminiviruses also accumulate at varying levels in Agrobacterium. Geminiviral DNA constructs that lacked the ability to replicate in Agrobacterium were rendered replication-competent by changing their configuration so that two copies of the viral ori were present. Furthermore, we report that low-level replication of TLCV DNA can occur in Escherichia coli containing a dimeric TLCV construct in a high copy number plasmid. These findings were reinforced by expression studies using beta-glucuronidase which revealed that all six TLCV promoters are active in Agrobacterium, and two are functional in E. coli.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader.

PubMed

Rock, Jeremy M; Lang, Ulla F; Chase, Michael R; Ford, Christopher B; Gerrick, Elias R; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah M; Lamers, Meindert H

2015-06-01

The DNA replication machinery is an important target for antibiotic development in increasingly drug-resistant bacteria, including Mycobacterium tuberculosis. Although blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In Escherichia coli, the proofreading subunit of the replisome, the ɛ exonuclease, is essential for high-fidelity DNA replication; however, we find that the corresponding subunit is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase DnaE1 itself encodes an editing function that proofreads DNA replication, mediated by an intrinsic 3'-5' exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by more than 3,000-fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP domain-mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader.

DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader

PubMed Central

Rock, Jeremy M.; Lang, Ulla F.; Chase, Michael R.; Ford, Christopher B.; Gerrick, Elias R.; Gawande, Richa; Coscolla, Mireia; Gagneux, Sebastien; Fortune, Sarah M.; Lamers, Meindert H.

2015-01-01

The DNA replication machinery is an important target for antibiotic development for increasingly drug resistant bacteria including Mycobacterium tuberculosis1. While blocking DNA replication leads to cell death, disrupting the processes used to ensure replication fidelity can accelerate mutation and the evolution of drug resistance. In E. coli, the proofreading subunit of the replisome, the ε-exonuclease, is essential for high fidelity DNA replication2; however, we find that it is completely dispensable in M. tuberculosis. Rather, the mycobacterial replicative polymerase, DnaE1, encodes a novel editing function that proofreads DNA replication, mediated by an intrinsic 3′-5′ exonuclease activity within its PHP domain. Inactivation of the DnaE1 PHP domain increases the mutation rate by greater than 3,000 fold. Moreover, phylogenetic analysis of DNA replication proofreading in the bacterial kingdom suggests that E. coli is a phylogenetic outlier and that PHP-domain mediated proofreading is widely conserved and indeed may be the ancestral prokaryotic proofreader. PMID:25894501

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

PubMed

Osipiuk, J; Zylicz, M

1991-01-01

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

New Therapeutic Strategies for Antibiotic-Resistant Select Agents

DTIC Science & Technology

2007-12-31

activity possibly due to inherent differences between mesophilic versus thermophilic replication enzymes (Figure 7A). E. coli helicase stimulated Y...primase activity possibly due to inherent differences between mesophilic versus thermophilic replication enzymes (Figure 7A). B E. coli...either lower the enzyme and template to 20 nM, or use higher compound concentrations that are the same as the enzyme and template. A series of

Secretome Biomarkers for the Identification and Differentiation of Enterohemorrhagic and Enteropathogenic Escherichia coli Strains

DTIC Science & Technology

2013-09-01

SbBS512_E4084 Shigella byodii /EC NC101 ND ND ND EC: E. coli ND: not determined 8 Table 2. Common Strain-Unique Proteins from Replicate...E24377A- Escherichia coli str. K-12 substr. MG1655- Escherichia coli SE11- Escherichia coli- W3110 Shigella boy dii CDC 3083-94- Shigella boy dii Sb227

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector▿

PubMed Central

Lee, Ju-Hoon; Halgerson, Jamie S.; Kim, Jeong-Hwan; O'Sullivan, Daniel J.

2007-01-01

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria. PMID:17526779

GeneGuard: A modular plasmid system designed for biosafety.

PubMed

Wright, Oliver; Delmans, Mihails; Stan, Guy-Bart; Ellis, Tom

2015-03-20

Synthetic biology applications in biosensing, bioremediation, and biomining envision the use of engineered microbes beyond a contained laboratory. Deployment of such microbes in the environment raises concerns of unchecked cellular proliferation or unwanted spread of synthetic genes. While antibiotic-resistant plasmids are the most utilized vectors for introducing synthetic genes into bacteria, they are also inherently insecure, acting naturally to propagate DNA from one cell to another. To introduce security into bacterial synthetic biology, we here took on the task of completely reformatting plasmids to be dependent on their intended host strain and inherently disadvantageous for others. Using conditional origins of replication, rich-media compatible auxotrophies, and toxin-antitoxin pairs we constructed a mutually dependent host-plasmid platform, called GeneGuard. In this, replication initiators for the R6K or ColE2-P9 origins are provided in trans by a specified host, whose essential thyA or dapA gene is translocated from a genomic to a plasmid location. This reciprocal arrangement is stable for at least 100 generations without antibiotic selection and is compatible for use in LB medium and soil. Toxin genes ζ or Kid are also employed in an auxiliary manner to make the vector disadvantageous for strains not expressing their antitoxins. These devices, in isolation and in concert, severely reduce unintentional plasmid propagation in E. coli and B. subtilis and do not disrupt the intended E. coli host's growth dynamics. Our GeneGuard system comprises several versions of modular cargo-ready vectors, along with their requisite genomic integration cassettes, and is demonstrated here as an efficient vector for heavy-metal biosensors.

Recombinational Repair of DNA Damage in Escherichia coli and Bacteriophage λ

PubMed Central

Kuzminov, Andrei

1999-01-01

Although homologous recombination and DNA repair phenomena in bacteria were initially extensively studied without regard to any relationship between the two, it is now appreciated that DNA repair and homologous recombination are related through DNA replication. In Escherichia coli, two-strand DNA damage, generated mostly during replication on a template DNA containing one-strand damage, is repaired by recombination with a homologous intact duplex, usually the sister chromosome. The two major types of two-strand DNA lesions are channeled into two distinct pathways of recombinational repair: daughter-strand gaps are closed by the RecF pathway, while disintegrated replication forks are reestablished by the RecBCD pathway. The phage λ recombination system is simpler in that its major reaction is to link two double-stranded DNA ends by using overlapping homologous sequences. The remarkable progress in understanding the mechanisms of recombinational repair in E. coli over the last decade is due to the in vitro characterization of the activities of individual recombination proteins. Putting our knowledge about recombinational repair in the broader context of DNA replication will guide future experimentation. PMID:10585965

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response.

PubMed

Mori, Tetsuya; Nakamura, Tatsuro; Okazaki, Naoto; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2012-01-01

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.

Homology between Escherichia coli plasmids ColE1 and p15A.

PubMed Central

Bird, R E

1981-01-01

The location and extent of the homology between plasmids ColE1 and p15A were determined by analysis of heteroduplexes formed between them as well as with a related plasmid, pBR322, and by hybridization of radioactive deoxyribonucleic acids to restriction fragments of p15A and ColE1. The homology between the plasmids contained the entire region of ColE1 required for its replication as well as an additional 400 base pairs downstream from the origin of replication. This region on p15A, which was 980 +/- 43 base pairs, started at 0.1 of the molecular length from one end formed by cleavage with the restriction endonuclease BglI and extended to 0.54 of the molecular length from the same end. Restriction cleavage maps for the enzymes BglI, HpaI, HaeII, HaeIII, and HincII are also presented. Images PMID:6259130

Plasmid Vectors for Xylella fastidiosa Utilizing a Toxin-Antitoxin System for Stability in the Absence of Antibiotic Selection.

PubMed

Burbank, Lindsey P; Stenger, Drake C

2016-08-01

The phytopathogen Xylella fastidiosa causes disease in a variety of important crop and landscape plants. Functional genetic studies have led to a broader understanding of virulence mechanisms used by this pathogen in the grapevine host. Plasmid shuttle vectors are important tools in studies of bacterial genetics but there are only a limited number of plasmid vectors available that replicate in X. fastidiosa, and even fewer that are retained without antibiotic selection. Two plasmids are described here that show stable replication in X. fastidiosa and are effective for gene complementation both in vitro and in planta. Plasmid maintenance is facilitated by incorporation of the PemI/PemK plasmid addiction system, consisting of PemK, an endoribonuclease toxin, and its cognate antitoxin, PemI. Vector pXf20pemIK utilizes a native X. fastidiosa replication origin as well as a high-copy-number pUC origin for propagation in Escherichia coli cloning strains. Broad-host-range vector pBBR5pemIK is a medium- to low-copy-number plasmid based on the pBBR1 backbone. Both plasmids are maintained for extended periods of time in the absence of antibiotic selection, as well as up to 14 weeks in grapevine, without affecting bacterial fitness. These plasmids present an alternative to traditional complementation and expression vectors which rely on antibiotic selection for plasmid retention.

Structure-Function Aspects of Membrane Associated Prokaryotic DNA replication

DTIC Science & Technology

1994-09-01

Membrane associated DNA replication in prokaryotes has been studied intensively using two model systems, Bacillus subtilis and plasmid RK2 cultured...in its Escherichia coli host. In the former a new membrane protein that had previously been found to act as an inhibitor of DNA replication was...prior to a round of DNA replication . In the latter, plasmid DNA replication has been found to be associated with the inner but not outer membrane of

Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase

PubMed Central

Afonso, José P.; Chintakayala, Kiran; Suwannachart, Chatrudee; Sedelnikova, Svetlana; Giles, Kevin; Hoyes, John B.; Soultanas, Panos; Rafferty, John B.; Oldham, Neil J.

2013-01-01

The clamp-loader complex plays a crucial role in DNA replication by loading the β-clamp onto primed DNA to be used by the replicative polymerase. Relatively little is known about the stoichiometry, structure and assembly pathway of this complex, and how it interacts with the replicative helicase, in Gram-positive organisms. Analysis of full and partial complexes by mass spectrometry revealed that a hetero-pentameric τ3-δ-δ′ Bacillus subtilis clamp-loader assembles via multiple pathways, which differ from those exhibited by the Gram-negative model Escherichia coli. Based on this information, a homology model of the B. subtilis τ3-δ-δ′ complex was constructed, which revealed the spatial positioning of the full C-terminal τ domain. The structure of the δ subunit was determined by X-ray crystallography and shown to differ from that of E. coli in the nature of the amino acids comprising the τ and δ′ binding regions. Most notably, the τ-δ interaction appears to be hydrophilic in nature compared with the hydrophobic interaction in E. coli. Finally, the interaction between τ3 and the replicative helicase DnaB was driven by ATP/Mg2+ conformational changes in DnaB, and evidence is provided that hydrolysis of one ATP molecule by the DnaB hexamer is sufficient to stabilize its interaction with τ3. PMID:23525462

Recombinase and translesion DNA polymerase decrease the speed of replication fork progression during the DNA damage response in Escherichia coli cells

PubMed Central

Tan, Kang Wei; Pham, Tuan Minh; Furukohri, Asako; Maki, Hisaji; Akiyama, Masahiro Tatsumi

2015-01-01

The SOS response is a DNA damage response pathway that serves as a general safeguard of genome integrity in bacteria. Extensive studies of the SOS response in Escherichia coli have contributed to establishing the key concepts of cellular responses to DNA damage. However, how the SOS response impacts on the dynamics of DNA replication fork movement remains unknown. We found that inducing the SOS response decreases the mean speed of individual replication forks by 30–50% in E. coli cells, leading to a 20–30% reduction in overall DNA synthesis. dinB and recA belong to a group of genes that are upregulated during the SOS response, and encode the highly conserved proteins DinB (also known as DNA polymerase IV) and RecA, which, respectively, specializes in translesion DNA synthesis and functions as the central recombination protein. Both genes were independently responsible for the SOS-dependent slowdown of replication fork progression. Furthermore, fork speed was reduced when each gene was ectopically expressed in SOS-uninduced cells to the levels at which they are expressed in SOS-induced cells. These results clearly indicate that the increased expression of dinB and recA performs a novel role in restraining the progression of an unperturbed replication fork during the SOS response. PMID:25628359

Identification of Proteins Required for Repair of Double-Strand Chromosome Breaks, a Predisposing Factor in Breast Cancer

DTIC Science & Technology

2001-06-01

enzymatic apparatus needed to initiate DNA replication on recombination intermediates. Escherichia coli PriA protein was found to play a critical function in...the transition from recombination to DNA replication . PriA specifically binds to forked DNA structures created by recombination or replication fork

Rv0004 is a new essential member of the mycobacterial DNA replication machinery

PubMed Central

Hooppaw, Anna J.; Richardson, Kirill; Lee, Hark Joon; Kimmey, Jacqueline M.; Aldridge, Bree B.

2017-01-01

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004’s DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes. PMID:29176877

Rv0004 is a new essential member of the mycobacterial DNA replication machinery.

PubMed

Mann, Katherine M; Huang, Deborah L; Hooppaw, Anna J; Logsdon, Michelle M; Richardson, Kirill; Lee, Hark Joon; Kimmey, Jacqueline M; Aldridge, Bree B; Stallings, Christina L

2017-11-01

DNA replication is fundamental for life, yet a detailed understanding of bacterial DNA replication is limited outside the organisms Escherichia coli and Bacillus subtilis. Many bacteria, including mycobacteria, encode no identified homologs of helicase loaders or regulators of the initiator protein DnaA, despite these factors being essential for DNA replication in E. coli and B. subtilis. In this study we discover that a previously uncharacterized protein, Rv0004, from the human pathogen Mycobacterium tuberculosis is essential for bacterial viability and that depletion of Rv0004 leads to a block in cell cycle progression. Using a combination of genetic and biochemical approaches, we found that Rv0004 has a role in DNA replication, interacts with DNA and the replicative helicase DnaB, and affects DnaB-DnaA complex formation. We also identify a conserved domain in Rv0004 that is predicted to structurally resemble the N-terminal protein-protein interaction domain of DnaA. Mutation of a single conserved tryptophan within Rv0004's DnaA N-terminal-like domain leads to phenotypes similar to those observed upon Rv0004 depletion and can affect the association of Rv0004 with DnaB. In addition, using live cell imaging during depletion of Rv0004, we have uncovered a previously unappreciated role for DNA replication in coordinating mycobacterial cell division and cell size. Together, our data support that Rv0004 encodes a homolog of the recently identified DciA family of proteins found in most bacteria that lack the DnaC-DnaI helicase loaders in E. coli and B. subtilis. Therefore, the mechanisms of Rv0004 elucidated here likely apply to other DciA homologs and reveal insight into the diversity of bacterial strategies in even the most conserved biological processes.

R-loop-mediated genomic instability is caused by impairment of replication fork progression

PubMed Central

Gan, Wenjian; Guan, Zhishuang; Liu, Jie; Gui, Ting; Shen, Keng; Manley, James L.; Li, Xialu

2011-01-01

Transcriptional R loops are anomalous RNA:DNA hybrids that have been detected in organisms from bacteria to humans. These structures have been shown in eukaryotes to result in DNA damage and rearrangements; however, the mechanisms underlying these effects have remained largely unknown. To investigate this, we first show that R-loop formation induces chromosomal DNA rearrangements and recombination in Escherichia coli, just as it does in eukaryotes. More importantly, we then show that R-loop formation causes DNA replication fork stalling, and that this in fact underlies the effects of R loops on genomic stability. Strikingly, we found that attenuation of replication strongly suppresses R-loop-mediated DNA rearrangements in both E. coli and HeLa cells. Our findings thus provide a direct demonstration that R-loop formation impairs DNA replication and that this is responsible for the deleterious effects of R loops on genome stability from bacteria to humans. PMID:21979917

Chk1 promotes replication fork progression by controlling replication initiation

PubMed Central

Petermann, Eva; Woodcock, Mick; Helleday, Thomas

2010-01-01

DNA replication starts at initiation sites termed replication origins. Metazoan cells contain many more potential origins than are activated (fired) during each S phase. Origin activation is controlled by the ATR checkpoint kinase and its downstream effector kinase Chk1, which suppresses origin firing in response to replication blocks and during normal S phase by inhibiting the cyclin-dependent kinase Cdk2. In addition to increased origin activation, cells deficient in Chk1 activity display reduced rates of replication fork progression. Here we investigate the causal relationship between increased origin firing and reduced replication fork progression. We use the Cdk inhibitor roscovitine or RNAi depletion of Cdc7 to inhibit origin firing in Chk1-inhibited or RNAi-depleted cells. We report that Cdk inhibition and depletion of Cdc7 can alleviate the slow replication fork speeds in Chk1-deficient cells. Our data suggest that increased replication initiation leads to slow replication fork progression and that Chk1 promotes replication fork progression during normal S phase by controlling replication origin activity. PMID:20805465

Isolation of a novel plasmid from Couchioplanes caeruleus and construction of two plasmid vectors for gene expression in Actinoplanes missouriensis.

PubMed

Jang, Moon-Sun; Fujita, Azusa; Ikawa, Satomi; Hanawa, Keitaro; Yamamura, Hideki; Tamura, Tomohiko; Hayakawa, Masayuki; Tezuka, Takeaki; Ohnishi, Yasuo

2015-01-01

To date, no plasmid vector has been developed for the rare actinomycete Actinoplanes missouriensis. Moreover, no small circular plasmid has been reported to exist in the genus Actinoplanes. Here, a novel plasmid, designated pCAZ1, was isolated from Couchioplanes caeruleus subsp. azureus via screening for small circular plasmids in Actinoplanes (57 strains) and Couchioplanes (2 strains). Nucleotide sequencing revealed that pCAZ1 is a 5845-bp circular molecule with a G + C content of 67.5%. The pCAZ1 copy number was estimated at 30 per chromosome. pCAZ1 contains seven putative open reading frames, one of which encodes a protein containing three motifs conserved among plasmid-encoded replication proteins that are involved in the rolling-circle mechanism of replication. Detection of single-stranded DNA intermediates in C. caeruleus confirmed that pCAZ1 replicates by this mechanism. The ColE1 origin from pBluescript SK(+) and the oriT sequence with the apramycin resistance gene aac(3)IV from pIJ773 were inserted together into pCAZ1, to construct the Escherichia coli-A. missouriensis shuttle vectors, pCAM1 and pCAM2, in which the foreign DNA fragment was inserted into pCAZ1 in opposite directions. pCAM1 and pCAM2 were successfully transferred to A. missouriensis through the E. coli-mediated conjugative transfer system. The copy numbers of pCAM1 and pCAM2 in A. missouriensis were estimated to be one and four per chromosome, respectively. Thus, these vectors can be used as effective genetic tools for homologous and heterologous gene expression studies in A. missouriensis. Copyright © 2014 Elsevier Inc. All rights reserved.

The activities of eukaryotic replication origins in chromatin.

PubMed

Weinreich, Michael; Palacios DeBeer, Madeleine A; Fox, Catherine A

2004-03-15

DNA replication initiates at chromosomal positions called replication origins. This review will focus on the activity, regulation and roles of replication origins in Saccharomyces cerevisiae. All eukaryotic cells, including S. cerevisiae, depend on the initiation (activity) of hundreds of replication origins during a single cell cycle for the duplication of their genomes. However, not all origins are identical. For example, there is a temporal order to origin activation with some origins firing early during the S-phase and some origins firing later. Recent studies provide evidence that posttranslational chromatin modifications, heterochromatin-binding proteins and nucleosome positioning can control the efficiency and/or timing of chromosomal origin activity in yeast. Many more origins exist than are necessary for efficient replication. The availability of excess replication origins leaves individual origins free to evolve distinct forms of regulation and/or roles in chromosomes beyond their fundamental role in DNA synthesis. We propose that some origins have acquired roles in controlling chromatin structure and/or gene expression. These roles are not linked obligatorily to replication origin activity per se, but instead exploit multi-subunit replication proteins with the potential to form context-dependent protein-protein interactions.

PSYCHOLOGY. Estimating the reproducibility of psychological science.

PubMed

2015-08-28

Reproducibility is a defining feature of science, but the extent to which it characterizes current research is unknown. We conducted replications of 100 experimental and correlational studies published in three psychology journals using high-powered designs and original materials when available. Replication effects were half the magnitude of original effects, representing a substantial decline. Ninety-seven percent of original studies had statistically significant results. Thirty-six percent of replications had statistically significant results; 47% of original effect sizes were in the 95% confidence interval of the replication effect size; 39% of effects were subjectively rated to have replicated the original result; and if no bias in original results is assumed, combining original and replication results left 68% with statistically significant effects. Correlational tests suggest that replication success was better predicted by the strength of original evidence than by characteristics of the original and replication teams. Copyright © 2015, American Association for the Advancement of Science.

Spatiotemporal coupling and decoupling of gene transcription with DNA replication origins during embryogenesis in C. elegans

PubMed Central

Pourkarimi, Ehsan; Bellush, James M; Whitehouse, Iestyn

2016-01-01

The primary task of developing embryos is genome replication, yet how DNA replication is integrated with the profound cellular changes that occur through development is largely unknown. Using an approach to map DNA replication at high resolution in C. elegans, we show that replication origins are marked with specific histone modifications that define gene enhancers. We demonstrate that the level of enhancer associated modifications scale with the efficiency at which the origin is utilized. By mapping replication origins at different developmental stages, we show that the positions and activity of origins is largely invariant through embryogenesis. Contrary to expectation, we find that replication origins are specified prior to the broad onset of zygotic transcription, yet when transcription initiates it does so in close proximity to the pre-defined replication origins. Transcription and DNA replication origins are correlated, but the association breaks down when embryonic cell division ceases. Collectively, our data indicate that replication origins are fundamental organizers and regulators of gene activity through embryonic development. DOI: http://dx.doi.org/10.7554/eLife.21728.001 PMID:28009254

Structure of nascent replicative form DNA of coliphage M13

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

Nascent replicative form type II (RFII) DNA of coliphage M13 synthesized in an Escherichia coli mutant deficient in the 5' ..-->.. 3' exonuclease associated with DNA polymerase I contains ribonucleotides that are retained in the covalently closed RFI DNA sealed in vitro by the joint action of T5 phage DNA polymerase and T4 phage DNA ligase. These RFI molecules are labile to alkali and RNase H, unlike the RFI produced either in vivo or from RFII with E. coli DNA polymerase I and E. coli DNA ligase. The ribonucleotides are located at one site and predominantly in one strand ofmore » the nascent RF DNA. Furthermore, these molecules contain multiple small gaps, randomly located, and one large gap in the intracistronic region.« less

The temporal program of chromosome replication: genomewide replication in clb5{Delta} Saccharomyces cerevisiae.

PubMed

McCune, Heather J; Danielson, Laura S; Alvino, Gina M; Collingwood, David; Delrow, Jeffrey J; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

2008-12-01

Temporal regulation of origin activation is widely thought to explain the pattern of early- and late-replicating domains in the Saccharomyces cerevisiae genome. Recently, single-molecule analysis of replication suggested that stochastic processes acting on origins with different probabilities of activation could generate the observed kinetics of replication without requiring an underlying temporal order. To distinguish between these possibilities, we examined a clb5Delta strain, where origin firing is largely limited to the first half of S phase, to ask whether all origins nonspecifically show decreased firing (as expected for disordered firing) or if only some origins ("late" origins) are affected. Approximately half the origins in the mutant genome show delayed replication while the remainder replicate largely on time. The delayed regions can encompass hundreds of kilobases and generally correspond to regions that replicate late in wild-type cells. Kinetic analysis of replication in wild-type cells reveals broad windows of origin firing for both early and late origins. Our results are consistent with a temporal model in which origins can show some heterogeneity in both time and probability of origin firing, but clustering of temporally like origins nevertheless yields a genome that is organized into blocks showing different replication times.

Modelling growth of Escherichia coli O157:H7 in fresh-cut lettuce submitted to commercial process conditions: chlorine washing and modified atmosphere packaging.

PubMed

Posada-Izquierdo, Guiomar D; Pérez-Rodríguez, Fernando; López-Gálvez, Francisco; Allende, Ana; Selma, María V; Gil, María I; Zurera, Gonzalo

2013-04-01

Fresh-cut iceberg lettuce inoculated with Escherichia coli O157:H7 was submitted to chlorine washing (150 mg/mL) and modified atmosphere packaging on laboratory scale. Populations of E. coli O157:H7 were assessed in fresh-cut lettuce stored at 4, 8, 13 and 16 °C using 6-8 replicates in each analysis point in order to capture experimental variability. The pathogen was able to grow at temperatures ≥8 °C, although at low temperatures, growth data presented a high variability between replicates. Indeed, at 8 °C after 15 days, some replicates did not show growth while other replicates did present an increase. A growth primary model was fitted to the raw growth data to estimate lag time and maximum growth rate. The prediction and confidence bands for the fitted growth models were estimated based on Monte-Carlo method. The estimated maximum growth rates (log cfu/day) corresponded to 0.14 (95% CI: 0.06-0.31), 0.55 (95% CI: 0.17-1.20) and 1.43 (95% CI: 0.82-2.15) for 8, 13 and 16 °C, respectively. A square-root secondary model was satisfactorily derived from the estimated growth rates (R(2) > 0.80; Bf = 0.97; Af = 1.46). Predictive models and data obtained in this study are intended to improve quantitative risk assessment studies for E. coli O157:H7 in leafy green products. Copyright © 2012. Published by Elsevier Ltd.

Collection methods and quality assessment for Esche-richia coli, water quality, and microbial source tracking data within Tumacácori National Historical Park and the upper Santa Cruz River, Arizona, 2015-16

USGS Publications Warehouse

Paretti, Nicholas; Coes, Alissa L.; Kephart, Christopher M.; Mayo, Justine

2018-03-05

Tumacácori National Historical Park protects the culturally important Mission, San José de Tumacácori, while also managing a portion of the ecologically diverse riparian corridor of the Santa Cruz River. This report describes the methods and quality assurance procedures used in the collection of water samples for the analysis of Escherichia coli (E. coli), microbial source tracking markers, suspended sediment, water-quality parameters, turbidity, and the data collection for discharge and stage; the process for data review and approval is also described. Finally, this report provides a quantitative assessment of the quality of the E. coli, microbial source tracking, and suspended sediment data.The data-quality assessment revealed that bias attributed to field and laboratory contamination was minimal, with E. coli detections in only 3 out of 33 field blank samples analyzed. Concentrations in the field blanks were several orders of magnitude lower than environmental concentrations. The microbial source tracking (MST) field blank was below the detection limit for all MST markers analyzed. Laboratory blanks for E. coli at the USGS Arizona Water Science Center and laboratory blanks for MST markers at the USGS Ohio Water Microbiology Laboratory were all below the detection limit. Irreplicate data for E. coli and suspended sediment indicated that bias was not introduced to the data by combining samples collected using discrete sampling methods with samples collected using automatic sampling methods.The split and sequential E. coli replicate data showed consistent analytical variability and a single equation was developed to explain the variability of E. coli concentrations. An additional analysis of analytical variability for E. coli indicated analytical variability around 18 percent relative standard deviation and no trend was observed in the concentration during the processing and analysis of multiple split-replicates. Two replicate samples were collected for MST and individual markers were compared for a base flow and flood sample. For the markers found in common between the two types of samples, the relative standard deviation for the base flow sample was more than 3 times greater than the markers in the flood sample. Sequential suspended sediment replicates had a relative standard deviation of about 1.3 percent, indicating that environmental and analytical variability was minimal.A holding time review and laboratory study analysis supported the extended holding times required for this investigation. Most concentrations for flood and base-flow samples were within the theoretical variability specified in the most probable number approach suggesting that extended hold times did not overly influence the final concentrations reported.

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

PubMed

Gibbs, P E; Kilbey, B J; Banerjee, S K; Lawrence, C W

1993-05-01

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.

Genetic Manipulation of Glycogen Allocation Affects Replicative Lifespan in E. coli

PubMed Central

Röösli, Thomas; Bigosch, Colette; Ackermann, Martin

2016-01-01

In bacteria, replicative aging manifests as a difference in growth or survival between the two cells emerging from division. One cell can be regarded as an aging mother with a decreased potential for future survival and division, the other as a rejuvenated daughter. Here, we aimed at investigating some of the processes involved in aging in the bacterium Escherichia coli, where the two types of cells can be distinguished by the age of their cell poles. We found that certain changes in the regulation of the carbohydrate metabolism can affect aging. A mutation in the carbon storage regulator gene, csrA, leads to a dramatically shorter replicative lifespan; csrA mutants stop dividing once their pole exceeds an age of about five divisions. These old-pole cells accumulate glycogen at their old cell poles; after their last division, they do not contain a chromosome, presumably because of spatial exclusion by the glycogen aggregates. The new-pole daughters produced by these aging mothers are born young; they only express the deleterious phenotype once their pole is old. These results demonstrate how manipulations of nutrient allocation can lead to the exclusion of the chromosome and limit replicative lifespan in E. coli, and illustrate how mutations can have phenotypic effects that are specific for cells with old poles. This raises the question how bacteria can avoid the accumulation of such mutations in their genomes over evolutionary times, and how they can achieve the long replicative lifespans that have recently been reported. PMID:27093302

Adaptive evolution of Escherichia coli to Ciprofloxacin in controlled stress environments: emergence of resistance in continuous and step-wise gradients

NASA Astrophysics Data System (ADS)

Deng, J.; Zhou, L.; Dong, Y.; Sanford, R. A.; Shechtman, L. A.; Alcalde, R.; Werth, C. J.; Fouke, B. W.

2017-12-01

Microorganisms in nature have evolved in response to a variety of environmental stresses, including gradients in pH, flow and chemistry. While environmental stresses are generally considered to be the driving force of adaptive evolution, the impact and extent of any specific stress needed to drive such changes has not been well characterized. In this study, a microfluidic diffusion chamber (MDC) and a batch culturing system were used to systematically study the effects of continuous versus step-wise stress increments on adaptation of E. coli to the antibiotic ciprofloxacin. In the MDC, a diffusion gradient of ciprofloxacin was established across a microfluidic well array to microscopically observe changes in Escherichia coli strain 307 replication and migration patterns that would indicate emergence of resistance due to genetic mutations. Cells recovered from the MDC only had resistance of 50-times the original minimum inhibition concentration (MICoriginal) of ciprofloxacin, although minimum exposure concentrations were over 80 × MICoriginal by the end of the experiment. In complementary batch experiments, E. coli 307 were exposed to step-wise daily increases of ciprofloxacin at rates equivalent to 0.1×, 0.2×, 0.4× or 0.8× times MICoriginal/day. Over a period of 18 days, E. coli cells were able to acquire resistance of up to 225 × MICoriginal, with exposure to ciprofloxacin concentration up to only 14.9 × MIC­original. The different levels of acquired resistance in the continuous MDC versus step-wise batch increment experiments suggests that the intrinsic rate of E. coli adaptation was exceeded in the MDC, while the step-wise experiments favor adaptation to the highest ciprofloxacin experiments. Genomic analyses of E. coli DNA extracted from the microfluidic cell and batch cultures indicated four single nucleotide polymorphism (SNP) mutations of amino acid 82, 83 and 87 in the gyrA gene. The progression of adaptation in the step-wise increments of ciprofloxacin indicate that the Ser83-Leu mutation gradually becomes dominant over other gyrA mutations with increased antibiotic resistance. Co-existence of the Ser83-Leu and Asp87—Gly mutations appear to provide the greatest level of resistance (i.e., 85 × to 225 × MICoriginal), and only emerged after the whole community acquired the Ser83—Leu mutation.

Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors

PubMed Central

Desomer, Jan; Dhaese, Patrick; Montagu, Marc Van

1990-01-01

The analysis of the virulence determinants of phytopathogenic Rhodococcus fascians has been hampered by the lack of a system for introducing exogenous DNA. We investigated the possibility of genetic transformation of R. fascians by high-voltage electroporation of intact bacterial cells in the presence of plasmid DNA. Electrotransformation in R. fascians D188 resulted in transformation frequencies ranging from 105/μg of DNA to 107/μg of DNA, depending on the DNA concentration. The effects of different electrical parameters and composition of electroporation medium on transformation efficiency are presented. By this transformation method, a cloning vector (pRF28) for R. fascians based on an indigenous 160-kilobase (chloramphenicol and cadmium resistance-encoding) plasmid pRF2 from strain NCPPB 1675 was developed. The origin of replication and the chloramphenicol resistance gene on pRF28 were used to construct cloning vectors that are capable of replication in R. fascians and Escherichia coli. The electroporation method presented was efficient enough to allow detection of the rare integration of replication-deficient pRF28 derivatives in the R. fascians D188 genome via either homologous or illegitimate recombination. Images PMID:16348290

The DnaA Tale

PubMed Central

Hansen, Flemming G.; Atlung, Tove

2018-01-01

More than 50 years have passed since the presentation of the Replicon Model which states that a positively acting initiator interacts with a specific site on a circular chromosome molecule to initiate DNA replication. Since then, the origin of chromosome replication, oriC, has been determined as a specific region that carries sequences required for binding of positively acting initiator proteins, DnaA-boxes and DnaA proteins, respectively. In this review we will give a historical overview of significant findings which have led to the very detailed knowledge we now possess about the initiation process in bacteria using Escherichia coli as the model organism, but emphasizing that virtually all bacteria have DnaA proteins that interacts with DnaA boxes to initiate chromosome replication. We will discuss the dnaA gene regulation, the special features of the dnaA gene expression, promoter strength, and translation efficiency, as well as, the DnaA protein, its concentration, its binding to DnaA-boxes, and its binding of ATP or ADP. Furthermore, we will discuss the different models for regulation of initiation which have been proposed over the years, with particular emphasis on the Initiator Titration Model. PMID:29541066

Intestinal colonization by enteroaggregative Escherichia coli supports long-term bacteriophage replication in mice.

PubMed

Maura, Damien; Morello, Eric; du Merle, Laurence; Bomme, Perrine; Le Bouguénec, Chantal; Debarbieux, Laurent

2012-08-01

Bacteriophages have been known to be present in the gut for many years, but studies of relationships between these viruses and their hosts in the intestine are still in their infancy. We isolated three bacteriophages specific for an enteroaggregative O104:H4 Escherichia coli (EAEC) strain responsible for diarrhoeal diseases in humans. We studied the replication of these bacteriophages in vitro and in vivo in a mouse model of gut colonization. Each bacteriophage was able to replicate in vitro in both aerobic and anaerobic conditions. Each bacteriophage individually reduced biofilms formed on plastic pegs and a cocktail of the three bacteriophages was found to be more efficient. The cocktail was also able to infect bacterial aggregates formed on the surface of epithelial cells. In the mouse intestine, bacteriophages replicated for at least 3 weeks, provided the host was present, with no change in host levels in the faeces. This model of stable and continuous viral replication provides opportunities for studying the long-term coevolution of virulent bacteriophages with their hosts within a mammalian polymicrobial ecosystem. © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.

The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, Jinlan; George, Nicholas P.; Duckett, Katrina L.

2010-05-25

Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 {angstrom} resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeaemore » and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.« less

Gene-targeted mice lacking the Trex1 (DNase III) 3'-->5' DNA exonuclease develop inflammatory myocarditis.

PubMed

Morita, Masashi; Stamp, Gordon; Robins, Peter; Dulic, Anna; Rosewell, Ian; Hrivnak, Geza; Daly, Graham; Lindahl, Tomas; Barnes, Deborah E

2004-08-01

TREX1, originally designated DNase III, was isolated as a major nuclear DNA-specific 3'-->5' exonuclease that is widely distributed in both proliferating and nonproliferating mammalian tissues. The cognate cDNA shows homology to the editing subunit of the Escherichia coli replicative DNA polymerase III holoenzyme and encodes an exonuclease which was able to serve a DNA-editing function in vitro, promoting rejoining of a 3' mismatched residue in a reconstituted DNA base excision repair system. Here we report the generation of gene-targeted Trex1(-/-) mice. The null mice are viable and do not show the increase in spontaneous mutation frequency or cancer incidence that would be predicted if Trex1 served an obligatory role of editing mismatched 3' termini generated during DNA repair or DNA replication in vivo. Unexpectedly, Trex1(-/-) mice exhibit a dramatically reduced survival and develop inflammatory myocarditis leading to progressive, often dilated, cardiomyopathy and circulatory failure.

RecQL4 is required for the association of Mcm10 and Ctf4 with replication origins in human cells

PubMed Central

Im, Jun-Sub; Park, Soon-Young; Cho, Won-Ho; Bae, Sung-Ho; Hurwitz, Jerard; Lee, Joon-Kyu

2015-01-01

Though RecQL4 was shown to be essential for the initiation of DNA replication in mammalian cells, its role in initiation is poorly understood. Here, we show that RecQL4 is required for the origin binding of Mcm10 and Ctf4, and their physical interactions and association with replication origins are controlled by the concerted action of both CDK and DDK activities. Although RecQL4-dependent binding of Mcm10 and Ctf4 to chromatin can occur in the absence of pre-replicative complex, their association with replication origins requires the presence of the pre-replicative complex and CDK and DDK activities. Their association with replication origins and physical interactions are also targets of the DNA damage checkpoint pathways which prevent initiation of DNA replication at replication origins. Taken together, the RecQL4-dependent association of Mcm10 and Ctf4 with replication origins appears to be the first important step controlled by S phase promoting kinases and checkpoint pathways for the initiation of DNA replication in human cells. PMID:25602958

Back to the Origin

PubMed Central

Evertts, Adam G.

2012-01-01

In bacteria, replication is a carefully orchestrated event that unfolds the same way for each bacterium and each cell division. The process of DNA replication in bacteria optimizes cell growth and coordinates high levels of simultaneous replication and transcription. In metazoans, the organization of replication is more enigmatic. The lack of a specific sequence that defines origins of replication has, until recently, severely limited our ability to define the organizing principles of DNA replication. This question is of particular importance as emerging data suggest that replication stress is an important contributor to inherited genetic damage and the genomic instability in tumors. We consider here the replication program in several different organisms including recent genome-wide analyses of replication origins in humans. We review recent studies on the role of cytosine methylation in replication origins, the role of transcriptional looping and gene gating in DNA replication, and the role of chromatin’s 3-dimensional structure in DNA replication. We use these new findings to consider several questions surrounding DNA replication in metazoans: How are origins selected? What is the relationship between replication and transcription? How do checkpoints inhibit origin firing? Why are there early and late firing origins? We then discuss whether oncogenes promote cancer through a role in DNA replication and whether errors in DNA replication are important contributors to the genomic alterations and gene fusion events observed in cancer. We conclude with some important areas for future experimentation. PMID:23634256

Phosphorylated SIRT1 associates with replication origins to prevent excess replication initiation and preserve genomic stability

PubMed Central

Utani, Koichi; Fu, Haiqing; Jang, Sang-Min; Marks, Anna B.; Smith, Owen K.; Zhang, Ya; Redon, Christophe E.; Shimizu, Noriaki

2017-01-01

Abstract Chromatin structure affects DNA replication patterns, but the role of specific chromatin modifiers in regulating the replication process is yet unclear. We report that phosphorylation of the human SIRT1 deacetylase on Threonine 530 (T530-pSIRT1) modulates DNA synthesis. T530-pSIRT1 associates with replication origins and inhibits replication from a group of ‘dormant’ potential replication origins, which initiate replication only when cells are subject to replication stress. Although both active and dormant origins bind T530-pSIRT1, active origins are distinguished from dormant origins by their unique association with an open chromatin mark, histone H3 methylated on lysine 4. SIRT1 phosphorylation also facilitates replication fork elongation. SIRT1 T530 phosphorylation is essential to prevent DNA breakage upon replication stress and cells harboring SIRT1 that cannot be phosphorylated exhibit a high prevalence of extrachromosomal elements, hallmarks of perturbed replication. These observations suggest that SIRT1 phosphorylation modulates the distribution of replication initiation events to insure genomic stability. PMID:28549174

Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor).

PubMed Central

Ishiai, M; Wada, C; Kawasaki, Y; Yura, T

1994-01-01

Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli. The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription. One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity. RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form. Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo. Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers. These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator. We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication. Images PMID:8170998

Oxidative DNA damage is instrumental in hyperreplication stress-induced inviability of Escherichia coli

PubMed Central

Charbon, Godefroid; Bjørn, Louise; Mendoza-Chamizo, Belén; Frimodt-Møller, Jakob; Løbner-Olesen, Anders

2014-01-01

In Escherichia coli, an increase in the ATP bound form of the DnaA initiator protein results in hyperinitiation and inviability. Here, we show that such replication stress is tolerated during anaerobic growth. In hyperinitiating cells, a shift from anaerobic to aerobic growth resulted in appearance of fragmented chromosomes and a decrease in terminus concentration, leading to a dramatic increase in ori/ter ratio and cessation of cell growth. Aerobic viability was restored by reducing the level of reactive oxygen species (ROS) or by deleting mutM (Fpg glycosylase). The double-strand breaks observed in hyperinitiating cells therefore results from replication forks encountering single-stranded DNA lesions generated while removing oxidized bases, primarily 8-oxoG, from the DNA. We conclude that there is a delicate balance between chromosome replication and ROS inflicted DNA damage so the number of replication forks can only increase when ROS formation is reduced or when the pertinent repair is compromised. PMID:25389264

Screening of E. coli β-clamp Inhibitors Revealed that Few Inhibit Helicobacter pylori More Effectively: Structural and Functional Characterization.

PubMed

Pandey, Preeti; Verma, Vijay; Dhar, Suman Kumar; Gourinath, Samudrala

2018-01-11

The characteristic of interaction with various enzymes and processivity-promoting nature during DNA replication makes β-clamp an important drug target. Helicobacter pylori ( H. pylori ) have several unique features in DNA replication machinery that makes it different from other microorganisms. To find out whether difference in DNA replication proteins behavior accounts for any difference in drug response when compared to E. coli , in the present study, we have tested E. coli β-clamp inhibitor molecules against H. pylori β-clamp. Various approaches were used to test the binding of inhibitors to H. pylori β-clamp including docking, surface competition assay, complex structure determination, as well as antimicrobial assay. Out of five shortlisted inhibitor molecules on the basis of docking score, three molecules, 5-chloroisatin, carprofen, and 3,4-difluorobenzamide were co-crystallized with H. pylori β-clamp and the structures show that they bind at the protein-protein interaction site as expected. In vivo studies showed only two molecules, 5-chloroisatin, and 3,4-difluorobenzamide inhibited the growth of the pylori with MIC values in micro molar range, which is better than the inhibitory effect of the same drugs on E. coli . Therefore, the evaluation of such drugs against H. pylori may explore the possibility to use to generate species-specific pharmacophore for development of new drugs against H. pylori .

Disintegration of Nascent Replication Bubbles during Thymine Starvation Triggers RecA- and RecBCD-dependent Replication Origin Destruction*

PubMed Central

Kuong, Kawai J.; Kuzminov, Andrei

2012-01-01

Thymineless death strikes cells unable to synthesize DNA precursor dTTP, with the nature of chromosomal damage still unclear. Thymine starvation stalls replication forks, whereas accumulating evidence indicates the replication origin is also affected. Using a novel DNA labeling technique, here we show that replication slowly continues in thymine-starved cells, but the newly synthesized DNA becomes fragmented and degraded. This degradation apparently releases enough thymine to sustain initiation of new replication bubbles from the chromosomal origin, which destabilizes the origin in a RecA-dependent manner. Marker frequency analysis with gene arrays 1) reveals destruction of the origin-centered chromosomal segment in RecA+ cells; 2) confirms origin accumulation in the recA mutants; and 3) identifies the sites around the origin where destruction initiates in the recBCD mutants. We propose that thymineless cells convert persistent single-strand gaps behind replication forks into double-strand breaks, using the released thymine for new initiations, whereas subsequent disintegration of small replication bubbles causes replication origin destruction. PMID:22621921

Replication profile of Saccharomyces cerevisiae chromosome VI.

PubMed

Friedman, K L; Brewer, B J; Fangman, W L

1997-11-01

An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae. Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early. As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.

Single molecule analysis of Trypanosoma brucei DNA replication dynamics

PubMed Central

Calderano, Simone Guedes; Drosopoulos, William C.; Quaresma, Marina Mônaco; Marques, Catarina A.; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L.; Elias, Maria Carolina

2015-01-01

Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5′ extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. PMID:25690894

Single molecule analysis of Trypanosoma brucei DNA replication dynamics.

PubMed

Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco; Marques, Catarina A; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L; Elias, Maria Carolina

2015-03-11

Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Genome-wide mapping reveals single-origin chromosome replication in Leishmania, a eukaryotic microbe.

PubMed

Marques, Catarina A; Dickens, Nicholas J; Paape, Daniel; Campbell, Samantha J; McCulloch, Richard

2015-10-19

DNA replication initiates on defined genome sites, termed origins. Origin usage appears to follow common rules in the eukaryotic organisms examined to date: all chromosomes are replicated from multiple origins, which display variations in firing efficiency and are selected from a larger pool of potential origins. To ask if these features of DNA replication are true of all eukaryotes, we describe genome-wide origin mapping in the parasite Leishmania. Origin mapping in Leishmania suggests a striking divergence in origin usage relative to characterized eukaryotes, since each chromosome appears to be replicated from a single origin. By comparing two species of Leishmania, we find evidence that such origin singularity is maintained in the face of chromosome fusion or fission events during evolution. Mapping Leishmania origins suggests that all origins fire with equal efficiency, and that the genomic sites occupied by origins differ from related non-origins sites. Finally, we provide evidence that origin location in Leishmania displays striking conservation with Trypanosoma brucei, despite the latter parasite replicating its chromosomes from multiple, variable strength origins. The demonstration of chromosome replication for a single origin in Leishmania, a microbial eukaryote, has implications for the evolution of origin multiplicity and associated controls, and may explain the pervasive aneuploidy that characterizes Leishmania chromosome architecture.

Heterogeneity of spontaneous DNA replication errors in single isogenic Escherichia coli cells

PubMed Central

2018-01-01

Despite extensive knowledge of the molecular mechanisms that control mutagenesis, it is not known how spontaneous mutations are produced in cells with fully operative mutation-prevention systems. By using a mutation assay that allows visualization of DNA replication errors and stress response transcriptional reporters, we examined populations of isogenic Escherichia coli cells growing under optimal conditions without exogenous stress. We found that spontaneous DNA replication errors in proliferating cells arose more frequently in subpopulations experiencing endogenous stresses, such as problems with proteostasis, genome maintenance, and reactive oxidative species production. The presence of these subpopulations of phenotypic mutators is not expected to affect the average mutation frequency or to reduce the mean population fitness in a stable environment. However, these subpopulations can contribute to overall population adaptability in fluctuating environments by serving as a reservoir of increased genetic variability.

DNA sequence analysis of the composite plasmid pTC conferring virulence and antimicrobial resistance for porcine enterotoxigenic Escherichia coli.

PubMed

Fekete, Péter Z; Brzuszkiewicz, Elzbieta; Blum-Oehler, Gabriele; Olasz, Ferenc; Szabó, Mónika; Gottschalk, Gerhard; Hacker, Jörg; Nagy, Béla

2012-01-01

In this study the plasmid pTC, a 90 kb self-conjugative virulence plasmid of the porcine enterotoxigenic Escherichia coli (ETEC) strain EC2173 encoding the STa and STb heat-stable enterotoxins and tetracycline resistance, has been sequenced in two steps. As a result we identified five main distinct regions of pTC: (i) the maintenance region responsible for the extreme stability of the plasmid, (ii) the TSL (toxin-specific locus comprising the estA and estB genes) which is unique and characteristic for pTC, (iii) a Tn10 transposon, encoding tetracycline resistance, (iv) the tra (plasmid transfer) region, and (v) the colE1-like origin of replication. It is concluded that pTC is a self-transmissible composite plasmid harbouring antibiotic resistance and virulence genes. pTC belongs to a group of large conjugative E. coli plasmids represented by NR1 with a widespread tra backbone which might have evolved from a common ancestor. This is the first report of a completely sequenced animal ETEC virulence plasmid containing an antimicrobial resistance locus, thereby representing a selection advantage for spread of pathogenicity in the presence of antimicrobials leading to increased disease potential. Copyright © 2011. Published by Elsevier GmbH.

Comparison of seven protocols to identify fecal contamination sources using Escherichia coli

USGS Publications Warehouse

Stoeckel, D.M.; Mathes, M.V.; Hyer, K.E.; Hagedorn, C.; Kator, H.; Lukasik, J.; O'Brien, T. L.; Fenger, T.W.; Samadpour, M.; Strickler, K.M.; Wiggins, B.A.

2004-01-01

Microbial source tracking (MST) uses various approaches to classify fecal-indicator microorganisms to source hosts. Reproducibility, accuracy, and robustness of seven phenotypic and genotypic MST protocols were evaluated by use of Escherichia coli from an eight-host library of known-source isolates and a separate, blinded challenge library. In reproducibility tests, measuring each protocol's ability to reclassify blinded replicates, only one (pulsed-field gel electrophoresis; PFGE) correctly classified all test replicates to host species; three protocols classified 48-62% correctly, and the remaining three classified fewer than 25% correctly. In accuracy tests, measuring each protocol's ability to correctly classify new isolates, ribotyping with EcoRI and PvuII approached 100% correct classification but only 6% of isolates were classified; four of the other six protocols (antibiotic resistance analysis, PFGE, and two repetitive-element PCR protocols) achieved better than random accuracy rates when 30-100% of challenge isolates were classified. In robustness tests, measuring each protocol's ability to recognize isolates from nonlibrary hosts, three protocols correctly classified 33-100% of isolates as "unknown origin," whereas four protocols classified all isolates to a source category. A relevance test, summarizing interpretations for a hypothetical water sample containing 30 challenge isolates, indicated that false-positive classifications would hinder interpretations for most protocols. Study results indicate that more representation in known-source libraries and better classification accuracy would be needed before field application. Thorough reliability assessment of classification results is crucial before and during application of MST protocols.

Open chromatin encoded in DNA sequence is the signature of ‘master’ replication origins in human cells

PubMed Central

Audit, Benjamin; Zaghloul, Lamia; Vaillant, Cédric; Chevereau, Guillaume; d'Aubenton-Carafa, Yves; Thermes, Claude; Arneodo, Alain

2009-01-01

For years, progress in elucidating the mechanisms underlying replication initiation and its coupling to transcriptional activities and to local chromatin structure has been hampered by the small number (approximately 30) of well-established origins in the human genome and more generally in mammalian genomes. Recent in silico studies of compositional strand asymmetries revealed a high level of organization of human genes around 1000 putative replication origins. Here, by comparing with recently experimentally identified replication origins, we provide further support that these putative origins are active in vivo. We show that regions ∼300-kb wide surrounding most of these putative replication origins that replicate early in the S phase are hypersensitive to DNase I cleavage, hypomethylated and present a significant enrichment in genomic energy barriers that impair nucleosome formation (nucleosome-free regions). This suggests that these putative replication origins are specified by an open chromatin structure favored by the DNA sequence. We discuss how this distinctive attribute makes these origins, further qualified as ‘master’ replication origins, priviledged loci for future research to decipher the human spatio-temporal replication program. Finally, we argue that these ‘master’ origins are likely to play a key role in genome dynamics during evolution and in pathological situations. PMID:19671527

How and why multiple MCMs are loaded at origins of DNA replication.

PubMed

Das, Shankar P; Rhind, Nicholas

2016-07-01

Recent work suggests that DNA replication origins are regulated by the number of multiple mini-chromosome maintenance (MCM) complexes loaded. Origins are defined by the loading of MCM - the replicative helicase which initiates DNA replication and replication kinetics determined by origin's location and firing times. However, activation of MCM is heterogeneous; different origins firing at different times in different cells. Also, more MCMs are loaded in G1 than are used in S phase. These aspects of MCM biology are explained by the observation that multiple MCMs are loaded at origins. Having more MCMs at early origins makes them more likely to fire, effecting differences in origin efficiency that define replication timing. Nonetheless, multiple MCM loading raises new questions, such as how they are loaded, where these MCMs reside at origins, and how their presence affects replication timing. In this review, we address these questions and discuss future avenues of research. © 2016 WILEY Periodicals, Inc.

Nucleosomes influence multiple steps during replication initiation

PubMed Central

Azmi, Ishara F; Watanabe, Shinya; Maloney, Michael F; Kang, Sukhyun; Belsky, Jason A; MacAlpine, David M; Peterson, Craig L; Bell, Stephen P

2017-01-01

Eukaryotic replication origin licensing, activation and timing are influenced by chromatin but a mechanistic understanding is lacking. Using reconstituted nucleosomal DNA replication assays, we assessed the impact of nucleosomes on replication initiation. To generate distinct nucleosomal landscapes, different chromatin-remodeling enzymes (CREs) were used to remodel nucleosomes on origin-DNA templates. Nucleosomal organization influenced two steps of replication initiation: origin licensing and helicase activation. Origin licensing assays showed that local nucleosome positioning enhanced origin specificity and modulated helicase loading by influencing ORC DNA binding. Interestingly, SWI/SNF- and RSC-remodeled nucleosomes were permissive for origin licensing but showed reduced helicase activation. Specific CREs rescued replication of these templates if added prior to helicase activation, indicating a permissive chromatin state must be established during origin licensing to allow efficient origin activation. Our studies show nucleosomes directly modulate origin licensing and activation through distinct mechanisms and provide insights into the regulation of replication initiation by chromatin. DOI: http://dx.doi.org/10.7554/eLife.22512.001 PMID:28322723

High community faecal carriage rates of CTX-M ESBL-producing Escherichia coli in a specific population group in Birmingham, UK.

PubMed

Wickramasinghe, Nimal H; Xu, Li; Eustace, Andrew; Shabir, Sahida; Saluja, Tranprit; Hawkey, Peter M

2012-05-01

To determine the proportion of E. coli carrying specific CTX-M extended-spectrum β-lactamase (ESBL) genotypes in a community population of East and North Birmingham. General practice and outpatient stool samples from 732 individuals submitted for examination for faecal pathogens in 2010 were screened for ESBL-producing E. coli using chromogenic agar. Multiplex PCR, denaturing HPLC, DNA sequencing and PFGE were used to determine the CTX-M genotype and clonal subtype. Isolates from people were assigned to 'Europe', 'Middle East/South Asia' (MESA) or 'uncategorized' groups using software to determine probable global origin based on the subject's full name. Prevalence of CTX-M carriage in the sample population was 11.3%. There was a statistically significant difference (P < 0.001) between carriage in the Europe group (8.1%) and the MESA group (22.8%). There was also a higher rate of carriage of CTX-M-15-producing E. coli (P < 0.001) in MESA subjects. The high community carriage rate and the significant difference in carriage between the Europe and MESA subjects may have important consequences for therapy. If the rising trend in carriage of bacteria producing ESBLs continues, guidelines for empirical therapy for patients presenting from the community may need to be modified. The findings also raise the concern that the pattern and routes of spread of CTX-M-15 may be replicated in the future by broader-spectrum β-lactamases, such as New Delhi metallo-β-lactamase ('NDM-1').

Scoring schemes of palindrome clusters for more sensitive prediction of replication origins in herpesviruses

PubMed Central

Chew, David S. H.; Choi, Kwok Pui; Leung, Ming-Ying

2005-01-01

Many empirical studies show that there are unusual clusters of palindromes, closely spaced direct and inverted repeats around the replication origins of herpesviruses. In this paper, we introduce two new scoring schemes to quantify the spatial abundance of palindromes in a genomic sequence. Based on these scoring schemes, a computational method to predict the locations of replication origins is developed. When our predictions are compared with 39 known or annotated replication origins in 19 herpesviruses, close to 80% of the replication origins are located within 2% of the genome length. A list of predicted locations of replication origins in all the known herpesviruses with complete genome sequences is reported. PMID:16141192

DNA replication origins—where do we begin?

PubMed Central

Prioleau, Marie-Noëlle; MacAlpine, David M.

2016-01-01

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. PMID:27542827

G4 motifs affect origin positioning and efficiency in two vertebrate replicators

PubMed Central

Valton, Anne-Laure; Hassan-Zadeh, Vahideh; Lema, Ingrid; Boggetto, Nicole; Alberti, Patrizia; Saintomé, Carole; Riou, Jean-François; Prioleau, Marie-Noëlle

2014-01-01

DNA replication ensures the accurate duplication of the genome at each cell cycle. It begins at specific sites called replication origins. Genome-wide studies in vertebrates have recently identified a consensus G-rich motif potentially able to form G-quadruplexes (G4) in most replication origins. However, there is no experimental evidence to demonstrate that G4 are actually required for replication initiation. We show here, with two model origins, that G4 motifs are required for replication initiation. Two G4 motifs cooperate in one of our model origins. The other contains only one critical G4, and its orientation determines the precise position of the replication start site. Point mutations affecting the stability of this G4 in vitro also impair origin function. Finally, this G4 is not sufficient for origin activity and must cooperate with a 200-bp cis-regulatory element. In conclusion, our study strongly supports the predicted essential role of G4 in replication initiation. PMID:24521668

The Spatiotemporal Program of Replication in the Genome of Lachancea kluyveri

PubMed Central

Agier, Nicolas; Romano, Orso Maria; Touzain, Fabrice; Cosentino Lagomarsino, Marco; Fischer, Gilles

2013-01-01

We generated a genome-wide replication profile in the genome of Lachancea kluyveri and assessed the relationship between replication and base composition. This species diverged from Saccharomyces cerevisiae before the ancestral whole genome duplication. The genome comprises eight chromosomes among which a chromosomal arm of 1 Mb has a G + C-content much higher than the rest of the genome. We identified 252 active replication origins in L. kluyveri and found considerable divergence in origin location with S. cerevisiae and with Lachancea waltii. Although some global features of S. cerevisiae replication are conserved: Centromeres replicate early, whereas telomeres replicate late, we found that replication origins both in L. kluyveri and L. waltii do not behave as evolutionary fragile sites. In L. kluyveri, replication timing along chromosomes alternates between regions of early and late activating origins, except for the 1 Mb GC-rich chromosomal arm. This chromosomal arm contains an origin consensus motif different from other chromosomes and is replicated early during S-phase. We showed that precocious replication results from the specific absence of late firing origins in this chromosomal arm. In addition, we found a correlation between GC-content and distance from replication origins as well as a lack of replication-associated compositional skew between leading and lagging strands specifically in this GC-rich chromosomal arm. These findings suggest that the unusual base composition in the genome of L. kluyveri could be linked to replication. PMID:23355306

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

PubMed Central

On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

2014-01-01

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

DNA replication origins-where do we begin?

PubMed

Prioleau, Marie-Noëlle; MacAlpine, David M

2016-08-01

For more than three decades, investigators have sought to identify the precise locations where DNA replication initiates in mammalian genomes. The development of molecular and biochemical approaches to identify start sites of DNA replication (origins) based on the presence of defining and characteristic replication intermediates at specific loci led to the identification of only a handful of mammalian replication origins. The limited number of identified origins prevented a comprehensive and exhaustive search for conserved genomic features that were capable of specifying origins of DNA replication. More recently, the adaptation of origin-mapping assays to genome-wide approaches has led to the identification of tens of thousands of replication origins throughout mammalian genomes, providing an unprecedented opportunity to identify both genetic and epigenetic features that define and regulate their distribution and utilization. Here we summarize recent advances in our understanding of how primary sequence, chromatin environment, and nuclear architecture contribute to the dynamic selection and activation of replication origins across diverse cell types and developmental stages. © 2016 Prioleau and MacAlpine; Published by Cold Spring Harbor Laboratory Press.

Replication Origins and Timing of Temporal Replication in Budding Yeast: How to Solve the Conundrum?

PubMed Central

Barberis, Matteo; Spiesser, Thomas W.; Klipp, Edda

2010-01-01

Similarly to metazoans, the budding yeast Saccharomyces cereviasiae replicates its genome with a defined timing. In this organism, well-defined, site-specific origins, are efficient and fire in almost every round of DNA replication. However, this strategy is neither conserved in the fission yeast Saccharomyces pombe, nor in Xenopus or Drosophila embryos, nor in higher eukaryotes, in which DNA replication initiates asynchronously throughout S phase at random sites. Temporal and spatial controls can contribute to the timing of replication such as Cdk activity, origin localization, epigenetic status or gene expression. However, a debate is going on to answer the question how individual origins are selected to fire in budding yeast. Two opposing theories were proposed: the “replicon paradigm” or “temporal program” vs. the “stochastic firing”. Recent data support the temporal regulation of origin activation, clustering origins into temporal blocks of early and late replication. Contrarily, strong evidences suggest that stochastic processes acting on origins can generate the observed kinetics of replication without requiring a temporal order. In mammalian cells, a spatiotemporal model that accounts for a partially deterministic and partially stochastic order of DNA replication has been proposed. Is this strategy the solution to reconcile the conundrum of having both organized replication timing and stochastic origin firing also for budding yeast? In this review we discuss this possibility in the light of our recent study on the origin activation, suggesting that there might be a stochastic component in the temporal activation of the replication origins, especially under perturbed conditions. PMID:21037857

Phage Lambda P Protein: Trans-Activation, Inhibition Phenotypes and their Suppression

PubMed Central

Hayes, Sidney; Erker, Craig; Horbay, Monique A.; Marciniuk, Kristen; Wang, Wen; Hayes, Connie

2013-01-01

The initiation of bacteriophage λ replication depends upon interactions between the oriλ DNA site, phage proteins O and P, and E. coli host replication proteins. P exhibits a high affinity for DnaB, the major replicative helicase for unwinding double stranded DNA. The concept of P-lethality relates to the hypothesis that P can sequester DnaB and in turn prevent cellular replication initiation from oriC. Alternatively, it was suggested that P-lethality does not involve an interaction between P and DnaB, but is targeted to DnaA. P-lethality is assessed by examining host cells for transformation by ColE1-type plasmids that can express P, and the absence of transformants is attributed to a lethal effect of P expression. The plasmid we employed enabled conditional expression of P, where under permissive conditions, cells were efficiently transformed. We observed that ColE1 replication and plasmid establishment upon transformation is extremely sensitive to P, and distinguish this effect from P-lethality directed to cells. We show that alleles of dnaB protect the variant cells from P expression. P-dependent cellular filamentation arose in ΔrecA or lexA[Ind-] cells, defective for SOS induction. Replication propagation and restart could represent additional targets for P interference of E. coli replication, beyond the oriC-dependent initiation step. PMID:23389467

Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria.

PubMed Central

Wolk, C P; Vonshak, A; Kehoe, P; Elhai, J

1984-01-01

Wild-type cyanobacteria of the genus Anabaena are capable of oxygenic photosynthesis, differentiation of cells called heterocysts at semiregular intervals along the cyanobacterial filaments, and aerobic nitrogen fixation by the heterocysts. To foster analysis of the physiological processes characteristic of these cyanobacteria, we have constructed a family of shuttle vectors capable of replication and selection in Escherichia coli and, in unaltered form, in several strains of Anabaena. Highly efficient conjugative transfer of these vectors from E. coli to Anabaena is dependent upon the presence of broad host-range plasmid RP-4 and of helper plasmids. The shuttle vectors contain portions of plasmid pBR322 required for replication and mobilization, with sites for Anabaena restriction enzymes deleted; cyanobacterial replicon pDU1, which lacks such sites; and determinants for resistance to chloramphenicol, streptomycin, neomycin, and erythromycin. Images PMID:6324204

Escherichia coli DnaE Polymerase Couples Pyrophosphatase Activity to DNA Replication

PubMed Central

Lapenta, Fabio; Montón Silva, Alejandro; Brandimarti, Renato; Lanzi, Massimiliano; Gratani, Fabio Lino; Vellosillo Gonzalez, Perceval; Perticarari, Sofia; Hochkoeppler, Alejandro

2016-01-01

DNA Polymerases generate pyrophosphate every time they catalyze a step of DNA elongation. This elongation reaction is generally believed as thermodynamically favoured by the hydrolysis of pyrophosphate, catalyzed by inorganic pyrophosphatases. However, the specific action of inorganic pyrophosphatases coupled to DNA replication in vivo was never demonstrated. Here we show that the Polymerase-Histidinol-Phosphatase (PHP) domain of Escherichia coli DNA Polymerase III α subunit features pyrophosphatase activity. We also show that this activity is inhibited by fluoride, as commonly observed for inorganic pyrophosphatases, and we identified 3 amino acids of the PHP active site. Remarkably, E. coli cells expressing variants of these catalytic residues of α subunit feature aberrant phenotypes, poor viability, and are subject to high mutation frequencies. Our findings indicate that DNA Polymerases can couple DNA elongation and pyrophosphate hydrolysis, providing a mechanism for the control of DNA extension rate, and suggest a promising target for novel antibiotics. PMID:27050298

A Natural Polymorphism in rDNA Replication Origins Links Origin Activation with Calorie Restriction and Lifespan

PubMed Central

Kwan, Elizabeth X.; Foss, Eric J.; Tsuchiyama, Scott; Alvino, Gina M.; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M. K.; Brewer, Bonita J.; Kennedy, Brian K.; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics. PMID:23505383

A natural polymorphism in rDNA replication origins links origin activation with calorie restriction and lifespan.

PubMed

Kwan, Elizabeth X; Foss, Eric J; Tsuchiyama, Scott; Alvino, Gina M; Kruglyak, Leonid; Kaeberlein, Matt; Raghuraman, M K; Brewer, Bonita J; Kennedy, Brian K; Bedalov, Antonio

2013-01-01

Aging and longevity are complex traits influenced by genetic and environmental factors. To identify quantitative trait loci (QTLs) that control replicative lifespan, we employed an outbred Saccharomyces cerevisiae model, generated by crossing a vineyard and a laboratory strain. The predominant QTL mapped to the rDNA, with the vineyard rDNA conferring a lifespan increase of 41%. The lifespan extension was independent of Sir2 and Fob1, but depended on a polymorphism in the rDNA origin of replication from the vineyard strain that reduced origin activation relative to the laboratory origin. Strains carrying vineyard rDNA origins have increased capacity for replication initiation at weak plasmid and genomic origins, suggesting that inability to complete genome replication presents a major impediment to replicative lifespan. Calorie restriction, a conserved mediator of lifespan extension that is also independent of Sir2 and Fob1, reduces rDNA origin firing in both laboratory and vineyard rDNA. Our results are consistent with the possibility that calorie restriction, similarly to the vineyard rDNA polymorphism, modulates replicative lifespan through control of rDNA origin activation, which in turn affects genome replication dynamics.

Initiation preference at a yeast origin of replication.

PubMed

Brewer, B J; Fangman, W L

1994-04-12

Replication origins in the yeast Saccharomyces cerevisiae are identified as autonomous replication sequence (ARS) elements. To examine the effect of origin density on replication initiation, we have analyzed the replication of a plasmid that contains two copies of the same origin, ARS1. The activation of origins and the direction that replication forks move through flanking sequences can be physically determined by analyzing replication intermediates on two-dimensional agarose gels. We find that only one of the two identical ARSs on the plasmid initiates replication on any given plasmid molecule; that is, this close spacing of ARSs results in an apparent interference between the potential origins. Moreover, in the particular plasmid that we constructed, one of the two identical copies of ARS1 is used four times more frequently than the other one. These results show that the plasmid context is critical for determining the preferred origin. This origin preference is also exhibited when the tandem copies of ARS1 are introduced into a yeast chromosome. The sequences responsible for establishing the origin preference have been identified by deletion analysis and are found to reside in a portion of the yeast URA3 gene.

Universal Temporal Profile of Replication Origin Activation in Eukaryotes

NASA Astrophysics Data System (ADS)

Goldar, Arach

2011-03-01

The complete and faithful transmission of eukaryotic genome to daughter cells involves the timely duplication of mother cell's DNA. DNA replication starts at multiple chromosomal positions called replication origin. From each activated replication origin two replication forks progress in opposite direction and duplicate the mother cell's DNA. While it is widely accepted that in eukaryotic organisms replication origins are activated in a stochastic manner, little is known on the sources of the observed stochasticity. It is often associated to the population variability to enter S phase. We extract from a growing Saccharomyces cerevisiae population the average rate of origin activation in a single cell by combining single molecule measurements and a numerical deconvolution technique. We show that the temporal profile of the rate of origin activation in a single cell is similar to the one extracted from a replicating cell population. Taking into account this observation we exclude the population variability as the origin of observed stochasticity in origin activation. We confirm that the rate of origin activation increases in the early stage of S phase and decreases at the latter stage. The population average activation rate extracted from single molecule analysis is in prefect accordance with the activation rate extracted from published micro-array data, confirming therefore the homogeneity and genome scale invariance of dynamic of replication process. All these observations point toward a possible role of replication fork to control the rate of origin activation.

Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome

PubMed Central

Guilbaud, Guillaume; Rappailles, Aurélien; Baker, Antoine; Chen, Chun-Long; Arneodo, Alain; Goldar, Arach; d'Aubenton-Carafa, Yves; Thermes, Claude; Audit, Benjamin; Hyrien, Olivier

2011-01-01

Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics. PMID:22219720

High-fidelity DNA replication in Mycobacterium tuberculosis relies on a trinuclear zinc center.

PubMed

Baños-Mateos, Soledad; van Roon, Anne-Marie M; Lang, Ulla F; Maslen, Sarah L; Skehel, J Mark; Lamers, Meindert H

2017-10-11

High-fidelity DNA replication depends on a proofreading 3'-5' exonuclease that is associated with the replicative DNA polymerase. The replicative DNA polymerase DnaE1 from the major pathogen Mycobacterium tuberculosis (Mtb) uses its intrinsic PHP-exonuclease that is distinct from the canonical DEDD exonucleases found in the Escherichia coli and eukaryotic replisomes. The mechanism of the PHP-exonuclease is not known. Here, we present the crystal structure of the Mtb DnaE1 polymerase. The PHP-exonuclease has a trinuclear zinc center, coordinated by nine conserved residues. Cryo-EM analysis reveals the entry path of the primer strand in the PHP-exonuclease active site. Furthermore, the PHP-exonuclease shows a striking similarity to E. coli endonuclease IV, which provides clues regarding the mechanism of action. Altogether, this work provides important insights into the PHP-exonuclease and reveals unique properties that make it an attractive target for novel anti-mycobacterial drugs.The polymerase and histidinol phosphatase (PHP) domain in the DNA polymerase DnaE1 is essential for mycobacterial high-fidelity DNA replication. Here, the authors determine the DnaE1 crystal structure, which reveals the PHP-exonuclease mechanism that can be exploited for antibiotic development.

Nucleosome occupancy as a novel chromatin parameter for replication origin functions

PubMed Central

Rodriguez, Jairo; Lee, Laura; Lynch, Bryony; Tsukiyama, Toshio

2017-01-01

Eukaryotic DNA replication initiates from multiple discrete sites in the genome, termed origins of replication (origins). Prior to S phase, multiple origins are poised to initiate replication by recruitment of the pre-replicative complex (pre-RC). For proper replication to occur, origin activation must be tightly regulated. At the population level, each origin has a distinct firing time and frequency of activation within S phase. Many studies have shown that chromatin can strongly influence initiation of DNA replication. However, the chromatin parameters that affect properties of origins have not been thoroughly established. We found that nucleosome occupancy in G1 varies greatly around origins across the S. cerevisiae genome, and nucleosome occupancy around origins significantly correlates with the activation time and efficiency of origins, as well as pre-RC formation. We further demonstrate that nucleosome occupancy around origins in G1 is established during transition from G2/M to G1 in a pre-RC-dependent manner. Importantly, the diminished cell-cycle changes in nucleosome occupancy around origins in the orc1-161 mutant are associated with an abnormal global origin usage profile, suggesting that proper establishment of nucleosome occupancy around origins is a critical step for regulation of global origin activities. Our work thus establishes nucleosome occupancy as a novel and key chromatin parameter for proper origin regulation. PMID:27895110

Roles of Type 1A Topoisomerases in Genome Maintenance in Escherichia coli

PubMed Central

Usongo, Valentine; Drolet, Marc

2014-01-01

In eukaryotes, type 1A topoisomerases (topos) act with RecQ-like helicases to maintain the stability of the genome. Despite having been the first type 1A enzymes to be discovered, much less is known about the involvement of the E. coli topo I (topA) and III (topB) enzymes in genome maintenance. These enzymes are thought to have distinct cellular functions: topo I regulates supercoiling and R-loop formation, and topo III is involved in chromosome segregation. To better characterize their roles in genome maintenance, we have used genetic approaches including suppressor screens, combined with microscopy for the examination of cell morphology and nucleoid shape. We show that topA mutants can suffer from growth-inhibitory and supercoiling-dependent chromosome segregation defects. These problems are corrected by deleting recA or recQ but not by deleting recJ or recO, indicating that the RecF pathway is not involved. Rather, our data suggest that RecQ acts with a type 1A topo on RecA-generated recombination intermediates because: 1-topo III overproduction corrects the defects and 2-recQ deletion and topo IIII overproduction are epistatic to recA deletion. The segregation defects are also linked to over-replication, as they are significantly alleviated by an oriC::aph suppressor mutation which is oriC-competent in topA null but not in isogenic topA+ cells. When both topo I and topo III are missing, excess supercoiling triggers growth inhibition that correlates with the formation of extremely long filaments fully packed with unsegregated and diffuse DNA. These phenotypes are likely related to replication from R-loops as they are corrected by overproducing RNase HI or by genetic suppressors of double topA rnhA mutants affecting constitutive stable DNA replication, dnaT::aph and rne::aph, which initiates from R-loops. Thus, bacterial type 1A topos maintain the stability of the genome (i) by preventing over-replication originating from oriC (topo I alone) and R-loops and (ii) by acting with RecQ. PMID:25102178

Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication.

PubMed

Beauregard, Arthur; Chalamcharla, Venkata R; Piazza, Carol Lyn; Belfort, Marlene; Coros, Colin J

2006-11-01

Group II introns are mobile genetic elements that invade their cognate intron-minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron-encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid-associated proteins H-NS, StpA and MukB, in variants of partitioning functions including the centromere-like sequence migS and the actin homologue MreB, as well as in the replication mutants DeltaoriC, seqA, tus and topoIV (ts). Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell.

Functional centromeres determine the activation time of pericentric origins of DNA replication in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Brewer, Bonita J; Raghuraman, M K

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.

Functional Centromeres Determine the Activation Time of Pericentric Origins of DNA Replication in Saccharomyces cerevisiae

PubMed Central

Pohl, Thomas J.; Brewer, Bonita J.; Raghuraman, M. K.

2012-01-01

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation. PMID:22589733

Origin Replication Complex Binding, Nucleosome Depletion Patterns, and a Primary Sequence Motif Can Predict Origins of Replication in a Genome with Epigenetic Centromeres

PubMed Central

Tsai, Hung-Ji; Baller, Joshua A.; Liachko, Ivan; Koren, Amnon; Burrack, Laura S.; Hickman, Meleah A.; Thevandavakkam, Mathuravani A.; Rusche, Laura N.

2014-01-01

ABSTRACT Origins of DNA replication are key genetic elements, yet their identification remains elusive in most organisms. In previous work, we found that centromeres contain origins of replication (ORIs) that are determined epigenetically in the pathogenic yeast Candida albicans. In this study, we used origin recognition complex (ORC) binding and nucleosome occupancy patterns in Saccharomyces cerevisiae and Kluyveromyces lactis to train a machine learning algorithm to predict the position of active arm (noncentromeric) origins in the C. albicans genome. The model identified bona fide active origins as determined by the presence of replication intermediates on nondenaturing two-dimensional (2D) gels. Importantly, these origins function at their native chromosomal loci and also as autonomously replicating sequences (ARSs) on a linear plasmid. A “mini-ARS screen” identified at least one and often two ARS regions of ≥100 bp within each bona fide origin. Furthermore, a 15-bp AC-rich consensus motif was associated with the predicted origins and conferred autonomous replicating activity to the mini-ARSs. Thus, while centromeres and the origins associated with them are epigenetic, arm origins are dependent upon critical DNA features, such as a binding site for ORC and a propensity for nucleosome exclusion. PMID:25182328

Function of BRCA1 at a DNA Replication Origin

DTIC Science & Technology

2004-07-01

origin of Epstein-Barr Virus DNA replication (Ori P). OriP replicates once and only once per cell cycle in synchrony with the cellular genome, and is...modifications, and to investigate its function at OriP in DNA replication and plasmid maintenance. We propose that these studies will provide valuable...information concerning the function of OriP at replication origins and in the control of DNA replication initiation and genome stability.

Ecology of coliphages in southern California coastal waters.

PubMed

Reyes, V C; Jiang, S C

2010-08-01

This study aims to investigate the ecology of coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of coliphages in coastal waters. Water samples from three locations in California's Newport Bay watershed were tested for the presence of coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The coliphages isolated on the environmental hosts had a broad host-range relative to the coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host-range may enable the proliferation of coliphages in the aquatic environment. Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host-range of coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic environment.

Exploring the roles of DNA methylation in the metal-reducing bacterium Shewanella oneidensis MR-1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bendall, Matthew L.; Luong, Khai; Wetmore, Kelly M.

2013-08-30

We performed whole genome analyses of DNA methylation in Shewanella 17 oneidensis MR-1 to examine its possible role in regulating gene expression and 18 other cellular processes. Single-Molecule Real Time (SMRT) sequencing 19 revealed extensive methylation of adenine (N6mA) throughout the 20 genome. These methylated bases were located in five sequence motifs, 21 including three novel targets for Type I restriction/modification enzymes. The 22 sequence motifs targeted by putative methyltranferases were determined via 23 SMRT sequencing of gene knockout mutants. In addition, we found S. 24 oneidensis MR-1 cultures grown under various culture conditions displayed 25 different DNA methylation patterns.more » However, the small number of differentially 26 methylated sites could not be directly linked to the much larger number of 27 differentially expressed genes in these conditions, suggesting DNA methylation is 28 not a major regulator of gene expression in S. oneidensis MR-1. The enrichment 29 of methylated GATC motifs in the origin of replication indicate DNA methylation 30 may regulate genome replication in a manner similar to that seen in Escherichia 31 coli. Furthermore, comparative analyses suggest that many 32 Gammaproteobacteria, including all members of the Shewanellaceae family, may 33 also utilize DNA methylation to regulate genome replication.« less

The dynamics of genome replication using deep sequencing

PubMed Central

Müller, Carolin A.; Hawkins, Michelle; Retkute, Renata; Malla, Sunir; Wilson, Ray; Blythe, Martin J.; Nakato, Ryuichiro; Komata, Makiko; Shirahige, Katsuhiko; de Moura, Alessandro P.S.; Nieduszynski, Conrad A.

2014-01-01

Eukaryotic genomes are replicated from multiple DNA replication origins. We present complementary deep sequencing approaches to measure origin location and activity in Saccharomyces cerevisiae. Measuring the increase in DNA copy number during a synchronous S-phase allowed the precise determination of genome replication. To map origin locations, replication forks were stalled close to their initiation sites; therefore, copy number enrichment was limited to origins. Replication timing profiles were generated from asynchronous cultures using fluorescence-activated cell sorting. Applying this technique we show that the replication profiles of haploid and diploid cells are indistinguishable, indicating that both cell types use the same cohort of origins with the same activities. Finally, increasing sequencing depth allowed the direct measure of replication dynamics from an exponentially growing culture. This is the first time this approach, called marker frequency analysis, has been successfully applied to a eukaryote. These data provide a high-resolution resource and methodological framework for studying genome biology. PMID:24089142

Identifying sites of replication initiation in yeast chromosomes: looking for origins in all the right places.

PubMed

van Brabant, A J; Hunt, S Y; Fangman, W L; Brewer, B J

1998-06-01

DNA fragments that contain an active origin of replication generate bubble-shaped replication intermediates with diverging forks. We describe two methods that use two-dimensional (2-D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificial Saccharomyces cerevisiae chromosomes. The first method uses 2-D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2-D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restriction map.

CedA is a novel Escherichia coli protein that activates the cell division inhibited by chromosomal DNA over-replication.

PubMed

Katayama, T; Takata, M; Sekimizu, K

1997-11-01

We isolated and characterized a new gene related to the control of cell division regulation in Escherichia coli. At 30 degrees C, the dnaAcos mutant causes over-replication of the chromosome, and colony formation is inhibited. We found that, at this temperature, the dnaAcos cells form filaments; therefore, septum formation is inhibited. This inhibition was independent of SfiA, an inhibitor of the septum-forming protein, FtsZ. To identify factors involved in this pathway of inhibition, we isolated seven multicopy suppressors for the cold-sensitive phenotype of the dnaAcos mutant. One of these proved to be a previously unknown gene, which we named cedA. This gene encoded a 12 kDa protein and resided at 38.9min on the E. coli genome map. A multicopy supply of the cedA gene to the dnaAcos cells did not repress over-replication of the chromosome but did stimulate cell division of the host, the result being growth of cells with an abnormally elevated chromosomal copy number. Therefore, the expression level of the cedA gene seems to be important for inhibiting cell division of the dnaAcos mutant at 30 degrees C. We propose that over-replication of the chromosome activates a pathway for inhibiting cell division and that the cedA gene modulates this division control. In the dnaA+ background, cedA also seems to affect cell division.

High-resolution mapping, characterization, and optimization of autonomously replicating sequences in yeast

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Keich, Uri; Dunham, Maitreya J.

2013-01-01

DNA replication origins are necessary for the duplication of genomes. In addition, plasmid-based expression systems require DNA replication origins to maintain plasmids efficiently. The yeast autonomously replicating sequence (ARS) assay has been a valuable tool in dissecting replication origin structure and function. However, the dearth of information on origins in diverse yeasts limits the availability of efficient replication origin modules to only a handful of species and restricts our understanding of origin function and evolution. To enable rapid study of origins, we have developed a sequencing-based suite of methods for comprehensively mapping and characterizing ARSs within a yeast genome. Our approach finely maps genomic inserts capable of supporting plasmid replication and uses massively parallel deep mutational scanning to define molecular determinants of ARS function with single-nucleotide resolution. In addition to providing unprecedented detail into origin structure, our data have allowed us to design short, synthetic DNA sequences that retain maximal ARS function. These methods can be readily applied to understand and modulate ARS function in diverse systems. PMID:23241746

A Model For Selecting An Environmentally Responsive Trait: Evaluating Micro-scale Fitness Through UV-C Resistance and Exposure in Escherichia coli.

NASA Astrophysics Data System (ADS)

Schenone, D. J.; Igama, S.; Marash-Whitman, D.; Sloan, C.; Okansinski, A.; Moffet, A.; Grace, J. M.; Gentry, D.

2015-12-01

Experimental evolution of microorganisms in controlled microenvironments serves as a powerful tool for understanding the relationship between micro-scale microbial interactions as well as local-to global-scale environmental factors. In response to iterative and targeted environmental pressures, mutagenesis drives the emergence of novel phenotypes. Current methods to induce expression of these phenotypes require repetitive and time intensive procedures and do not allow for the continuous monitoring of conditions such as optical density, pH and temperature. To address this shortcoming, an Automated Dynamic Directed Evolution Chamber is being developed. It will initially produce Escherichia coli cells with an elevated UV-C resistance phenotype that will ultimately be adapted for different organisms as well as studying environmental effects. A useful phenotype and environmental factor for examining this relationship is UV-C resistance and exposure. In order to build a baseline for the device's operational parameters, a UV-C assay was performed on six E. coli replicates with three exposure fluxes across seven iterations. The fluxes included a 0 second exposure (control), 6 seconds at 3.3 J/m2/s and 40 seconds at 0.5 J/m2/s. After each iteration the cells were regrown and tested for UV-C resistance. We sought to quantify the increase and variability of UV-C resistance among different fluxes, and observe changes in each replicate at each iteration in terms of variance. Under different fluxes, we observed that the 0s control showed no significant increase in resistance, while the 6s/40s fluxes showed increased resistance as the number of iterations increased. A one-million fold increase in survivability was observed after seven iterations. Through statistical analysis using Spearman's rank correlation, the 40s exposure showed signs of more consistently increased resistance, but seven iterations was insufficient to demonstrate statistical significance; to test this further, our experiments will include more iterations. Furthermore, we plan to sequence all the replicants. As adaptation dynamics under intense UV exposure leads to high rate of change, it would be useful to observe differences in tolerance-related and non-tolerance-related genes between the original and UV resistant strains.

Genomic mapping of single-stranded DNA in hydroxyurea-challenged yeasts identifies origins of replication.

PubMed

Feng, Wenyi; Collingwood, David; Boeck, Max E; Fox, Lindsay A; Alvino, Gina M; Fangman, Walton L; Raghuraman, Mosur K; Brewer, Bonita J

2006-02-01

During DNA replication one or both strands transiently become single stranded: first at the sites where initiation of DNA synthesis occurs (known as origins of replication) and subsequently on the lagging strands of replication forks as discontinuous Okazaki fragments are generated. We report a genome-wide analysis of single-stranded DNA (ssDNA) formation in the presence of hydroxyurea during DNA replication in wild-type and checkpoint-deficient rad53 Saccharomyces cerevisiae cells. In wild-type cells, ssDNA was first observed at a subset of replication origins and later 'migrated' bi-directionally, suggesting that ssDNA formation is associated with continuously moving replication forks. In rad53 cells, ssDNA was observed at virtually every known origin, but remained there over time, suggesting that replication forks stall. Telomeric regions seemed to be particularly sensitive to the loss of Rad53 checkpoint function. Replication origins in Schizosaccharomyces pombe were also mapped using our method.

The origin and early evolution of nucleic acid polymerases

NASA Technical Reports Server (NTRS)

Lazcano, A.; Cappello, R.; Valverde, V.; Llaca, V.; Oro, J.

1992-01-01

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA-polymerase beta-prime subunit and its homologues is discussed. It is shown that, in the DNA-dependent RNA polymerases from three cellular lineages, a very conserved sequence of eight amino acids, also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein, is present. The optimal conditions for the replicase activity of the avian-myeloblastosis-virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is discussed.

GC-rich DNA elements enable replication origin activity in the methylotrophic yeast Pichia pastoris.

PubMed

Liachko, Ivan; Youngblood, Rachel A; Tsui, Kyle; Bubb, Kerry L; Queitsch, Christine; Raghuraman, M K; Nislow, Corey; Brewer, Bonita J; Dunham, Maitreya J

2014-03-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins--a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation.

Examining a DNA Replication Requirement for Bacteriophage λ Red- and Rac Prophage RecET-Promoted Recombination in Escherichia coli.

PubMed

Thomason, Lynn C; Costantino, Nina; Court, Donald L

2016-09-13

Recombineering, in vivo genetic engineering with bacteriophage homologous recombination systems, is a powerful technique for making genetic modifications in bacteria. Two systems widely used in Escherichia coli are the Red system from phage λ and RecET from the defective Rac prophage. We investigated the in vivo dependence of recombineering on DNA replication of the recombining substrate using plasmid targets. For λ Red recombination, when DNA replication of a circular target plasmid is prevented, recombination with single-stranded DNA oligonucleotides is greatly reduced compared to that under replicating conditions. For RecET recombination, when DNA replication of the targeted plasmid is prevented, the recombination frequency is also reduced, to a level identical to that seen for the Red system in the absence of replication. The very low level of oligonucleotide recombination observed in the absence of any phage recombination functions is the same in the presence or absence of DNA replication. In contrast, both the Red and RecET systems recombine a nonreplicating linear dimer plasmid with high efficiency to yield a circular monomer. Therefore, the DNA replication requirement is substrate dependent. Our data are consistent with recombination by both the Red and RecET systems occurring predominately by single-strand annealing rather than by strand invasion. Bacteriophage homologous recombination systems are widely used for in vivo genetic engineering in bacteria. Single- or double-stranded linear DNA substrates containing short flanking homologies to chromosome targets are used to generate precise and accurate genetic modifications when introduced into bacteria expressing phage recombinases. Understanding the molecular mechanism of these recombination systems will facilitate improvements in the technology. Here, two phage-specific systems are shown to require exposure of complementary single-strand homologous targets for efficient recombination; these single-strand regions may be created during DNA replication or by single-strand exonuclease digestion of linear duplex DNA. Previously, in vitro studies reported that these recombinases promote the single-strand annealing of two complementary DNAs and also strand invasion of a single DNA strand into duplex DNA to create a three-stranded region. Here, in vivo experiments show that recombinase-mediated annealing of complementary single-stranded DNA is the predominant recombination pathway in E. coli. Copyright © 2016 Thomason et al.

Lambda Red Mediated Gap Repair Utilizes a Novel Replicative Intermediate in Escherichia coli

PubMed Central

Reddy, Thimma R.; Fevat, Léna M. S.; Munson, Sarah E.; Stewart, A. Francis; Cowley, Shaun M.

2015-01-01

The lambda phage Red recombination system can mediate efficient homologous recombination in Escherichia coli, which is the basis of the DNA engineering technique termed recombineering. Red mediated insertion of DNA requires DNA replication, involves a single-stranded DNA intermediate and is more efficient on the lagging strand of the replication fork. Lagging strand recombination has also been postulated to explain the Red mediated repair of gapped plasmids by an Okazaki fragment gap filling model. Here, we demonstrate that gap repair involves a different strand independent mechanism. Gap repair assays examining the strand asymmetry of recombination did not show a lagging strand bias. Directly testing an ssDNA plasmid showed lagging strand recombination is possible but dsDNA plasmids did not employ this mechanism. Insertional recombination combined with gap repair also did not demonstrate preferential lagging strand bias, supporting a different gap repair mechanism. The predominant recombination route involved concerted insertion and subcloning though other routes also operated at lower frequencies. Simultaneous insertion of DNA resulted in modification of both strands and was unaffected by mutations to DNA polymerase I, responsible for Okazaki fragment maturation. The lower efficiency of an alternate Red mediated ends-in recombination pathway and the apparent lack of a Holliday junction intermediate suggested that gap repair does not involve a different Red recombination pathway. Our results may be explained by a novel replicative intermediate in gap repair that does not involve a replication fork. We exploited these observations by developing a new recombineering application based on concerted insertion and gap repair, termed SPI (subcloning plus insertion). SPI selected against empty vector background and selected for correct gap repair recombinants. We used SPI to simultaneously insert up to four different gene cassettes in a single recombineering reaction. Consequently, our findings have important implications for the understanding of E. coli replication and Red recombination. PMID:25803509

A Molecular Toolbox to Engineer Site-Specific DNA Replication Perturbation.

PubMed

Larsen, Nicolai B; Hickson, Ian D; Mankouri, Hocine W

2018-01-01

Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.

Optimization of a one-step heat-inducible in vivo mini DNA vector production system.

PubMed

Nafissi, Nafiseh; Sum, Chi Hong; Wettig, Shawn; Slavcev, Roderick A

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called "Super Sequences" that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼ 90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics.

Optimization of a One-Step Heat-Inducible In Vivo Mini DNA Vector Production System

PubMed Central

Wettig, Shawn; Slavcev, Roderick A.

2014-01-01

While safer than their viral counterparts, conventional circular covalently closed (CCC) plasmid DNA vectors offer a limited safety profile. They often result in the transfer of unwanted prokaryotic sequences, antibiotic resistance genes, and bacterial origins of replication that may lead to unwanted immunostimulatory responses. Furthermore, such vectors may impart the potential for chromosomal integration, thus potentiating oncogenesis. Linear covalently closed (LCC), bacterial sequence free DNA vectors have shown promising clinical improvements in vitro and in vivo. However, the generation of such minivectors has been limited by in vitro enzymatic reactions hindering their downstream application in clinical trials. We previously characterized an in vivo temperature-inducible expression system, governed by the phage λ pL promoter and regulated by the thermolabile λ CI[Ts]857 repressor to produce recombinant protelomerase enzymes in E. coli. In this expression system, induction of recombinant protelomerase was achieved by increasing culture temperature above the 37°C threshold temperature. Overexpression of protelomerase led to enzymatic reactions, acting on genetically engineered multi-target sites called “Super Sequences” that serve to convert conventional CCC plasmid DNA into LCC DNA minivectors. Temperature up-shift, however, can result in intracellular stress responses and may alter plasmid replication rates; both of which may be detrimental to LCC minivector production. We sought to optimize our one-step in vivo DNA minivector production system under various induction schedules in combination with genetic modifications influencing plasmid replication, processing rates, and cellular heat stress responses. We assessed different culture growth techniques, growth media compositions, heat induction scheduling and temperature, induction duration, post-induction temperature, and E. coli genetic background to improve the productivity and scalability of our system, achieving an overall LCC DNA minivector production efficiency of ∼90%.We optimized a robust technology conferring rapid, scalable, one-step in vivo production of LCC DNA minivectors with potential application to gene transfer-mediated therapeutics. PMID:24586704

Effects of simulated Mars conditions on the survival and growth of Escherichia coli and Serratia liquefaciens.

PubMed

Berry, Bonnie J; Jenkins, David G; Schuerger, Andrew C

2010-04-01

Escherichia coli and Serratia liquefaciens, two bacterial spacecraft contaminants known to replicate under low atmospheric pressures of 2.5 kPa, were tested for growth and survival under simulated Mars conditions. Environmental stresses of high salinity, low temperature, and low pressure were screened alone and in combination for effects on bacterial survival and replication, and then cells were tested in Mars analog soils under simulated Mars conditions. Survival and replication of E. coli and S. liquefaciens cells in liquid medium were evaluated for 7 days under low temperatures (5, 10, 20, or 30 degrees C) with increasing concentrations (0, 5, 10, or 20%) of three salts (MgCl(2), MgSO(4), NaCl) reported to be present on the surface of Mars. Moderate to high growth rates were observed for E. coli and S. liquefaciens at 30 or 20 degrees C and in solutions with 0 or 5% salts. In contrast, cell densities of both species generally did not increase above initial inoculum levels under the highest salt concentrations (10 and 20%) and the four temperatures tested, with the exception that moderately higher cell densities were observed for both species at 10% MgSO(4) maintained at 20 or 30 degrees C. Growth rates of E. coli and S. liquefaciens in low salt concentrations were robust under all pressures (2.5, 10, or 101.3 kPa), exhibiting a general increase of up to 2.5 orders of magnitude above the initial inoculum levels of the assays. Vegetative E. coli cells were maintained in a Mars analog soil for 7 days under simulated Mars conditions that included temperatures between 20 and -50 degrees C for a day/night diurnal period, UVC irradiation (200 to 280 nm) at 3.6 W m(-2) for daytime operations (8 h), pressures held at a constant 0.71 kPa, and a gas composition that included the top five gases found in the martian atmosphere. Cell densities of E. coli failed to increase under simulated Mars conditions, and survival was reduced 1 to 2 orders of magnitude by the interactive effects of desiccation, UV irradiation, high salinity, and low pressure (in decreasing order of importance). Results suggest that E. coli may be able to survive, but not grow, in surficial soils on Mars.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

PubMed

Agarwal, Meetu; Bhowmick, Krishanu; Shah, Kushal; Krishnamachari, Annangarachari; Dhar, Suman Kumar

2017-08-01

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum. © 2017 Federation of European Biochemical Societies.

Cdc7 is required throughout the yeast S phase to activate replication origins.

PubMed

Donaldson, A D; Fangman, W L; Brewer, B J

1998-02-15

The long-standing conclusion that the Cdc7 kinase of Saccharomyces cerevisiae is required only to trigger S phase has been challenged by recent data that suggests it acts directly on individual replication origins. We tested the possibility that early- and late-activated origins have different requirements for Cdc7 activity. Cells carrying a cdc7(ts) allele were first arrested in G1 at the cdc7 block by incubation at 37 degrees C, and then were allowed to enter S phase by brief incubation at 23 degrees C. During the S phase, after return to 37 degrees C, early-firing replication origins were activated, but late origins failed to fire. Similarly, a plasmid with a late-activated origin was defective in replication. As a consequence of the origin activation defect, duplication of chromosomal sequences that are normally replicated from late origins was greatly delayed. Early-replicating regions of the genome duplicated at approximately their normal time. The requirements of early and late origins for Cdc7 appear to be temporally rather than quantitatively different, as reducing overall levels of Cdc7 by growth at semi-permissive temperature reduced activation at early and late origins approximately equally. Our results show that Cdc7 activates early and late origins separately, with late origins requiring the activity later in S phase to permit replication initiation.

The LE1 Bacteriophage Replicates as a Plasmid within Leptospira biflexa: Construction of an L. biflexa-Escherichia coli Shuttle Vector

PubMed Central

Girons, Isabelle Saint; Bourhy, Pascale; Ottone, Catherine; Picardeau, Mathieu; Yelton, David; Hendrix, Roger W.; Glaser, Philippe; Charon, Nyles

2000-01-01

We have discovered that LE1, one of the plaque-forming phages previously described as lytic for the Leptospira biflexa saprophytic spirochete (I. Saint Girons, D. Margarita, P. Amouriaux, and G. Baranton, Res. Microbiol. 141:1131–1138, 1990), was indeed temperate. LE1 was found to be unusual, as Southern blot analysis indicated that it is one of the few phages to replicate in the prophage state as a circular plasmid. The unavailability of such small endogenous replicons has hindered genetic experimentation in Leptospira. We have developed a shuttle vector with DNA derived from LE1. Random LE1 DNA fragments were cloned into a pGEM 7Zf(+) derivative devoid of most of the bla gene but carrying a kanamycin resistance marker from the gram-positive bacterium Enterococcus (Streptococcus) faecalis. These constructs were transformed into L. biflexa strain Patoc 1 by electroporation, giving rise to kanamycin-resistant transformants. A 2.2-kb fragment from LE1 was responsible for replication of the vector in L. biflexa. However, a larger region including an intact parA gene homologue was necessary for the stability of the shuttle vector. Direct repeats and AT-rich regions characterized the LE1 origin of replication. Our data indicate that the replicon derived from the LE1 leptophage, together with the kanamycin resistance gene, is a promising tool with which to develop the genetics of Leptospira species. PMID:11004167

Chromosomal context and replication properties of ARS plasmids in Schizosaccharomyces pombe.

PubMed

Pratihar, Aditya S; Tripathi, Vishnu P; Yadav, Mukesh P; Dubey, Dharani D

2015-12-01

Short, specific DNA sequences called as Autonomously Replicating Sequence (ARS) elements function as plasmid as well as chromosomal replication origins in yeasts. As compared to ARSs, different chromosomal origins vary greatly in their efficiency and timing of replication probably due to their wider chromosomal context. The two Schizosaccharomyces pombe ARS elements, ars727 and ars2004, represent two extremities in their chromosomal origin activity - ars727 is inactive and late replicating, while ars2004 is a highly active, early-firing origin. To determine the effect of chromosomal context on the activity of these ARS elements, we have cloned them with their extended chromosomal context as well as in the context of each other in both orientations and analysed their replication efficiency by ARS and plasmid stability assays. We found that these ARS elements retain their origin activity in their extended/altered context. However, deletion of a 133-bp region of the previously reported ars727- associated late replication enforcing element (LRE) caused advancement in replication timing of the resulting plasmid. These results confirm the role of LRE in directing plasmid replication timing and suggest that the plasmid origin efficiency of ars2004 or ars727 remains unaltered by the extended chromosomal context.

The Spectrum of Replication Errors in the Absence of Error Correction Assayed Across the Whole Genome of Escherichia coli.

PubMed

Niccum, Brittany A; Lee, Heewook; MohammedIsmail, Wazim; Tang, Haixu; Foster, Patricia L

2018-06-15

When the DNA polymerase that replicates the Escherichia coli chromosome, DNA Pol III, makes an error, there are two primary defenses against mutation: proofreading by the epsilon subunit of the holoenzyme and mismatch repair. In proofreading deficient strains, mismatch repair is partially saturated and the cell's response to DNA damage, the SOS response, may be partially induced. To investigate the nature of replication errors, we used mutation accumulation experiments and whole genome sequencing to determine mutation rates and mutational spectra across the entire chromosome of strains deficient in proofreading, mismatch repair, and the SOS response. We report that a proofreading-deficient strain has a mutation rate 4,000-fold greater than wild-type strains. While the SOS response may be induced in these cells, it does not contribute to the mutational load. Inactivating mismatch repair in a proofreading-deficient strain increases the mutation rate another 1.5-fold. DNA polymerase has a bias for converting G:C to A:T base pairs, but proofreading reduces the impact of these mutations, helping to maintain the genomic G:C content. These findings give an unprecedented view of how polymerase and error-correction pathways work together to maintain E. coli' s low mutation rate of 1 per thousand generations. Copyright © 2018, Genetics.

Maintaining replication origins in the face of genomic change.

PubMed

Di Rienzi, Sara C; Lindstrom, Kimberly C; Mann, Tobias; Noble, William S; Raghuraman, M K; Brewer, Bonita J

2012-10-01

Origins of replication present a paradox to evolutionary biologists. As a collection, they are absolutely essential genomic features, but individually are highly redundant and nonessential. It is therefore difficult to predict to what extent and in what regard origins are conserved over evolutionary time. Here, through a comparative genomic analysis of replication origins and chromosomal replication patterns in the budding yeasts Saccharomyces cerevisiae and Lachancea waltii, we assess to what extent replication origins survived genomic change produced from 150 million years of evolution. We find that L. waltii origins exhibit a core consensus sequence and nucleosome occupancy pattern highly similar to those of S. cerevisiae origins. We further observe that the overall progression of chromosomal replication is similar between L. waltii and S. cerevisiae. Nevertheless, few origins show evidence of being conserved in location between the two species. Among the conserved origins are those surrounding centromeres and adjacent to histone genes, suggesting that proximity to an origin may be important for their regulation. We conclude that, over evolutionary time, origins maintain sequence, structure, and regulation, but are continually being created and destroyed, with the result that their locations are generally not conserved.

Maintaining replication origins in the face of genomic change

PubMed Central

Di Rienzi, Sara C.; Lindstrom, Kimberly C.; Mann, Tobias; Noble, William S.; Raghuraman, M.K.; Brewer, Bonita J.

2012-01-01

Origins of replication present a paradox to evolutionary biologists. As a collection, they are absolutely essential genomic features, but individually are highly redundant and nonessential. It is therefore difficult to predict to what extent and in what regard origins are conserved over evolutionary time. Here, through a comparative genomic analysis of replication origins and chromosomal replication patterns in the budding yeasts Saccharomyces cerevisiae and Lachancea waltii, we assess to what extent replication origins survived genomic change produced from 150 million years of evolution. We find that L. waltii origins exhibit a core consensus sequence and nucleosome occupancy pattern highly similar to those of S. cerevisiae origins. We further observe that the overall progression of chromosomal replication is similar between L. waltii and S. cerevisiae. Nevertheless, few origins show evidence of being conserved in location between the two species. Among the conserved origins are those surrounding centromeres and adjacent to histone genes, suggesting that proximity to an origin may be important for their regulation. We conclude that, over evolutionary time, origins maintain sequence, structure, and regulation, but are continually being created and destroyed, with the result that their locations are generally not conserved. PMID:22665441

Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1

PubMed Central

Bröker, Daniel; Arenskötter, Matthias; Legatzki, Antje; Nies, Dietrich H.; Steinbüchel, Alexander

2004-01-01

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1. PMID:14679241

Mechanisms of bacterial DNA replication restart

PubMed Central

Windgassen, Tricia A; Wessel, Sarah R; Bhattacharyya, Basudeb

2018-01-01

Abstract Multi-protein DNA replication complexes called replisomes perform the essential process of copying cellular genetic information prior to cell division. Under ideal conditions, replisomes dissociate only after the entire genome has been duplicated. However, DNA replication rarely occurs without interruptions that can dislodge replisomes from DNA. Such events produce incompletely replicated chromosomes that, if left unrepaired, prevent the segregation of full genomes to daughter cells. To mitigate this threat, cells have evolved ‘DNA replication restart’ pathways that have been best defined in bacteria. Replication restart requires recognition and remodeling of abandoned replication forks by DNA replication restart proteins followed by reloading of the replicative DNA helicase, which subsequently directs assembly of the remaining replisome subunits. This review summarizes our current understanding of the mechanisms underlying replication restart and the proteins that drive the process in Escherichia coli (PriA, PriB, PriC and DnaT). PMID:29202195

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

2016-01-01

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq)

PubMed Central

Langley, Alexander R.; Gräf, Stefan; Smith, James C.; Krude, Torsten

2016-01-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. PMID:27587586

Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq).

PubMed

Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten

2016-12-01

Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Varicella-zoster virus (VZV) origin of DNA replication oriS influences origin-dependent DNA replication and flanking gene transcription.

PubMed

Khalil, Mohamed I; Sommer, Marvin H; Hay, John; Ruyechan, William T; Arvin, Ann M

2015-07-01

The VZV genome has two origins of DNA replication (oriS), each of which consists of an AT-rich sequence and three origin binding protein (OBP) sites called Box A, C and B. In these experiments, the mutation in the core sequence CGC of the Box A and C not only inhibited DNA replication but also inhibited both ORF62 and ORF63 expression in reporter gene assays. In contrast the Box B mutation did not influence DNA replication or flanking gene transcription. These results suggest that efficient DNA replication enhances ORF62 and ORF63 transcription. Recombinant viruses carrying these mutations in both sites and one with a deletion of the whole oriS were constructed. Surprisingly, the recombinant virus lacking both copies of oriS retained the capacity to replicate in melanoma and HELF cells suggesting that VZV has another origin of DNA replication. Copyright © 2015 Elsevier Inc. All rights reserved.

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mannik, Jaana; Castillo, Daniel E.; Yang, Da

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

The role of MatP, ZapA and ZapB in chromosomal organization and dynamics in Escherichia coli

DOE PAGES

Mannik, Jaana; Castillo, Daniel E.; Yang, Da; ...

2016-01-13

Despite extensive research over several decades, a comprehensive view of how the Escherichia coli chromosome is organized within the nucleoid, and how two daughter chromosomes segregate has yet to emerge. Here we investigate the role of the MatP, ZapA and ZapB proteins in organizing the replication terminus (Ter) region and in the chromosomal segregation process. Quantitative image analysis of the fluorescently labeled Ter region shows that the replication terminus attaches to the divisome in a single segment along the perimeter of the cell in a MatP, ZapA and ZapB-dependent manner. The attachment does not significantly affect the bulk chromosome segregationmore » in slow growth conditions. With or without the attachment, two chromosomal masses separate from each other at a speed comparable to the cell growth. The separation starts even before the replication terminus region positions itself at the center of the nucleoid. Modeling of the segregation based on conformational entropy correctly predicts the positioning of the replication terminus region within the nucleoid. Furthermore, the model produces a distinctly different chromosomal density distribution than the experiment, indicating that the conformational entropy plays a limited role in segregating the chromosomes in the late stages of replication.« less

Origins of DNA Replication and Amplification in the Breast Cancer Genome

DTIC Science & Technology

2012-09-01

W81XWH-10-1-0463 TITLE: Origins of DNA Replication and Amplification in the...2. REPORT TYPE Final 3. DATES COVERED 1 Sep 2010 – 31 Aug 2012 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Origins of DNA Replication and...described in the DOD funded parent grant, to test our hypothesis we need to map origins of DNA replication in the genome and ask which of these

GC-Rich DNA Elements Enable Replication Origin Activity in the Methylotrophic Yeast Pichia pastoris

PubMed Central

Liachko, Ivan; Youngblood, Rachel A.; Tsui, Kyle; Bubb, Kerry L.; Queitsch, Christine; Raghuraman, M. K.; Nislow, Corey; Brewer, Bonita J.; Dunham, Maitreya J.

2014-01-01

The well-studied DNA replication origins of the model budding and fission yeasts are A/T-rich elements. However, unlike their yeast counterparts, both plant and metazoan origins are G/C-rich and are associated with transcription start sites. Here we show that an industrially important methylotrophic budding yeast, Pichia pastoris, simultaneously employs at least two types of replication origins—a G/C-rich type associated with transcription start sites and an A/T-rich type more reminiscent of typical budding and fission yeast origins. We used a suite of massively parallel sequencing tools to map and dissect P. pastoris origins comprehensively, to measure their replication dynamics, and to assay the global positioning of nucleosomes across the genome. Our results suggest that some functional overlap exists between promoter sequences and G/C-rich replication origins in P. pastoris and imply an evolutionary bifurcation of the modes of replication initiation. PMID:24603708

Variability of Photodynamic Killing in Escherichia coli and Avoidance of Variability with Agar

PubMed Central

O'Bryan, Corliss; Harrison, Arthur P.

1971-01-01

Photodynamic killing of Escherichia coli in acridine orange is influenced by the composition of the containing vessel, and after high kill the variance between replicate suspensions is greater than attributable solely to sampling and plating. Addition of agar minimizes both phenomena, but a higher illumination dose is required to produce the same degree of killing. PMID:4934057

Mechanisms and regulation of DNA replication initiation in eukaryotes

PubMed Central

Parker, Matthew W.; Botchan, Michael R.; Berger, James M.

2017-01-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a given cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the Origin Recognition Complex (ORC), and subsequent activation of the helicase by incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms. PMID:28094588

Mechanisms and regulation of DNA replication initiation in eukaryotes.

PubMed

Parker, Matthew W; Botchan, Michael R; Berger, James M

2017-04-01

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation - from selecting replication start sites to replicative helicase loading and activation - and describe how these events are often distinctly regulated across different eukaryotic model organisms.

Mapping replication origins in yeast chromosomes.

PubMed

Brewer, B J; Fangman, W L

1991-07-01

The replicon hypothesis, first proposed in 1963 by Jacob and Brenner, states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms.

The eukaryotic bell-shaped temporal rate of DNA replication origin firing emanates from a balance between origin activation and passivation.

PubMed

Arbona, Jean-Michel; Goldar, Arach; Hyrien, Olivier; Arneodo, Alain; Audit, Benjamin

2018-06-01

The time-dependent rate I(t) of origin firing per length of unreplicated DNA presents a universal bell shape in eukaryotes that has been interpreted as the result of a complex time-evolving interaction between origins and limiting firing factors. Here we show that a normal diffusion of replication fork components towards localized potential replication origins (p-oris) can more simply account for the I(t) universal bell shape, as a consequence of a competition between the origin firing time and the time needed to replicate DNA separating two neighboring p-oris . We predict the I(t) maximal value to be the product of the replication fork speed with the squared p-ori density. We show that this relation is robustly observed in simulations and in experimental data for several eukaryotes. Our work underlines that fork-component recycling and potential origins localization are sufficient spatial ingredients to explain the universality of DNA replication kinetics. © 2018, Arbona et al.

Regulatory Mechanisms That Prevent Re-initiation of DNA Replication Can Be Locally Modulated at Origins by Nearby Sequence Elements

PubMed Central

Richardson, Christopher D.; Li, Joachim J.

2014-01-01

Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ∼60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others. PMID:24945837

Checkpoint independence of most DNA replication origins in fission yeast

PubMed Central

Mickle, Katie L; Ramanathan, Sunita; Rosebrock, Adam; Oliva, Anna; Chaudari, Amna; Yompakdee, Chulee; Scott, Donna; Leatherwood, Janet; Huberman, Joel A

2007-01-01

Background In budding yeast, the replication checkpoint slows progress through S phase by inhibiting replication origin firing. In mammals, the replication checkpoint inhibits both origin firing and replication fork movement. To find out which strategy is employed in the fission yeast, Schizosaccharomyces pombe, we used microarrays to investigate the use of origins by wild-type and checkpoint-mutant strains in the presence of hydroxyurea (HU), which limits the pool of deoxyribonucleoside triphosphates (dNTPs) and activates the replication checkpoint. The checkpoint-mutant cells carried deletions either of rad3 (which encodes the fission yeast homologue of ATR) or cds1 (which encodes the fission yeast homologue of Chk2). Results Our microarray results proved to be largely consistent with those independently obtained and recently published by three other laboratories. However, we were able to reconcile differences between the previous studies regarding the extent to which fission yeast replication origins are affected by the replication checkpoint. We found (consistent with the three previous studies after appropriate interpretation) that, in surprising contrast to budding yeast, most fission yeast origins, including both early- and late-firing origins, are not significantly affected by checkpoint mutations during replication in the presence of HU. A few origins (~3%) behaved like those in budding yeast: they replicated earlier in the checkpoint mutants than in wild type. These were located primarily in the heterochromatic subtelomeric regions of chromosomes 1 and 2. Indeed, the subtelomeric regions defined by the strongest checkpoint restraint correspond precisely to previously mapped subtelomeric heterochromatin. This observation implies that subtelomeric heterochromatin in fission yeast differs from heterochromatin at centromeres, in the mating type region, and in ribosomal DNA, since these regions replicated at least as efficiently in wild-type cells as in checkpoint-mutant cells. Conclusion The fact that ~97% of fission yeast replication origins – both early and late – are not significantly affected by replication checkpoint mutations in HU-treated cells suggests that (i) most late-firing origins are restrained from firing in HU-treated cells by at least one checkpoint-independent mechanism, and (ii) checkpoint-dependent slowing of S phase in fission yeast when DNA is damaged may be accomplished primarily by the slowing of replication forks. PMID:18093330

β-lactam antibiotics promote bacterial mutagenesis via an RpoS-mediated reduction in replication fidelity

PubMed Central

Gutierrez, A.; Laureti, L.; Crussard, S.; Abida, H.; Rodríguez-Rojas, A.; Blázquez, J.; Baharoglu, Z.; Mazel, D.; Darfeuille, F.; Vogel, J.; Matic, I.

2013-01-01

Regardless of their targets and modes of action, subinhibitory concentrations of antibiotics can have an impact on cell physiology and trigger a large variety of cellular responses in different bacterial species. Subinhibitory concentrations of β-lactam antibiotics cause reactive oxygen species production and induce PolIV-dependent mutagenesis in Escherichia coli. Here we show that subinhibitory concentrations of β-lactam antibiotics induce the RpoS regulon. RpoS-regulon induction is required for PolIV-dependent mutagenesis because it diminishes the control of DNA-replication fidelity by depleting MutS in E. coli, Vibrio cholerae and Pseudomonas aeruginosa. We also show that in E. coli, the reduction in mismatch-repair activity is mediated by SdsR, the RpoS-controlled small RNA. In summary, we show that mutagenesis induced by subinhibitory concentrations of antibiotics is a genetically controlled process. Because this mutagenesis can generate mutations conferring antibiotic resistance, it should be taken into consideration for the development of more efficient antimicrobial therapeutic strategies. PMID:23511474

Origin of life in a digital microcosm

NASA Astrophysics Data System (ADS)

C G, Nitash; LaBar, Thomas; Hintze, Arend; Adami, Christoph

2017-11-01

While all organisms on Earth share a common descent, there is no consensus on whether the origin of the ancestral self-replicator was a one-off event or whether it only represented the final survivor of multiple origins. Here, we use the digital evolution system Avida to study the origin of self-replicating computer programs. By using a computational system, we avoid many of the uncertainties inherent in any biochemical system of self-replicators (while running the risk of ignoring a fundamental aspect of biochemistry). We generated the exhaustive set of minimal-genome self-replicators and analysed the network structure of this fitness landscape. We further examined the evolvability of these self-replicators and found that the evolvability of a self-replicator is dependent on its genomic architecture. We also studied the differential ability of replicators to take over the population when competed against each other, akin to a primordial-soup model of biogenesis, and found that the probability of a self-replicator outcompeting the others is not uniform. Instead, progenitor (most-recent common ancestor) genotypes are clustered in a small region of the replicator space. Our results demonstrate how computational systems can be used as test systems for hypotheses concerning the origin of life. This article is part of the themed issue 'Reconceptualizing the origins of life'.

Respirator Filter Efficiency Testing Against Particulate and Biological Aerosols Under Moderate to High Flow Rates

DTIC Science & Technology

2006-08-01

Biotech QCount® Colony Counter G.2 MS2 Phage G.2.1 Growth of E . coli E . co/i serves as the host for MS2 replication and was needed for the MS2...quantification assay. Before culturing, the E . coli (American Type Culture Collection [ATCC] No. 15597, Rockville, MD) stock was tested for purity by streaking on...pure, a working solution of E . coli was prepared by inoculating nutrient broth (NB) media and incubating in a shaking incubator at 37°C and 150

Two subunits of human ORC are dispensable for DNA replication and proliferation.

PubMed

Shibata, Etsuko; Kiran, Manjari; Shibata, Yoshiyuki; Singh, Samarendra; Kiran, Shashi; Dutta, Anindya

2016-12-01

The six-subunit Origin Recognition Complex (ORC) is believed to be an essential eukaryotic ATPase that binds to origins of replication as a ring-shaped heterohexamer to load MCM2-7 and initiate DNA replication. We have discovered that human cell lines in culture proliferate with intact chromosomal origins of replication after disruption of both alleles of ORC2 or of the ATPase subunit, ORC1 . The ORC1 or ORC2 -depleted cells replicate with decreased chromatin loading of MCM2-7 and become critically dependent on another ATPase, CDC6, for survival and DNA replication. Thus, either the ORC ring lacking a subunit, even its ATPase subunit, can load enough MCM2-7 in partnership with CDC6 to initiate DNA replication, or cells have an ORC-independent, CDC6-dependent mechanism to load MCM2-7 on origins of replication.

Intragenic origins due to short G1 phases underlie oncogene-induced DNA replication stress.

PubMed

Macheret, Morgane; Halazonetis, Thanos D

2018-03-01

Oncogene-induced DNA replication stress contributes critically to the genomic instability that is present in cancer. However, elucidating how oncogenes deregulate DNA replication has been impeded by difficulty in mapping replication initiation sites on the human genome. Here, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 and MYC. Remarkably, both oncogenes induced firing of a novel set of DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase, before all genic regions had been transcribed, allowed firing of origins within genes in cells with activated oncogenes. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-stranded break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of intragenic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

Re-wiring of energy metabolism promotes viability during hyperreplication stress in E. coli

PubMed Central

Campion, Christopher; Weimann, Allan

2017-01-01

Chromosome replication in Escherichia coli is initiated by DnaA. DnaA binds ATP which is essential for formation of a DnaA-oriC nucleoprotein complex that promotes strand opening, helicase loading and replisome assembly. Following initiation, DnaAATP is converted to DnaAADP primarily by the Regulatory Inactivation of DnaA process (RIDA). In RIDA deficient cells, DnaAATP accumulates leading to uncontrolled initiation of replication and cell death by accumulation of DNA strand breaks. Mutations that suppress RIDA deficiency either dampen overinitiation or permit growth despite overinitiation. We characterize mutations of the last group that have in common that distinct metabolic routes are rewired resulting in the redirection of electron flow towards the cytochrome bd-1. We propose a model where cytochrome bd-1 lowers the formation of reactive oxygen species and hence oxidative damage to the DNA in general. This increases the processivity of replication forks generated by overinitiation to a level that sustains viability. PMID:28129339

Genomics and structure/function studies of Rhabdoviridae proteins involved in replication and transcription.

PubMed

Assenberg, R; Delmas, O; Morin, B; Graham, S C; De Lamballerie, X; Laubert, C; Coutard, B; Grimes, J M; Neyts, J; Owens, R J; Brandt, B W; Gorbalenya, A; Tucker, P; Stuart, D I; Canard, B; Bourhy, H

2010-08-01

Some mammalian rhabdoviruses may infect humans, and also infect invertebrates, dogs, and bats, which may act as vectors transmitting viruses among different host species. The VIZIER programme, an EU-funded FP6 program, has characterized viruses that belong to the Vesiculovirus, Ephemerovirus and Lyssavirus genera of the Rhabdoviridae family to perform ground-breaking research on the identification of potential new drug targets against these RNA viruses through comprehensive structural characterization of the replicative machinery. The contribution of VIZIER programme was of several orders. First, it contributed substantially to research aimed at understanding the origin, evolution and diversity of rhabdoviruses. This diversity was then used to obtain further structural information on the proteins involved in replication. Two strategies were used to produce recombinant proteins by expression of both full length or domain constructs in either E. coli or insect cells, using the baculovirus system. In both cases, parallel cloning and expression screening at small-scale of multiple constructs based on different viruses including the addition of fusion tags, was key to the rapid generation of expression data. As a result, some progress has been made in the VIZIER programme towards dissecting the multi-functional L protein into components suitable for structural and functional studies. However, the phosphoprotein polymerase co-factor and the structural matrix protein, which play a number of roles during viral replication and drives viral assembly, have both proved much more amenable to structural biology. Applying the multi-construct/multi-virus approach central to protein production processes in VIZIER has yielded new structural information which may ultimately be exploitable in the derivation of novel ways of intervening in viral replication. Copyright 2010 Elsevier B.V. All rights reserved.

Structure, replication efficiency and fragility of yeast ARS elements.

PubMed

Dhar, Manoj K; Sehgal, Shelly; Kaul, Sanjana

2012-05-01

DNA replication in eukaryotes initiates at specific sites known as origins of replication, or replicators. These replication origins occur throughout the genome, though the propensity of their occurrence depends on the type of organism. In eukaryotes, zones of initiation of replication spanning from about 100 to 50,000 base pairs have been reported. The characteristics of eukaryotic replication origins are best understood in the budding yeast Saccharomyces cerevisiae, where some autonomously replicating sequences, or ARS elements, confer origin activity. ARS elements are short DNA sequences of a few hundred base pairs, identified by their efficiency at initiating a replication event when cloned in a plasmid. ARS elements, although structurally diverse, maintain a basic structure composed of three domains, A, B and C. Domain A is comprised of a consensus sequence designated ACS (ARS consensus sequence), while the B domain has the DNA unwinding element and the C domain is important for DNA-protein interactions. Although there are ∼400 ARS elements in the yeast genome, not all of them are active origins of replication. Different groups within the genus Saccharomyces have ARS elements as components of replication origin. The present paper provides a comprehensive review of various aspects of ARSs, starting from their structural conservation to sequence thermodynamics. All significant and conserved functional sequence motifs within different types of ARS elements have been extensively described. Issues like silencing at ARSs, their inherent fragility and factors governing their replication efficiency have also been addressed. Progress in understanding crucial components associated with the replication machinery and timing at these ARS elements is discussed in the section entitled "The replicon revisited". Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Origins of DNA Replication and Amplification in the Breast Cancer Genome

DTIC Science & Technology

2011-09-01

AD_________________ Award Number: W81XWH-10-1-0463 TITLE: Origins of DNA Replication and...hypothesis we need to map origins of DNA replication in the genome and ask which of these coincide with sites of DNA amplification and with ER...Spring Harbor DNA Replication meetings this summer/earlyfall. Figures from the posters and also the abstracts are attached. The samples have been

The Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification

DTIC Science & Technology

2013-09-01

Replication Origins in Breast Cancer DNA Amplification PRINCIPAL INVESTIGATOR: Cinzia Casella CONTRACTING ORGANIZATION: Brown...Interplay Between Estrogen and Replication Origins in Breast Cancer DNA Amplification 5b. GRANT NUMBER W81XWH-11-1-0599 5c. PROGRAM ELEMENT NUMBER 6... amplification and oncogenes activation in breast cancer cells? This project aims to understand the role of estrogen in inducing re-replication, thus

Initiation at closely spaced replication origins in a yeast chromosome.

PubMed

Brewer, B J; Fangman, W L

1993-12-10

Replication of eukaryotic chromosomes involves initiation at origins spaced an average of 50 to 100 kilobase pairs. In yeast, potential origins can be recognized as autonomous replication sequences (ARSs) that allow maintenance of plasmids. However, there are more ARS elements than active chromosomal origins. The possibility was examined that close spacing of ARSs can lead to inactive origins. Two ARSs located 6.5 kilobase pairs apart can indeed interfere with each other. Replication is initiated from one or the other ARS with equal probability, but rarely (< 5%) from both ARSs on the same DNA molecule.

The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

PubMed Central

Patel, Prasanta K.; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John

2008-01-01

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency. PMID:18799612

“Direct cloning in Lactobacillus plantarum: Electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete”

PubMed Central

2012-01-01

Background Lactic acid bacteria (LAB) play an important role in agricultural as well as industrial biotechnology. Development of improved LAB strains using e.g. library approaches is often limited by low transformation efficiencies wherefore one reason could be differences in the DNA methylation patterns between the Escherichia coli intermediate host for plasmid amplification and the final LAB host. In the present study, we examined the influence of DNA methylation on transformation efficiency in LAB and developed a direct cloning approach for Lactobacillus plantarum CD033. Therefore, we propagated plasmid pCD256 in E. coli strains with different dam/dcm-methylation properties. The obtained plasmid DNA was purified and transformed into three different L. plantarum strains and a selection of other LAB species. Results Best transformation efficiencies were obtained using the strain L. plantarum CD033 and non-methylated plasmid DNA. Thereby we achieved transformation efficiencies of ~ 109 colony forming units/μg DNA in L. plantarum CD033 which is in the range of transformation efficiencies reached with E. coli. Based on these results, we directly transformed recombinant expression vectors received from PCR/ligation reactions into L. plantarum CD033, omitting plasmid amplification in E. coli. Also this approach was successful and yielded a sufficient number of recombinant clones. Conclusions Transformation efficiency of L. plantarum CD033 was drastically increased when non-methylated plasmid DNA was used, providing the possibility to generate expression libraries in this organism. A direct cloning approach, whereby ligated PCR-products where successfully transformed directly into L. plantarum CD033, obviates the construction of shuttle vectors containing E. coli-specific sequences, as e.g. a ColEI origin of replication, and makes amplification of these vectors in E. coli obsolete. Thus, plasmid constructs become much smaller and occasional structural instability or mutagenesis during E. coli propagation is excluded. The results of our study provide new genetic tools for L. plantarum which will allow fast, forward and systems based genetic engineering of this species. PMID:23098256

Optical tweezers reveal how proteins alter replication

NASA Astrophysics Data System (ADS)

Chaurasiya, Kathy

Single molecule force spectroscopy is a powerful method that explores the DNA interaction properties of proteins involved in a wide range of fundamental biological processes such as DNA replication, transcription, and repair. We use optical tweezers to capture and stretch a single DNA molecule in the presence of proteins that bind DNA and alter its mechanical properties. We quantitatively characterize the DNA binding mechanisms of proteins in order to provide a detailed understanding of their function. In this work, we focus on proteins involved in replication of Escherichia coli (E. coli ), endogenous eukaryotic retrotransposons Ty3 and LINE-1, and human immunodeficiency virus (HIV). DNA polymerases replicate the entire genome of the cell, and bind both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) during DNA replication. The replicative DNA polymerase in the widely-studied model system E. coli is the DNA polymerase III subunit alpha (DNA pol III alpha). We use optical tweezers to determine that UmuD, a protein that regulates bacterial mutagenesis through its interactions with DNA polymerases, specifically disrupts alpha binding to ssDNA. This suggests that UmuD removes alpha from its ssDNA template to allow DNA repair proteins access to the damaged DNA, and to facilitate exchange of the replicative polymerase for an error-prone translesion synthesis (TLS) polymerase that inserts nucleotides opposite the lesions, so that bacterial DNA replication may proceed. This work demonstrates a biophysical mechanism by which E. coli cells tolerate DNA damage. Retroviruses and retrotransposons reproduce by copying their RNA genome into the nuclear DNA of their eukaryotic hosts. Retroelements encode proteins called nucleic acid chaperones, which rearrange nucleic acid secondary structure and are therefore required for successful replication. The chaperone activity of these proteins requires strong binding affinity for both single- and double-stranded nucleic acids. We use single molecule DNA stretching to show that the nucleocapsid protein (NC) of the yeast retrotransposon Ty3, which is likely to be an ancestor of HIV NC, has optimal nucleic acid chaperone activity with only a single zinc finger. We also show that the chaperone activity of the ORF1 protein is responsible for successful replication of the mouse LINE-1 retrotransposon. LINE-1 is also 17% of the human genome, where it generates insertion mutations and alters gene expression. Retrotransposons such as LINE-1 and Ty3 are likely to be ancestors of retroviruses such as HIV. Human APOBEC3G (A3G) inhibits HIV-1 replication via cytidine deamination of the viral ssDNA genome, as well as via a distinct deamination-independent mechanism. Efficient deamination requires rapid on-off binding kinetics, but a slow dissociation rate is required for the proposed deaminase-independent mechanism. We resolve this apparent contradiction with a new quantitative single molecule method, which shows that A3G initially binds ssDNA with fast on-off rates and subsequently converts to a slow binding mode. This suggests that oligomerization transforms A3G from a fast enzyme to a slow binding protein, which is the biophysical mechanism that allows A3G to inhibit HIV replication. A complete understanding of the mechanism of A3G-mediated antiviral activity is required to design drugs that disrupt the viral response to A3G, enhance A3G packaging inside the viral core, and other potential strategies for long-term treatment of HIV infection. We use single molecule biophysics to explore the function of proteins involved in bacterial DNA replication, endogenous retrotransposition of retroelements in eukaryotic hosts such yeast and mice, and HIV replication in human cells. Our quantitative results provide insight into protein function in a range of complex biological systems and have wide-ranging implications for human health.

DNA Replication Origins and Fork Progression at Mammalian Telomeres

PubMed Central

Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

2017-01-01

Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

PubMed

Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

2016-04-07

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

Electron Microscopic Analysis of the Products of DNA Synthesis by DNA Polymerases from Calf Thymus and Herpes Simplex Virus Type I

DTIC Science & Technology

1988-10-03

DNA replication showed an average of 2.5 primers per M13 DNA circle. The measurement of the double stranded length from individual replicative intermediates by electron microscopy was within the accuracy of 10% standard deviation. The product length distribution obtained from the HSV-1 DNA polymerase catalyzed replication of M13 DNA primed with a specific pentadecamer and in the presence of E. Coli SSB protein showed a near Poisson distribution. Replication of the same primer-template system or DNA primase primed M13 DNA template by calf thymus DNA polymerase a showed a

The DNA repair endonuclease Mus81 facilitates fast DNA replication in the absence of exogenous damage

PubMed Central

Fu, Haiqing; Martin, Melvenia M.; Regairaz, Marie; Huang, Liang; You, Yang; Lin, Chi-Mei; Ryan, Michael; Kim, RyangGuk; Shimura, Tsutomu; Pommier, Yves; Aladjem, Mirit I.

2015-01-01

The Mus81 endonuclease resolves recombination intermediates and mediates cellular responses to exogenous replicative stress. Here, we show that Mus81 also regulates the rate of DNA replication during normal growth by promoting replication fork progression while reducing the frequency of replication initiation events. In the absence of Mus81 endonuclease activity, DNA synthesis is slowed and replication initiation events are more frequent. In addition, Mus81 deficient cells fail to recover from exposure to low doses of replication inhibitors and cell viability is dependent on the XPF endonuclease. Despite an increase in replication initiation frequency, cells lacking Mus81 use the same pool of replication origins as Mus81-expressing cells. Therefore, decelerated DNA replication in Mus81 deficient cells does not initiate from cryptic or latent origins not used during normal growth. These results indicate that Mus81 plays a key role in determining the rate of DNA replication without activating a novel group of replication origins. PMID:25879486

Thermoadaptation of a mesophilic hygromycin B phosphotransferase by directed evolution in hyperthermophilic Archaea: selection of a stable genetic marker for DNA transfer into Sulfolobus solfataricus.

PubMed

Cannio, R; Contursi, P; Rossi, M; Bartolucci, S

2001-06-01

A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency.

Evolution of Shiga toxin-producing Escherichia coli O157: eight major lineages of human and cattle origin strain signature genotypes

USDA-ARS?s Scientific Manuscript database

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli O157 (STEC O157) and harbor genetic subtypes that do not all associate with human disease. STEC O157 evolved from an E. coli O55:H7 progenitor, however, depauperate nucleotide polymorphism discovery from cattle and human origin...

Mechanisms involved in regulating DNA replication origins during the cell cycle and in response to DNA damage.

PubMed Central

Early, Anne; Drury, Lucy S; Diffley, John F X

2004-01-01

Replication origins in eukaryotic cells never fire more than once in a given S phase. Here, we summarize the role of cyclin-dependent kinases in limiting DNA replication origin usage to once per cell cycle in the budding yeast Saccharomyces cerevisiae. We have examined the role of different cyclins in the phosphorylation and regulation of several replication/regulatory factors including Cdc6, Sic1, ORC and DNA polymerase alpha-primase. In addition to being regulated by the cell cycle machinery, replication origins are also regulated by the genome integrity checkpoint kinases, Mec1 and Rad53. In response to DNA damage or drugs which interfere with the progression of replication forks, the activation of late-firing replication origins is inhibited. There is evidence indicating that the temporal programme of origin firing depends upon the local histone acetylation state. We have attempted to test the possibility that checkpoint regulation of late-origin firing operates through the regulation of the acetylation state. We found that overexpression of the essential histone acetylase, Esal, cannot override checkpoint regulation of origin firing. We have also constructed a temperature-sensitive esa1 mutant. This mutant is unable to resume cell cycle progression after alpha-factor arrest. This can be overcome by overexpression of the G1 cyclin, Cln2, revealing a novel role for Esal in regulating Start. PMID:15065654

Rif1 Binding and Control of Chromosome-Internal DNA Replication Origins Is Limited by Telomere Sequestration.

PubMed

Hafner, Lukas; Lezaja, Aleksandra; Zhang, Xu; Lemmens, Laure; Shyian, Maksym; Albert, Benjamin; Follonier, Cindy; Nunes, Jose Manuel; Lopes, Massimo; Shore, David; Mattarocci, Stefano

2018-04-24

The Saccharomyces cerevisiae telomere-binding protein Rif1 plays an evolutionarily conserved role in control of DNA replication timing by promoting PP1-dependent dephosphorylation of replication initiation factors. However, ScRif1 binding outside of telomeres has never been detected, and it has thus been unclear whether Rif1 acts directly on the replication origins that it controls. Here, we show that, in unperturbed yeast cells, Rif1 primarily regulates late-replicating origins within 100 kb of a telomere. Using the chromatin endogenous cleavage ChEC-seq technique, we robustly detect Rif1 at late-replicating origins that we show are targets of its inhibitory action. Interestingly, abrogation of Rif1 telomere association by mutation of its Rap1-binding module increases Rif1 binding and origin inhibition elsewhere in the genome. Our results indicate that Rif1 inhibits replication initiation by interacting directly with origins and suggest that Rap1-dependent sequestration of Rif1 increases its effective concentration near telomeres, while limiting its action at chromosome-internal sites. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

What Should Researchers Expect When They Replicate Studies? A Statistical View of Replicability in Psychological Science.

PubMed

Patil, Prasad; Peng, Roger D; Leek, Jeffrey T

2016-07-01

A recent study of the replicability of key psychological findings is a major contribution toward understanding the human side of the scientific process. Despite the careful and nuanced analysis reported, the simple narrative disseminated by the mass, social, and scientific media was that in only 36% of the studies were the original results replicated. In the current study, however, we showed that 77% of the replication effect sizes reported were within a 95% prediction interval calculated using the original effect size. Our analysis suggests two critical issues in understanding replication of psychological studies. First, researchers' intuitive expectations for what a replication should show do not always match with statistical estimates of replication. Second, when the results of original studies are very imprecise, they create wide prediction intervals-and a broad range of replication effects that are consistent with the original estimates. This may lead to effects that replicate successfully, in that replication results are consistent with statistical expectations, but do not provide much information about the size (or existence) of the true effect. In this light, the results of the Reproducibility Project: Psychology can be viewed as statistically consistent with what one might expect when performing a large-scale replication experiment. © The Author(s) 2016.

Role of DNA polymerase I-associated 5'-exonuclease in replication of coliphage M13 replicative-form DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

The conversion of both parental- and progeny-nascent open circular M13 RF DNA into covalently closed RF I is drastically reduced in an E. coli mutant deficient in the 5' ..-->.. 3' exonuclease associated with DNA polymerase I. The nascent progeny RF DNA also contains a significant proportion of fragments of smaller than unit length.

Multiple determinants controlling activation of yeast replication origins late in S phase.

PubMed

Friedman, K L; Diller, J D; Ferguson, B M; Nyland, S V; Brewer, B J; Fangman, W L

1996-07-01

Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.

Novel Type of Fimbriae Encoded by the Large Plasmid of Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H−

PubMed Central

Brunder, Werner; Khan, A. Salam; Hacker, Jörg; Karch, Helge

2001-01-01

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H−, pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H− strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H−. PMID:11401985

Structural and Thermodynamic Signatures of DNA Recognition by Mycobacterium tuberculosis DnaA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsodikov, Oleg V.; Biswas, Tapan

An essential protein, DnaA, binds to 9-bp DNA sites within the origin of replication oriC. These binding events are prerequisite to forming an enigmatic nucleoprotein scaffold that initiates replication. The number, sequences, positions, and orientations of these short DNA sites, or DnaA boxes, within the oriCs of different bacteria vary considerably. To investigate features of DnaA boxes that are important for binding Mycobacterium tuberculosis DnaA (MtDnaA), we have determined the crystal structures of the DNA binding domain (DBD) of MtDnaA bound to a cognate MtDnaA-box (at 2.0 {angstrom} resolution) and to a consensus Escherichia coli DnaA-box (at 2.3 {angstrom}). Thesemore » structures, complemented by calorimetric equilibrium binding studies of MtDnaA DBD in a series of DnaA-box variants, reveal the main determinants of DNA recognition and establish the [T/C][T/A][G/A]TCCACA sequence as a high-affinity MtDnaA-box. Bioinformatic and calorimetric analyses indicate that DnaA-box sequences in mycobacterial oriCs generally differ from the optimal binding sequence. This sequence variation occurs commonly at the first 2 bp, making an in vivo mycobacterial DnaA-box effectively a 7-mer and not a 9-mer. We demonstrate that the decrease in the affinity of these MtDnaA-box variants for MtDnaA DBD relative to that of the highest-affinity box TTGTCCACA is less than 10-fold. The understanding of DnaA-box recognition by MtDnaA and E. coli DnaA enables one to map DnaA-box sequences in the genomes of M. tuberculosis and other eubacteria.« less

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

PubMed

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

Relationship Between Radiation Response and the Deoxyribonucleic Acid Replication Cycle in Bacteria: Dependence on the Excision-Repair System

PubMed Central

Billen, Daniel; Bruns, Laura

1970-01-01

Prestarvation of Escherichia coli for required amino acids results in a marked enhancement in both ultraviolet light (UV) or X-ray resistance for selective strains. Preventing protein synthesis by starvation for required amino acids results in completion of the cycle of chromosomal replication then underway. We have investigated the relationship between starvation-induced resistance enhancement (SIRE) and the excision-repair (Hcr) system in several E. coli strains including E. coli B/r hcr+ and its isogenic mutant E. coli B/r hcr−. The following observations were made. (i) The Hcr system is the major component of SIRE in UV-irradiated strain B/r. By using the Hcr+ strain, SIRE increases the 10% survival dose from ∼400 ergs to ∼1,200 ergs/mm2. With the Hcr cells, the increase is from ∼45 ergs to 60 ergs/mm2. (ii) Although prestarvation leads to a moderate enhancement of resistance to X irradiation, this effect is not dependent on the Hcr system. (iii) The double mutant, E. coli Bs–1 (hcr−exr−) is completely unable to express SIRE whether studied with UV or X irradiation. It is concluded that the Hcr system is the major system responsible for SIRE in UV-treated cells, whereas Exr (resistance to X rays) may be involved to a minor extent. The Exr character appears to be required for SIRE expression in X-ray exposed cells. PMID:4914566

Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins

PubMed Central

Samson, Rachel Y.; Abeyrathne, Priyanka D.; Bell, Stephen D.

2015-01-01

Summary Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins. PMID:26725007

Different nucleosomal architectures at early and late replicating origins in Saccharomyces cerevisiae.

PubMed

Soriano, Ignacio; Morafraile, Esther C; Vázquez, Enrique; Antequera, Francisco; Segurado, Mónica

2014-09-13

Eukaryotic genomes are replicated during S phase according to a temporal program. Several determinants control the timing of origin firing, including the chromatin environment and epigenetic modifications. However, how chromatin structure influences the timing of the activation of specific origins is still poorly understood. By performing high-resolution analysis of genome-wide nucleosome positioning we have identified different chromatin architectures at early and late replication origins. These different patterns are already established in G1 and are tightly correlated with the organization of adjacent transcription units. Moreover, specific early and late nucleosomal patterns are fixed robustly, even in rpd3 mutants in which histone acetylation and origin timing have been significantly altered. Nevertheless, higher histone acetylation levels correlate with the local modulation of chromatin structure, leading to increased origin accessibility. In addition, we conducted parallel analyses of replication and nucleosome dynamics that revealed that chromatin structure at origins is modulated during origin activation. Our results show that early and late replication origins present distinctive nucleosomal configurations, which are preferentially associated to different genomic regions. Our data also reveal that origin structure is dynamic and can be locally modulated by histone deacetylation, as well as by origin activation. These data offer novel insight into the contribution of chromatin structure to origin selection and firing in budding yeast.

Trace detection of specific viable bacteria using tetracysteine-tagged bacteriophages.

PubMed

Wu, Lina; Luan, Tian; Yang, Xiaoting; Wang, Shuo; Zheng, Yan; Huang, Tianxun; Zhu, Shaobin; Yan, Xiaomei

2014-01-07

Advanced methods are urgently needed to determine the identity and viability of trace amounts of pathogenic bacteria in a short time. Existing approaches either fall short in the accurate assessment of microbial viability or lack specificity in bacterial identification. Bacteriophages (or phages for short) are viruses that exclusively infect bacterial host cells with high specificity. As phages infect and replicate only in living bacterial hosts, here we exploit the strategy of using tetracysteine (TC)-tagged phage in combination with biarsenical dye to the discriminative detection of viable target bacteria from dead target cells and other viable but nontarget bacterial cells. Using recombinant M13KE-TC phage and Escherichia coli ER2738 as a model system, distinct differentiation between individual viable target cells from dead target cells was demonstrated by flow cytometry and fluorescence microscopy. As few as 1% viable E. coli ER2738 can be accurately quantified in a mix with dead E. coli ER2738 by flow cytometry. With fluorescence microscopic measurement, specific detection of as rare as 1 cfu/mL original viable target bacteria was achieved in the presence of a large excess of dead target cells and other viable but nontarget bacterial cells in 40 mL artificially contaminated drinking water sample in less than 3 h. This TC-phage-FlAsH approach is sensitive, specific, rapid, and simple, and thus shows great potential in water safety monitoring, health surveillance, and clinical diagnosis of which trace detection and identification of viable bacterial pathogens is highly demanded.

Dcm methylation is detrimental to plasmid transformation in Clostridium thermocellum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guss, Adam M; Olson, Daniel G.; Caiazza, Nicky

2012-01-01

BACKGROUND: Industrial production of biofuels and other products by cellulolytic microorganisms is of interest but hindered by the nascent state of genetic tools. Although a genetic system for Clostridium thermocellum DSM1313 has recently been developed, available methods achieve relatively low efficiency and similar plasmids can transform C. thermocellum at dramatically different efficiencies. RESULTS: We report an increase in transformation efficiency of C. thermocellum for a variety of plasmids by using DNA that has been methylated by Escherichia coli Dam but not Dcm methylases. When isolated from a dam+ dcm+ E. coli strain, pAMG206 transforms C. thermocellum 100-fold better than themore » similar plasmid pAMG205, which contains an additional Dcm methylation site in the pyrF gene. Upon removal of Dcm methylation, transformation with pAMG206 showed a four- to seven-fold increase in efficiency; however, transformation efficiency of pAMG205 increased 500-fold. Removal of the Dcm methylation site from the pAM205 pyrF gene via silent mutation resulted in increased transformation efficiencies equivalent to that of pAMG206. Upon proper methylation, transformation efficiency of plasmids bearing the pMK3 and pB6A origins of replication increased ca. three orders of magnitude. CONCLUSION: E. coli Dcm methylation decreases transformation efficiency in C. thermocellum DSM1313. The use of properly methylated plasmid DNA should facilitate genetic manipulation of this industrially relevant bacterium.« less

Nuclear Mitochondrial DNA Activates Replication in Saccharomyces cerevisiae

PubMed Central

Chatre, Laurent; Ricchetti, Miria

2011-01-01

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in. PMID:21408151

Nuclear mitochondrial DNA activates replication in Saccharomyces cerevisiae.

PubMed

Chatre, Laurent; Ricchetti, Miria

2011-03-08

The nuclear genome of eukaryotes is colonized by DNA fragments of mitochondrial origin, called NUMTs. These insertions have been associated with a variety of germ-line diseases in humans. The significance of this uptake of potentially dangerous sequences into the nuclear genome is unclear. Here we provide functional evidence that sequences of mitochondrial origin promote nuclear DNA replication in Saccharomyces cerevisiae. We show that NUMTs are rich in key autonomously replicating sequence (ARS) consensus motifs, whose mutation results in the reduction or loss of DNA replication activity. Furthermore, 2D-gel analysis of the mrc1 mutant exposed to hydroxyurea shows that several NUMTs function as late chromosomal origins. We also show that NUMTs located close to or within ARS provide key sequence elements for replication. Thus NUMTs can act as independent origins, when inserted in an appropriate genomic context or affect the efficiency of pre-existing origins. These findings show that migratory mitochondrial DNAs can impact on the replication of the nuclear region they are inserted in.

Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome

PubMed Central

Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H.; Liskovykh, Mikhail; Earnshaw, William C.; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I.; Larionov, Vladimir

2014-01-01

In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. PMID:25228468

Tolerance of Sir1p/Origin Recognition Complex-Dependent Silencing for Enhanced Origin Firing at HMRa

PubMed Central

McConnell, Kristopher H.; Müller, Philipp; Fox, Catherine A.

2006-01-01

The HMR-E silencer is a DNA element that directs the formation of silent chromatin at the HMRa locus in Saccharomyces cerevisiae. Sir1p is one of four Sir proteins required for silent chromatin formation at HMRa. Sir1p functions by binding the origin recognition complex (ORC), which binds to HMR-E, and recruiting the other Sir proteins (Sir2p to -4p). ORCs also bind to hundreds of nonsilencer positions distributed throughout the genome, marking them as replication origins, the sites for replication initiation. HMR-E also acts as a replication origin, but compared to many origins in the genome, it fires extremely inefficiently and late during S phase. One postulate to explain this observation is that ORC's role in origin firing is incompatible with its role in binding Sir1p and/or the formation of silent chromatin. Here we examined a mutant HMR-E silencer and fusions between robust replication origins and HMR-E for HMRa silencing, origin firing, and replication timing. Origin firing within HMRa and from the HMR-E silencer itself could be significantly enhanced, and the timing of HMRa replication during an otherwise normal S phase advanced, without a substantial reduction in SIR1-dependent silencing. However, although the robust origin/silencer fusions silenced HMRa quite well, they were measurably less effective than a comparable silencer containing HMR-E's native ORC binding site. PMID:16479013

Sliding Clamp–DNA Interactions Are Required for Viability and Contribute to DNA Polymerase Management in Escherichia coli

DOE Office of Scientific and Technical Information (OSTI.GOV)

Heltzel, J.; Scouten Ponticelli, S; Sanders, L

2009-01-01

Sliding clamp proteins topologically encircle DNA and play vital roles in coordinating the actions of various DNA replication, repair, and damage tolerance proteins. At least three distinct surfaces of the Escherichia coli {beta} clamp interact physically with the DNA that it topologically encircles. We utilized mutant {beta} clamp proteins bearing G66E and G174A substitutions ({beta}159), affecting the single-stranded DNA-binding region, or poly-Ala substitutions in place of residues 148-HQDVR-152 ({beta}148-152), affecting the double-stranded DNA binding region, to determine the biological relevance of clamp-DNA interactions. As part of this work, we solved the X-ray crystal structure of {beta}148-152, which verified that themore » poly-Ala substitutions failed to significantly alter the tertiary structure of the clamp. Based on functional assays, both {beta}159 and {beta}148-152 were impaired for loading and retention on a linear primed DNA in vitro. In the case of {beta}148-152, this defect was not due to altered interactions with the DnaX clamp loader, but rather was the result of impaired {beta}148-152-DNA interactions. Once loaded, {beta}148-152 was proficient for DNA polymerase III (Pol III) replication in vitro. In contrast, {beta}148-152 was severely impaired for Pol II and Pol IV replication and was similarly impaired for direct physical interactions with these Pols. Despite its ability to support Pol III replication in vitro, {beta}148-152 was unable to support viability of E. coli. Nevertheless, physiological levels of {beta}148-152 expressed from a plasmid efficiently complemented the temperature-sensitive growth phenotype of a strain expressing {beta}159 (dnaN159), provided that Pol II and Pol IV were inactivated. Although this strain was impaired for Pol V-dependent mutagenesis, inactivation of Pol II and Pol IV restored the Pol V mutator phenotype. Taken together, these results support a model in which a sophisticated combination of competitive clamp-DNA, clamp-partner, and partner-DNA interactions serve to manage the actions of the different E. coli Pols in vivo.« less

3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression

PubMed Central

Löb, D.; Lengert, N.; Chagin, V. O.; Reinhart, M.; Casas-Delucchi, C. S.; Cardoso, M. C.; Drossel, B.

2016-01-01

DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase. PMID:27052359

Spatial distribution and specification of mammalian replication origins during G1 phase

PubMed Central

Li, Feng; Chen, Jianhua; Solessio, Eduardo; Gilbert, David M.

2003-01-01

We have examined the distribution of early replicating origins on stretched DNA fibers when nuclei from CHO cells synchronized at different times during G1 phase initiate DNA replication in Xenopus egg extracts. Origins were differentially labeled in vivo versus in vitro to allow a comparison of their relative positions and spacing. With nuclei isolated in the first hour of G1 phase, in vitro origins were distributed throughout a larger number of DNA fibers and did not coincide with in vivo origins. With nuclei isolated 1 h later, a similar total number of in vitro origins were clustered within a smaller number of DNA fibers but still did not coincide with in vivo origins. However, with nuclei isolated later in G1 phase, the positions of many in vitro origins coincided with in vivo origin sites without further change in origin number or density. These results highlight two distinct G1 steps that establish a spatial and temporal program for replication. PMID:12707307

Addressing the "Replication Crisis": Using Original Studies to Design Replication Studies with Appropriate Statistical Power.

PubMed

Anderson, Samantha F; Maxwell, Scott E

2017-01-01

Psychology is undergoing a replication crisis. The discussion surrounding this crisis has centered on mistrust of previous findings. Researchers planning replication studies often use the original study sample effect size as the basis for sample size planning. However, this strategy ignores uncertainty and publication bias in estimated effect sizes, resulting in overly optimistic calculations. A psychologist who intends to obtain power of .80 in the replication study, and performs calculations accordingly, may have an actual power lower than .80. We performed simulations to reveal the magnitude of the difference between actual and intended power based on common sample size planning strategies and assessed the performance of methods that aim to correct for effect size uncertainty and/or bias. Our results imply that even if original studies reflect actual phenomena and were conducted in the absence of questionable research practices, popular approaches to designing replication studies may result in a low success rate, especially if the original study is underpowered. Methods correcting for bias and/or uncertainty generally had higher actual power, but were not a panacea for an underpowered original study. Thus, it becomes imperative that 1) original studies are adequately powered and 2) replication studies are designed with methods that are more likely to yield the intended level of power.

Spatial organization of transcription machinery and its segregation from the replisome in fast-growing bacterial cells

PubMed Central

Cagliero, Cedric; Zhou, Yan Ning; Jin, Ding Jun

2014-01-01

In a fast-growing Escherichia coli cell, most RNA polymerase (RNAP) is allocated to rRNA synthesis forming transcription foci at clusters of rrn operons or bacterial nucleolus, and each of the several nascent nucleoids contains multiple pairs of replication forks. The composition of transcription foci has not been determined. In addition, how the transcription machinery is three-dimensionally organized to promote cell growth in concord with replication machinery in the nucleoid remains essentially unknown. Here, we determine the spatial and functional landscapes of transcription and replication machineries in fast-growing E. coli cells using super-resolution-structured illumination microscopy. Co-images of RNAP and DNA reveal spatial compartmentation and duplication of the transcription foci at the surface of the bacterial chromosome, encompassing multiple nascent nucleoids. Transcription foci cluster with NusA and NusB, which are the rrn anti-termination system and are associated with nascent rRNAs. However, transcription foci tend to separate from SeqA and SSB foci, which track DNA replication forks and/or the replisomes, demonstrating that transcription machinery and replisome are mostly located in different chromosomal territories to maintain harmony between the two major cellular functions in fast-growing cells. Our study suggests that bacterial chromosomes are spatially and functionally organized, analogous to eukaryotes. PMID:25416798

Role of Escherichia coli dnaA gene and its integrative suppression in M13 Coliphage DNA synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mitra, S.; Stallions, D.R.

An F/sup +/ derivative of Escherichia coli E508 thermosensitive in dnaA function (involved in DNA synthesis initiation), its revertant and an Hfr derivative of E508(ts) in which the temperature-sensitive phenotype is suppressed by integrative suppression have been compared for their ability to support M13 phage DNA synthesis at the nonpermissive temperature. Upon infection at the nonpermissive temperature, both the revertant and the Hfr strain support normal phage replication while the temperature-sensitive mutant does not. However, when infection is carried out at a permissive temperature and the temperature is shifted up after infection, phage synthesis occurs in the temperature-sensitive mutant also,more » but in lesser quantity than in the revertant strain. Analysis of intracellular labeled phage DNA indicates: (a) parental replicative form DNA synthesis is not dependent on dnaA function; (b) progeny replicative form DNA synthesis is strongly inhibited in the temperature-sensitive dnaA mutant at the nonpermissive temperature; (c) progeny single-strand DNA synthesis does not absolutely require dnaA function; (d) progeny single-strand DNA is present in the circular form. The implication of the host DNA replication in M13 DNA synthesis is discussed.« less

Purification, crystallization and X-ray diffraction analysis of the C-terminal protease domain of Venezuelan equine encephalitis virus nsP2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russo, Andrew T.; Watowich, Stanley J., E-mail: watowich@xray.utmb.edu

2006-06-01

The C-terminal protease domain of Venezuelan equine encephalitis virus (VEEV) nsP2 has been overexpressed in E. coli, purified and successfully crystallized. Native crystals diffract to beyond 2.5 Å resolution and isomorphous heavy-atom derivatives suitable for phase analysis have been identified. The C-terminal region of Venezuelan equine encephalitis virus (VEEV) nsP2 is responsible for proteolytic processing of the VEEV polyprotein replication complex. This action regulates the activity of the replication complex and is essential for viral replication, thus making nsP2 a very attractive target for development of VEEV therapeutics. The 338-amino-acid C-terminal region of VEEV nsP2 has been overexpressed in Escherichiamore » coli, purified and crystallized. Crystals diffract to beyond 2.5 Å resolution and belong to the orthorhombic space group P2{sub 1}2{sub 1}2{sub 1}. Isomorphous heavy-atom derivatives suitable for phase analysis have been obtained and work on building a complete structural model is under way.« less

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms

PubMed Central

Abberton, Cathy L.; Bereschenko, Ludmila; van der Wielen, Paul W. J. J.

2016-01-01

ABSTRACT Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T90 (time taken for a reduction in cell number of 1 log10 unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm−2; BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli. The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. IMPORTANCE The provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely. PMID:27342552

Survival, Biofilm Formation, and Growth Potential of Environmental and Enteric Escherichia coli Strains in Drinking Water Microcosms.

PubMed

Abberton, Cathy L; Bereschenko, Ludmila; van der Wielen, Paul W J J; Smith, Cindy J

2016-09-01

Escherichia coli is the most commonly used indicator for fecal contamination in drinking water distribution systems (WDS). The assumption is that E. coli bacteria are of enteric origin and cannot persist for long outside their host and therefore act as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water, specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric isolates and one soil isolate) in sterile and nonsterile drinking water microcosms at 8 and 17°C, with T90 (time taken for a reduction in cell number of 1 log10 unit) values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (reverse transcription-)quantitative PCR [(RT-)Q-PCR] assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dynes [dyn] cm(-2); BioFlux system; Fluxion); however, cell numbers did not increase when drinking water flowed over the biofilm (P > 0.05 by t test). Finally, E. coli regrowth within drinking water microcosms containing polyethylene PE-100 pipe wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely. The provision of clean, safe drinking water is fundamental to society. WDS deliver water to consumers via a vast network of pipes. E. coli is used as an indicator organism for recent contamination events based on the premise that it cannot survive for long outside its host. A key public health concern therefore arises around the fate of E. coli on entering a WDS; its survival, ability to form a biofilm, and potential for regrowth. In particular, if E. coli bacteria have the ability to incorporate and regrow within the pipe wall biofilm of a WDS, they could reinoculate the water at a later stage. This study sheds light on the fate of environmental and enteric strains of E. coli in drinking water showing extended survival, the potential for biofilm formation under shear stress, and importantly, that regrowth in the presence of an indigenous microbial community is unlikely. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

ATR prohibits replication catastrophe by preventing global exhaustion of RPA.

PubMed

Toledo, Luis Ignacio; Altmeyer, Matthias; Rask, Maj-Britt; Lukas, Claudia; Larsen, Dorthe Helena; Povlsen, Lou Klitgaard; Bekker-Jensen, Simon; Mailand, Niels; Bartek, Jiri; Lukas, Jiri

2013-11-21

ATR, activated by replication stress, protects replication forks locally and suppresses origin firing globally. Here, we show that these functions of ATR are mechanistically coupled. Although initially stable, stalled forks in ATR-deficient cells undergo nucleus-wide breakage after unscheduled origin firing generates an excess of single-stranded DNA that exhausts the nuclear pool of RPA. Partial reduction of RPA accelerated fork breakage, and forced elevation of RPA was sufficient to delay such "replication catastrophe" even in the absence of ATR activity. Conversely, unscheduled origin firing induced breakage of stalled forks even in cells with active ATR. Thus, ATR-mediated suppression of dormant origins shields active forks against irreversible breakage via preventing exhaustion of nuclear RPA. This study elucidates how replicating genomes avoid destabilizing DNA damage. Because cancer cells commonly feature intrinsically high replication stress, this study also provides a molecular rationale for their hypersensitivity to ATR inhibitors. Copyright © 2013 Elsevier Inc. All rights reserved.

Conversion of commensal Escherichia coli K-12 to an invasive form via expression of a mutant histone-like protein.

PubMed

Koli, Preeti; Sudan, Sudhanshu; Fitzgerald, David; Adhya, Sankar; Kar, Sudeshna

2011-01-01

The HUα(E38K, V42L) mutant of the bacterial histone-like protein HU causes a major change in the transcription profile of the commensal organism Escherichia coli K-12 (Kar S, Edgar R, Adhya S, Proc. Natl. Acad. Sci. U. S. A. 102:16397-16402, 2005). Among the upregulated genes are several related to pathogenic interactions with mammalian cells, as evidenced by the expression of curli fibers, Ivy, and hemolysin E. When E. coli K-12/ HUα(E38K, V42L) was added to Int-407 cells, there was host cell invasion, phagosomal disruption, and intracellular replication. The invasive trait was also retained in a murine ileal loop model and intestinal explant assays. In addition to invasion, the internalized bacteria caused a novel subversion of host cell apoptosis through modification and regulation of the BH3-only proteins Bim(EL) and Puma. Changes in the transcription profile were attributed to positive supercoiling of DNA leading to the altered availability of relevant promoters. Using the E. coli K-12/HUα(E38K, V42L) variant as a model, we propose that traditional commensal E. coli can adopt an invasive lifestyle through reprogramming its cellular transcription, without gross genetic changes. Escherichia coli K-12 is well established as a benign laboratory strain and a human intestinal commensal. Recent evidences, however, indicate that the typical noninvasive nature of resident E. coli can be reversed under specific circumstances even in the absence of any major genomic flux. We previously engineered an E. coli strain with a mutant histone-like protein, HU, which exhibited significant changes in nucleoid organization and global transcription. Here we showed that the changes induced by the mutant HU have critical functional consequences: from a strict extracellular existence, the mutant E. coli adopts an almost obligate intracellular lifestyle. The internalized E. coli exhibits many of the prototypical characteristics of traditional intracellular bacteria, like phagosomal escape, intracellular replication, and subversion of host cell apoptosis. We suggest that E. coli K-12 can switch between widely divergent lifestyles in relation to mammalian host cells by reprogramming its cellular transcription program and without gross changes in its genomic content.

Replication, falsification, and the crisis of confidence in social psychology

PubMed Central

Earp, Brian D.; Trafimow, David

2015-01-01

The (latest) crisis in confidence in social psychology has generated much heated discussion about the importance of replication, including how it should be carried out as well as interpreted by scholars in the field. For example, what does it mean if a replication attempt “fails”—does it mean that the original results, or the theory that predicted them, have been falsified? And how should “failed” replications affect our belief in the validity of the original research? In this paper, we consider the replication debate from a historical and philosophical perspective, and provide a conceptual analysis of both replication and falsification as they pertain to this important discussion. Along the way, we highlight the importance of auxiliary assumptions (for both testing theories and attempting replications), and introduce a Bayesian framework for assessing “failed” replications in terms of how they should affect our confidence in original findings. PMID:26042061

Activation of a yeast replication origin near a double-stranded DNA break.

PubMed

Raghuraman, M K; Brewer, B J; Fangman, W L

1994-03-01

Irradiation in the G1 phase of the cell cycle delays the onset of DNA synthesis and transiently inhibits the activation of replication origins in mammalian cells. It has been suggested that this inhibition is the result of the loss of torsional tension in the DNA after it has been damaged. Because irradiation causes DNA damage at an undefined number of nonspecific sites in the genome, it is not known how cells respond to limited DNA damage, and how replication origins in the immediate vicinity of a damage site would behave. Using the sequence-specific HO endonuclease, we have created a defined double-stranded DNA break in a centromeric plasmid in G1-arrested cells of the yeast Saccharomyces cerevisiae. We show that replication does initiate at the origin on the cut plasmid, and that the plasmid replicates early in the S phase after linearization in vivo. These observations suggest that relaxation of a supercoiled DNA domain in yeast need not inactivate replication origins within that domain. Furthermore, these observations rule out the possibility that the late replication context associated with chromosomal termini is a consequence of DNA ends.

Least-Squares Support Vector Machine Approach to Viral Replication Origin Prediction

PubMed Central

Cruz-Cano, Raul; Chew, David S.H.; Kwok-Pui, Choi; Ming-Ying, Leung

2010-01-01

Replication of their DNA genomes is a central step in the reproduction of many viruses. Procedures to find replication origins, which are initiation sites of the DNA replication process, are therefore of great importance for controlling the growth and spread of such viruses. Existing computational methods for viral replication origin prediction have mostly been tested within the family of herpesviruses. This paper proposes a new approach by least-squares support vector machines (LS-SVMs) and tests its performance not only on the herpes family but also on a collection of caudoviruses coming from three viral families under the order of caudovirales. The LS-SVM approach provides sensitivities and positive predictive values superior or comparable to those given by the previous methods. When suitably combined with previous methods, the LS-SVM approach further improves the prediction accuracy for the herpesvirus replication origins. Furthermore, by recursive feature elimination, the LS-SVM has also helped find the most significant features of the data sets. The results suggest that the LS-SVMs will be a highly useful addition to the set of computational tools for viral replication origin prediction and illustrate the value of optimization-based computing techniques in biomedical applications. PMID:20729987

Least-Squares Support Vector Machine Approach to Viral Replication Origin Prediction.

PubMed

Cruz-Cano, Raul; Chew, David S H; Kwok-Pui, Choi; Ming-Ying, Leung

2010-06-01

Replication of their DNA genomes is a central step in the reproduction of many viruses. Procedures to find replication origins, which are initiation sites of the DNA replication process, are therefore of great importance for controlling the growth and spread of such viruses. Existing computational methods for viral replication origin prediction have mostly been tested within the family of herpesviruses. This paper proposes a new approach by least-squares support vector machines (LS-SVMs) and tests its performance not only on the herpes family but also on a collection of caudoviruses coming from three viral families under the order of caudovirales. The LS-SVM approach provides sensitivities and positive predictive values superior or comparable to those given by the previous methods. When suitably combined with previous methods, the LS-SVM approach further improves the prediction accuracy for the herpesvirus replication origins. Furthermore, by recursive feature elimination, the LS-SVM has also helped find the most significant features of the data sets. The results suggest that the LS-SVMs will be a highly useful addition to the set of computational tools for viral replication origin prediction and illustrate the value of optimization-based computing techniques in biomedical applications.

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

PubMed

Zannis-Hadjopoulos, M; Kaufmann, G; Martin, R G

1984-11-15

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Occurrence and sources of Escherichia coli in metropolitan St. Louis streams, October 2004 through September 2007

USGS Publications Warehouse

Wilkison, Donald H.; Davis, Jerri V.

2010-01-01

The occurrence and sources of Escherichia coli (E. coli), one of several fecal indicator bacteria, in metropolitan St. Louis streams known to receive nonpoint source runoff, occasional discharges from combined and sanitary sewers, and treated wastewater effluent were investigated from October 2004 through September 2007. Three Missouri River sites, five Mississippi River sites, and six small basin tributary stream sites were sampled during base flow and storm events for the presence of E. coli and their sources. E. coli host-source determinations were conducted using local library based genotypic methods. Human fecal contamination in stream samples was additionally confirmed by the presence of Bacteroides thetaiotaomicron, an anaerobic, enteric bacterium with a high occurrence in, and specificity to, humans. Missouri River E. coli densities and loads during base flow were approximately 10 times greater than those in the Mississippi River above its confluence with the Missouri River. Although substantial amounts of E. coli originated from within the study area during base flow and storm events, considerable amounts of E. coli in the Missouri River, as well as in the middle Mississippi River sections downstream from its confluence with the Missouri River, originated in Missouri River reaches upstream from the study area. In lower Mississippi River reaches, bacteria contributions from the numerous combined and sanitary sewer overflows within the study area, as well as contributions from nonpoint source runoff, greatly increased instream E. coli densities. Although other urban factors cannot be discounted, average E. coli densities in streams were strongly correlated with the number of upstream combined and sanitary sewer overflow points, and the percentage of upstream impervious cover. Small basin sites with the greatest number of combined and sanitary sewer overflows (Maline Creek and the River des Peres) had larger E. coli densities, larger loads, and a greater percentage of E. coli attributable to humans than other small basin sites; however, even though small basin E. coli densities typically were much larger than in large river receiving streams, small basins contributed, on average, only a small part (a maximum of 16 percent) of the total E. coli load to larger rivers. On average, approximately one-third of E. coli in metropolitan St. Louis streams was identified as originating from humans. Another one-third of the E. coli was determined to have originated from unidentified sources; dogs and geese contributed lesser amounts, 10 and 20 percent, of the total instream bacteria. Sources of E. coli were largely independent of hydrologic conditions-an indication that sources remained relatively consistent with time.

A Bayesian Perspective on the Reproducibility Project: Psychology

PubMed Central

Etz, Alexander; Vandekerckhove, Joachim

2016-01-01

We revisit the results of the recent Reproducibility Project: Psychology by the Open Science Collaboration. We compute Bayes factors—a quantity that can be used to express comparative evidence for an hypothesis but also for the null hypothesis—for a large subset (N = 72) of the original papers and their corresponding replication attempts. In our computation, we take into account the likely scenario that publication bias had distorted the originally published results. Overall, 75% of studies gave qualitatively similar results in terms of the amount of evidence provided. However, the evidence was often weak (i.e., Bayes factor < 10). The majority of the studies (64%) did not provide strong evidence for either the null or the alternative hypothesis in either the original or the replication, and no replication attempts provided strong evidence in favor of the null. In all cases where the original paper provided strong evidence but the replication did not (15%), the sample size in the replication was smaller than the original. Where the replication provided strong evidence but the original did not (10%), the replication sample size was larger. We conclude that the apparent failure of the Reproducibility Project to replicate many target effects can be adequately explained by overestimation of effect sizes (or overestimation of evidence against the null hypothesis) due to small sample sizes and publication bias in the psychological literature. We further conclude that traditional sample sizes are insufficient and that a more widespread adoption of Bayesian methods is desirable. PMID:26919473

A Bayesian Perspective on the Reproducibility Project: Psychology.

PubMed

Etz, Alexander; Vandekerckhove, Joachim

2016-01-01

We revisit the results of the recent Reproducibility Project: Psychology by the Open Science Collaboration. We compute Bayes factors-a quantity that can be used to express comparative evidence for an hypothesis but also for the null hypothesis-for a large subset (N = 72) of the original papers and their corresponding replication attempts. In our computation, we take into account the likely scenario that publication bias had distorted the originally published results. Overall, 75% of studies gave qualitatively similar results in terms of the amount of evidence provided. However, the evidence was often weak (i.e., Bayes factor < 10). The majority of the studies (64%) did not provide strong evidence for either the null or the alternative hypothesis in either the original or the replication, and no replication attempts provided strong evidence in favor of the null. In all cases where the original paper provided strong evidence but the replication did not (15%), the sample size in the replication was smaller than the original. Where the replication provided strong evidence but the original did not (10%), the replication sample size was larger. We conclude that the apparent failure of the Reproducibility Project to replicate many target effects can be adequately explained by overestimation of effect sizes (or overestimation of evidence against the null hypothesis) due to small sample sizes and publication bias in the psychological literature. We further conclude that traditional sample sizes are insufficient and that a more widespread adoption of Bayesian methods is desirable.

Adenovirus sequences required for replication in vivo.

PubMed Central

Wang, K; Pearson, G D

1985-01-01

We have studied the in vivo replication properties of plasmids carrying deletion mutations within cloned adenovirus terminal sequences. Deletion mapping located the adenovirus DNA replication origin entirely within the first 67 bp of the adenovirus inverted terminal repeat. This region could be further subdivided into two functional domains: a minimal replication origin and an adjacent auxillary region which boosted the efficiency of replication by more than 100-fold. The minimal origin occupies the first 18 to 21 bp and includes sequences conserved between all adenovirus serotypes. The adjacent auxillary region extends past nucleotide 36 but not past nucleotide 67 and contains the binding site for nuclear factor I. Images PMID:2991857

Unveiling the mystery of mitochondrial DNA replication in yeasts.

PubMed

Chen, Xin Jie; Clark-Walker, George Desmond

2018-01-01

Conventional DNA replication is initiated from specific origins and requires the synthesis of RNA primers for both the leading and lagging strands. In contrast, the replication of yeast mitochondrial DNA is origin-independent. The replication of the leading strand is likely primed by recombinational structures and proceeded by a rolling circle mechanism. The coexistent linear and circular DNA conformers facilitate the recombination-based initiation. The replication of the lagging strand is poorly understood. Re-evaluation of published data suggests that the rolling circle may also provide structures for the synthesis of the lagging-strand by mechanisms such as template switching. Thus, the coupling of recombination with rolling circle replication and possibly, template switching, may have been selected as an economic replication mode to accommodate the reductive evolution of mitochondria. Such a replication mode spares the need for conventional replicative components, including those required for origin recognition/remodelling, RNA primer synthesis and lagging-strand processing. Copyright © 2017 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

The actin-like MreB cytoskeleton organizes viral DNA replication in bacteria.

PubMed

Muñoz-Espín, Daniel; Daniel, Richard; Kawai, Yoshikazu; Carballido-López, Rut; Castilla-Llorente, Virginia; Errington, Jeff; Meijer, Wilfried J J; Salas, Margarita

2009-08-11

Little is known about the organization or proteins involved in membrane-associated replication of prokaryotic genomes. Here we show that the actin-like MreB cytoskeleton of the distantly related bacteria Escherichia coli and Bacillus subtilis is required for efficient viral DNA replication. Detailed analyses of B. subtilis phage ϕ29 showed that the MreB cytoskeleton plays a crucial role in organizing phage DNA replication at the membrane. Thus, phage double-stranded DNA and components of the ϕ29 replication machinery localize in peripheral helix-like structures in a cytoskeleton-dependent way. Importantly, we show that MreB interacts directly with the ϕ29 membrane-protein p16.7, responsible for attaching viral DNA at the cell membrane. Altogether, the results reveal another function for the MreB cytoskeleton and describe a mechanism by which viral DNA replication is organized at the bacterial membrane.

Epigenetically-inherited centromere and neocentromere DNA replicates earliest in S-phase.

PubMed

Koren, Amnon; Tsai, Hung-Ji; Tirosh, Itay; Burrack, Laura S; Barkai, Naama; Berman, Judith

2010-08-19

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position.

Mechanism of Origin DNA Recognition and Assembly of an Initiator-Helicase Complex by SV40 Large Tumor Antigen

PubMed Central

Chang, Y. Paul; Xu, Meng; Machado, Ana Carolina Dantas; Yu, Xian Jessica; Rohs, Remo; Chen, Xiaojiang S.

2013-01-01

SUMMARY The DNA tumor virus Simian virus 40 (SV40) is a model system for studying eukaryotic replication. SV40 large tumor antigen (LTag) is the initiator/helicase that is essential for genome replication. LTag recognizes and assembles at the viral replication origin. We determined the structure of two multidomain LTag subunits bound to origin DNA. The structure reveals that the origin binding domains (OBDs) and Zn and AAA+ domains are involved in origin recognition and assembly. Notably, the OBDs recognize the origin in an unexpected manner. The histidine residues of the AAA+ domains insert into a narrow minor groove region with enhanced negative electrostatic potential. Computational analysis indicates that this region is intrinsically narrow, demonstrating the role of DNA shape readout in origin recognition. Our results provide important insights into the assembly of the LTag initiator/ helicase at the replication origin and suggest that histidine contacts with the minor groove serve as a mechanism of DNA shape readout. PMID:23545501

Histone Modification Associated with Initiation of DNA Replication | Center for Cancer Research

Cancer.gov

Before cells are able to divide, they must first duplicate their chromosomes accurately. DNA replication and packaging of DNA into chromosomes by histone proteins need to be coordinated by the cell to ensure proper transmission of genetic and epigenetic information to the next generation. Mammalian DNA replication begins at specific chromosomal sites, called replication origins, which are located throughout the genome. The replication origins are tightly regulated to start replication only once per cell division so that genomic stability is maintained and cancer development is prevented.

Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

PubMed Central

Verma, Subhash C.; Lu, Jie; Cai, Qiliang; Kosiyatrakul, Settapong; McDowell, Maria E.; Schildkraut, Carl L.; Robertson, Erle S.

2011-01-01

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences. PMID:22072974

Histone acetylation regulates the time of replication origin firing.

PubMed

Vogelauer, Maria; Rubbi, Liudmilla; Lucas, Isabelle; Brewer, Bonita J; Grunstein, Michael

2002-11-01

The temporal firing of replication origins throughout S phase in yeast depends on unknown determinants within the adjacent chromosomal environment. We demonstrate here that the state of histone acetylation of surrounding chromatin is an important regulator of temporal firing. Deletion of RPD3 histone deacetylase causes earlier origin firing and concurrent binding of the replication factor Cdc45p to origins. In addition, increased acetylation of histones in the vicinity of the late origin ARS1412 by recruitment of the histone acetyltransferase Gcn5p causes ARS1412 alone to fire earlier. These data indicate that histone acetylation is a direct determinant of the timing of origin firing.

Causation and the origin of life. Metabolism or replication first?

PubMed

Pross, Addy

2004-06-01

The conceptual gulf that separates the 'metabolism first' and 'replication first' mechanisms for the emergence of life continues to cloud the origin of life debate. In the present paper we analyze this aspect of the origin of life problem and offer arguments in favor of the 'replication first' school. Utilizing Wicken's two-tier approach to causation we argue that a causal connection between replication and metabolism can only be demonstrated if replication would have preceded metabolism. In conjunction with existing empirical evidence and theoretical reasoning, our analysis concludes that there is no substantive evidence for a 'metabolism first' mechanism for life's emergence, while a coherent case can be made for the 'replication first' group of mechanisms. The analysis reaffirms our conviction that life is an extreme expression of kinetic control, and that the emergence of metabolic pathways can be understood by considering life as a manifestation of 'replicative chemistry'.

Transcriptionally Driven DNA Replication Program of the Human Parasite Leishmania major.

PubMed

Lombraña, Rodrigo; Álvarez, Alba; Fernández-Justel, José Miguel; Almeida, Ricardo; Poza-Carrión, César; Gomes, Fábia; Calzada, Arturo; Requena, José María; Gómez, María

2016-08-09

Faithful inheritance of eukaryotic genomes requires the orchestrated activation of multiple DNA replication origins (ORIs). Although origin firing is mechanistically conserved, how origins are specified and selected for activation varies across different model systems. Here, we provide a complete analysis of the nucleosomal landscape and replication program of the human parasite Leishmania major, building on a better evolutionary understanding of replication organization in Eukarya. We found that active transcription is a driving force for the nucleosomal organization of the L. major genome and that both the spatial and the temporal program of DNA replication can be explained as associated to RNA polymerase kinetics. This simple scenario likely provides flexibility and robustness to deal with the environmental changes that impose alterations in the genetic programs during parasitic life cycle stages. Our findings also suggest that coupling replication initiation to transcription elongation could be an ancient solution used by eukaryotic cells for origin maintenance. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

The histone acetyltransferases CBP and Chameau integrate developmental and DNA replication programs in Drosophila ovarian follicle cells

PubMed Central

McConnell, Kristopher H.; Dixon, Michael; Calvi, Brian R.

2012-01-01

DNA replication origin activity changes during development. Chromatin modifications are known to influence the genomic location of origins and the time during S phase that they initiate replication in different cells. However, how chromatin regulates origins in concert with cell differentiation remains poorly understood. Here, we use developmental gene amplification in Drosophila ovarian follicle cells as a model to investigate how chromatin modifiers regulate origins in a developmental context. We find that the histone acetyltransferase (HAT) Chameau (Chm) binds to amplicon origins and is partially required for their function. Depletion of Chm had relatively mild effects on origins during gene amplification and genomic replication compared with previous knockdown of its ortholog HBO1 in human cells, which has severe effects on origin function. We show that another HAT, CBP (Nejire), also binds amplicon origins and is partially required for amplification. Knockdown of Chm and CBP together had a more severe effect on nucleosome acetylation and amplicon origin activity than knockdown of either HAT alone, suggesting that these HATs collaborate in origin regulation. In addition to their local function at the origin, we show that Chm and CBP also globally regulate the developmental transition of follicle cells into the amplification stages of oogenesis. Our results reveal a complexity of origin epigenetic regulation by multiple HATs during development and suggest that chromatin modifiers are a nexus that integrates differentiation and DNA replication programs. PMID:22951641

Replication landscape of the human genome

PubMed Central

Petryk, Nataliya; Kahli, Malik; d'Aubenton-Carafa, Yves; Jaszczyszyn, Yan; Shen, Yimin; Silvain, Maud; Thermes, Claude; Chen, Chun-Long; Hyrien, Olivier

2016-01-01

Despite intense investigation, human replication origins and termini remain elusive. Existing data have shown strong discrepancies. Here we sequenced highly purified Okazaki fragments from two cell types and, for the first time, quantitated replication fork directionality and delineated initiation and termination zones genome-wide. Replication initiates stochastically, primarily within non-transcribed, broad (up to 150 kb) zones that often abut transcribed genes, and terminates dispersively between them. Replication fork progression is significantly co-oriented with the transcription. Initiation and termination zones are frequently contiguous, sometimes separated by regions of unidirectional replication. Initiation zones are enriched in open chromatin and enhancer marks, even when not flanked by genes, and often border ‘topologically associating domains' (TADs). Initiation zones are enriched in origin recognition complex (ORC)-binding sites and better align to origins previously mapped using bubble-trap than λ-exonuclease. This novel panorama of replication reveals how chromatin and transcription modulate the initiation process to create cell-type-specific replication programs. PMID:26751768

Interactions and Localization of Escherichia coli Error-Prone DNA Polymerase IV after DNA Damage.

PubMed

Mallik, Sarita; Popodi, Ellen M; Hanson, Andrew J; Foster, Patricia L

2015-09-01

Escherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication. DNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication.

PubMed

Wang, Weiping; Manna, David; Simmons, Daniel T

2007-05-01

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.

Best practices for mapping replication origins in eukaryotic chromosomes.

PubMed

Besnard, Emilie; Desprat, Romain; Ryan, Michael; Kahli, Malik; Aladjem, Mirit I; Lemaitre, Jean-Marc

2014-09-02

Understanding the regulatory principles ensuring complete DNA replication in each cell division is critical for deciphering the mechanisms that maintain genomic stability. Recent advances in genome sequencing technology facilitated complete mapping of DNA replication sites and helped move the field from observing replication patterns at a handful of single loci to analyzing replication patterns genome-wide. These advances address issues, such as the relationship between replication initiation events, transcription, and chromatin modifications, and identify potential replication origin consensus sequences. This unit summarizes the technological and fundamental aspects of replication profiling and briefly discusses novel insights emerging from mining large datasets, published in the last 3 years, and also describes DNA replication dynamics on a whole-genome scale. Copyright © 2014 John Wiley & Sons, Inc.

Escherichia coli K-12 pathogenicity in the pea aphid, Acyrthosiphon pisum, reveals reduced antibacterial defense in aphids.

PubMed

Altincicek, Boran; Ter Braak, Bas; Laughton, Alice M; Udekwu, Klas I; Gerardo, Nicole M

2011-10-01

To better understand the molecular basis underlying aphid immune tolerance to beneficial bacteria and immune defense to pathogenic bacteria, we characterized how the pea aphid Acyrthosiphon pisum responds to Escherichia coli K-12 infections. E. coli bacteria, usually cleared in the hemolymph of other insect species, were capable of growing exponentially and killing aphids within a few days. Red fluorescence protein expressing E. coli K-12 laboratory strain multiplied in the aphid hemolymph as well as in the digestive tract, resulting in death of infected aphids. Selected gene deletion mutants of the E. coli K-12 predicted to have reduced virulence during systemic infections showed no difference in either replication or killing rate when compared to the wild type E. coli strain. Of note, however, the XL1-Blue E. coli K-12 strain exhibited a significant lag phase before multiplying and killing aphids. This bacterial strain has recently been shown to be more sensitive to oxidative stress than other E. coli K-12 strains, revealing a potential role for reactive oxygen species-mediated defenses in the otherwise reduced aphid immune system. Copyright © 2011 Elsevier Ltd. All rights reserved.

DNA sequence templates adjacent nucleosome and ORC sites at gene amplification origins in Drosophila

PubMed Central

Liu, Jun; Zimmer, Kurt; Rusch, Douglas B.; Paranjape, Neha; Podicheti, Ram; Tang, Haixu; Calvi, Brian R.

2015-01-01

Eukaryotic origins of DNA replication are bound by the origin recognition complex (ORC), which scaffolds assembly of a pre-replicative complex (pre-RC) that is then activated to initiate replication. Both pre-RC assembly and activation are strongly influenced by developmental changes to the epigenome, but molecular mechanisms remain incompletely defined. We have been examining the activation of origins responsible for developmental gene amplification in Drosophila. At a specific time in oogenesis, somatic follicle cells transition from genomic replication to a locus-specific replication from six amplicon origins. Previous evidence indicated that these amplicon origins are activated by nucleosome acetylation, but how this affects origin chromatin is unknown. Here, we examine nucleosome position in follicle cells using micrococcal nuclease digestion with Ilumina sequencing. The results indicate that ORC binding sites and other essential origin sequences are nucleosome-depleted regions (NDRs). Nucleosome position at the amplicons was highly similar among developmental stages during which ORC is or is not bound, indicating that being an NDR is not sufficient to specify ORC binding. Importantly, the data suggest that nucleosomes and ORC have opposite preferences for DNA sequence and structure. We propose that nucleosome hyperacetylation promotes pre-RC assembly onto adjacent DNA sequences that are disfavored by nucleosomes but favored by ORC. PMID:26227968

Evaluation of the methods for enumerating coliform bacteria from water samples using precise reference standards.

PubMed

Wohlsen, T; Bates, J; Vesey, G; Robinson, W A; Katouli, M

2006-04-01

To use BioBall cultures as a precise reference standard to evaluate methods for enumeration of Escherichia coli and other coliform bacteria in water samples. Eight methods were evaluated including membrane filtration, standard plate count (pour and spread plate methods), defined substrate technology methods (Colilert and Colisure), the most probable number method and the Petrifilm disposable plate method. Escherichia coli and Enterobacter aerogenes BioBall cultures containing 30 organisms each were used. All tests were performed using 10 replicates. The mean recovery of both bacteria varied with the different methods employed. The best and most consistent results were obtained with Petrifilm and the pour plate method. Other methods either yielded a low recovery or showed significantly high variability between replicates. The BioBall is a very suitable quality control tool for evaluating the efficiency of methods for bacterial enumeration in water samples.

Assessment of berberine as a multi-target antimicrobial: a multi-omics study for drug discovery and repositioning.

PubMed

Karaosmanoglu, Kubra; Sayar, Nihat Alpagu; Kurnaz, Isil Aksan; Akbulut, Berna Sariyar

2014-01-01

Postgenomics drug development is undergoing major transformation in the age of multi-omics studies and drug repositioning. Rather than applications solely in personalized medicine, omics science thus additionally offers a better understanding of a broader range of drug targets and drug repositioning. Berberine is an isoquinoline alkaloid found in many medicinal plants. We report here a whole genome microarray study in tandem with proteomics techniques for mining the plethora of targets that are putatively involved in the antimicrobial activity of berberine against Escherichia coli. We found DNA replication/repair and transcription to be triggered by berberine, indicating that nucleic acids, in general, are among its targets. Our combined transcriptomics and proteomics multi-omics findings underscore that, in the presence of berberine, cell wall or cell membrane transport and motility-related functions are also specifically regulated. We further report a general decline in metabolism, as seen by repression of genes in carbohydrate and amino acid metabolism, energy production, and conversion. An involvement of multidrug efflux pumps, as well as reduced membrane permeability for developing resistance against berberine in E. coli was noted. Collectively, these findings offer original and significant leads for omics-guided drug discovery and future repositioning approaches in the postgenomics era, using berberine as a multi-omics case study.

Diversity, Replication, Pathogenicity and Cell Biology of Crimean Congo Hemorrhagic Fever Virus

DTIC Science & Technology

2006-10-01

recombinant protein produced and purified in E . coli was able to bind to ssRNA in a dot blot filter binding assay. In order to identify domains in the N...Fig. 1. RNA binding domains of the N protein of CCHFV. The depicted GST-fusion proteins were expressed in E . coli and purified using a...detected and NSm protein produced after cleavage of the glycoprotein precursor in virus infected cells. The NSm is stable and transported to the Golgi

Electrostatic interactions during acidic phospholipid reactivation of DnaA protein, the Escherichia coli initiator of chromosomal replication.

PubMed

Kitchen, J L; Li, Z; Crooke, E

1999-05-11

The initiation of Escherichia coli chromosomal replication by DnaA protein is strongly influenced by the tight binding of the nucleotides ATP and ADP. Anionic phospholipids in a fluid bilayer promote the conversion of inactive ADP-DnaA protein to replicatively active ATP-DnaA protein in vitro, and thus likely play a key role in regulating DnaA activity. Previous studies have revealed that, during this reactivation, a specific region of DnaA protein inserts into the hydrophobic portion of the lipid bilayer in an acidic phospholipid-dependent manner. To elucidate the requirement for acidic phospholipids in the reactivation process, the contribution of electrostatic forces in the interaction of DnaA and lipid was examined. DnaA-lipid binding required anionic phospholipids, and DnaA-lipid binding as well as lipid-mediated release of DnaA-bound nucleotide were inhibited by increased ionic strength, suggesting the involvement of electrostatic interactions in these processes. As the vesicular content of acidic phospholipids was increased, both nucleotide release and DnaA-lipid binding increased in a linear, parallel manner. Given that DnaA-membrane binding, the insertion of DnaA into the membrane, and the consequent nucleotide release all require anionic phospholipids, the acidic headgroup may be necessary to recruit DnaA protein to the membrane for insertion and subsequent reactivation for replication.

A possible role for chromium(III) in genotoxicity.

PubMed

Snow, E T

1991-05-01

Chromium is found in the environment in two major forms: reduced CrIII and CrVI, or chromate. Chromate, the most biologically active species, is readily taken up by living cells and reduced intracellularly, via reactive intermediates, to stable CrIII species. CrIII, the most abundant form of chromium in the environment, does not readily cross cell membranes and is relatively inactive in vivo. However, intracellular CrIII can react slowly with both nucleic acids and proteins and can be genotoxic. We have investigated the genotoxicity of CrIII in vitro using a DNA replication assay and in vivo by CaCl2-mediated transfection of chromium-treated DNA into Escherichia coli. When DNA replication was measured on a CrIII-treated template using purified DNA polymerases (either bacterial or mammalian), both the rate of DNA replication and the amount of incorporation per polymerase binding event (processivity) were greatly increased relative to controls. When transfected into E. coli, CrIII-treated M13mp2 bacteriophage DNA showed a dose-dependent increase in mutation frequency. These results suggest that CrIII alters the interaction between the DNA template and the polymerase such that the binding strength of the DNA polymerase is increased and the fidelity of DNA replication is decreased. These interactions may contribute to the mutagenicity of chromium ions in vivo and suggest that CrIII can contribute to chromium-mediated carcinogenesis.

Mutant DnaAs of Escherichia coli that are refractory to negative control

PubMed Central

Chodavarapu, Sundari; Felczak, Magdalena M.; Simmons, Lyle A.; Murillo, Alec; Kaguni, Jon M.

2013-01-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli. PMID:23990329

Mutant DnaAs of Escherichia coli that are refractory to negative control.

PubMed

Chodavarapu, Sundari; Felczak, Magdalena M; Simmons, Lyle A; Murillo, Alec; Kaguni, Jon M

2013-12-01

DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli.

Ease of articulation: A replication.

PubMed

Shuster, Linda I; Cottrill, Claire

2015-01-01

Researchers, as well as the lay public and the popular press, have become increasingly concerned about the lack of reproducibility of research findings. Despite this concern, research has shown that replications of previously published work comprise a very small proportion of published studies. Moreover, there are fewer published direct replications of research studies by independent investigators, and this type of replication is much less likely to confirm the results of the original research than are replications by the original investigator or conceptual replications. A search of the communication disorders research literature reveals that direct replications by independent investigators are virtually non-existent. The purpose of this project was to describe the major issues related to research reproducibility and report the results of a direct replication of a study by Locke (1972) regarding ease of articulation. Two methods for rating ease of articulation were employed. We were able to reproduce the results of the original study for the first method, obtaining a moderate positive correlation between our rankings of phoneme difficulty and Locke's rankings. We obtained a very high positive correlation between our phoneme rankings and rankings obtained in the original study for the second method. Moreover, we found a higher correlation between difficulty rankings and order of speech sound acquisition for American English than was found in the original study. Direct replication is not necessary for all studies in communication disorders, but should be considered for high impact studies, treatment studies, and those that provide data to support models and theories. The reader will be able to: (1) describe the major concerns related to the replicability of research findings; (2) describe the status of research replications in communication disorders; (3) describe how ease of articulation may relate to the order of speech sound acquisition in children; (4) list some types/areas of research that might be candidates for replication in the field of communication disorders. Copyright © 2015 Elsevier Inc. All rights reserved.

Crystallization and preliminary crystallographic characterization of the origin-binding domain of the bacteriophage λ O replication initiator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Struble, E. B., E-mail: evi.struble@nist.gov; Department of Biochemistry and Molecular Biology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; Center for Advanced Research in Biotechnology/NIST, 9600 Gudelsky Drive, Rockville, MD 20850

2007-06-01

Crystallization and preliminary diffraction data of the N-terminal 19–139 fragment of the origin-binding domain of bacteriophage λ O replication initiator are reported. The bacteriophage λ O protein binds to the λ replication origin (oriλ) and serves as the primary replication initiator for the viral genome. The binding energy derived from the binding of O to oriλ is thought to help drive DNA opening to facilitate initiation of DNA replication. Detailed understanding of this process is severely limited by the lack of high-resolution structures of O protein or of any lambdoid phage-encoded paralogs either with or without DNA. The production ofmore » crystals of the origin-binding domain of λ O that diffract to 2.5 Å is reported. Anomalous dispersion methods will be used to solve this structure.« less

How MCM loading and spreading specify eukaryotic DNA replication initiation sites.

PubMed

Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes.

Mapping yeast origins of replication via single-stranded DNA detection.

PubMed

Feng, Wenyi; Raghuraman, M K; Brewer, Bonita J

2007-02-01

Studies in th Saccharomyces cerevisiae have provided a framework for understanding how eukaryotic cells replicate their chromosomal DNA to ensure faithful transmission of genetic information to their daughter cells. In particular, S. cerevisiae is the first eukaryote to have its origins of replication mapped on a genomic scale, by three independent groups using three different microarray-based approaches. Here we describe a new technique of origin mapping via detection of single-stranded DNA in yeast. This method not only identified the majority of previously discovered origins, but also detected new ones. We have also shown that this technique can identify origins in Schizosaccharomyces pombe, illustrating the utility of this method for origin mapping in other eukaryotes.

Histone H4K20 tri-methylation at late-firing origins ensures timely heterochromatin replication.

PubMed

Brustel, Julien; Kirstein, Nina; Izard, Fanny; Grimaud, Charlotte; Prorok, Paulina; Cayrou, Christelle; Schotta, Gunnar; Abdelsamie, Alhassan F; Déjardin, Jérôme; Méchali, Marcel; Baldacci, Giuseppe; Sardet, Claude; Cadoret, Jean-Charles; Schepers, Aloys; Julien, Eric

2017-09-15

Among other targets, the protein lysine methyltransferase PR-Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4-20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila , partially impairs S-phase progression and protects from DNA re-replication induced by stabilization of PR-Set7. Using Epstein-Barr virus-derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4-20h is not sufficient to define an efficient origin per se , but rather serves as an enhancer for MCM2-7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4-20h-mediated H4K20 tri-methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1-associated origins, which ensure proper replication timing of late-replicating heterochromatin domains. Altogether, these results reveal Suv4-20h-mediated H4K20 tri-methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes. © 2017 The Authors.

Comparison of specific binding sites for Escherichia coli RNA polymerase with naturally occurring hairpin regions in single-stranded DNA of coliphage M13. [Aspergillus oryzae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Niyogi, S.K.; Mitra, S.

Escherichia coli RNA polymerase binds specifically to the single-stranded circular DNA of coliphage M13 in the presence of a saturating concentration of the bacterial DNA binding protein presumably as an essential step in the synthesis of the RNA primer required for synthesizing the complementary DNA strand in parental replicative-form DNA. The RNA polymerase-protected DNA regions were isolated after extensive digestion with pancreatic DNase, S1 endonuclease of Aspergillus oryzae, and exonuclease I of E. coli. The physicochemical properties of the RNA polymerase-protected segments (called PI and PII) were compared with those of the naturally occurring hairpin regions.

Nucleosomes in the neighborhood

PubMed Central

Dorn, Elizabeth Suzanne

2011-01-01

The importance of local chromatin structure in regulating replication initiation has become increasingly apparent. Most recently, histone methylation and nucleosome positioning have been added to the list of modifications demonstrated to regulate origins. In particular, the methylation states of H3K4, H3K36 and H4K20 have been associated with establishing active, repressed or poised origins depending on the timing and extent of methylation. The stability and precise positioning of nucleosomes has also been demonstrated to affect replication efficiency. Although it is not yet clear how these modifications alter the behavior of specific replication factors, ample evidence establishes their role in maintaining coordinated replication. This review will summarize recent advances in understanding these aspects of chromatin structure in DNA replication origin control. PMID:21364325

Comparative genome analysis of a thermotolerant Escherichia coli obtained by Genome Replication Engineering Assisted Continuous Evolution (GREACE) and its parent strain provides new understanding of microbial heat tolerance.

PubMed

Luan, Guodong; Bao, Guanhui; Lin, Zhao; Li, Yang; Chen, Zugen; Li, Yin; Cai, Zhen

2015-12-25

Heat tolerance of microbes is of great importance for efficient biorefinery and bioconversion. However, engineering and understanding of microbial heat tolerance are difficult and insufficient because it is a complex physiological trait which probably correlates with all gene functions, genetic regulations, and cellular metabolisms and activities. In this work, a novel strain engineering approach named Genome Replication Engineering Assisted Continuous Evolution (GREACE) was employed to improve the heat tolerance of Escherichia coli. When the E. coli strain carrying a mutator was cultivated under gradually increasing temperature, genome-wide mutations were continuously generated during genome replication and the mutated strains with improved thermotolerance were autonomously selected. A thermotolerant strain HR50 capable of growing at 50°C on LB agar plate was obtained within two months, demonstrating the efficiency of GREACE in improving such a complex physiological trait. To understand the improved heat tolerance, genomes of HR50 and its wildtype strain DH5α were sequenced. Evenly distributed 361 mutations covering all mutation types were found in HR50. Closed material transportations, loose genome conformation, and possibly altered cell wall structure and transcription pattern were the main differences of HR50 compared with DH5α, which were speculated to be responsible for the improved heat tolerance. This work not only expanding our understanding of microbial heat tolerance, but also emphasizing that the in vivo continuous genome mutagenesis method, GREACE, is efficient in improving microbial complex physiological trait. Copyright © 2015 Elsevier B.V. All rights reserved.

Sequential folding of UmuC by the Hsp70 and Hsp60 chaperone complexes of Escherichia coli.

PubMed

Petit, M A; Bedale, W; Osipiuk, J; Lu, C; Rajagopalan, M; McInerney, P; Goodman, M F; Echols, H

1994-09-23

Replication-blocking lesions generate a signal in Escherichia coli that leads to the induction of the multigene SOS response. Among the SOS-induced genes are umuD and umuC, whose products are necessary for the increased mutation rate in induced bacteria. The mutations are likely to result from replication across the DNA lesion, and such a bypass event has been reconstituted in vitro (Rajagopalan, M., L, C., Woodgate, R., O'Donnel, M., Goodman, M. F., Echols, H. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 10777-10781). In this work, we show that the chaperone proteins promote the proper folding of UmuC protein in vitro. We treated purified and inactive UmuC with Hsp70 and Hsp60. After Hsp70 treatment, the DNA binding activity of UmuC was recovered, but the ability to promote replication across DNA lesions was not. However, lesion bypass activity was recovered upon further treatment with Hsp60. The biological significance of such a folding pathway for UmuC protein is strengthened by in vivo evidence for a role of DnaK in UV-induced mutagenesis.

The Bombyx mori nucleopolyhedrovirus Bm111 affects virulence but not virus replication.

PubMed

Han, Yingying; Xia, Hengchuan; Tang, Qi; Lü, Peng; Ma, Shangshang; Yang, Yanhua; Shao, Dandan; Ma, Quanbing; Chen, Keping

2014-07-01

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication.

Ultrastructure of Deoxyribonucleic Acid-Membrane Associations in Escherichia coli

PubMed Central

Altenburg, B. C.; Suit, Joan C.; Brinkley, B. R.

1970-01-01

Areas of contact between deoxyribonucleic acid (DNA) and intracytoplasmic membrane are frequently seen in the “extra” membrane-forming strain Escherichia coli 0111a1. By examination of serial sections, it has been estimated that these DNA-membrane associations occur in at least 60% of the extra membrane-containing cells. Most of the DNA masses contained only one contact area. Several cells in which the DNA had been stretched revealed individual fibers connecting to the membrane, suggesting a firm attachment of DNA to membrane. The areas of membrane associated with DNA fibers were usually between 100 and 500 nm in diameter, although some smaller areas were seen. Electron microscopic autoradiography of cells in which the replication forks were labeled showed grains over 24% of the profiles containing a contact area, whereas there were grains over only 16% of the profiles without a contact area. Data from autoradiographs of cells in which the label was “chased” away from the replication fork showed the reverse labeling pattern. These data indicate that the areas of contact between DNA and intracytoplasmic membranes seen in electron micrographs contain the DNA replication forks. Images PMID:4919755

Stable transformation of a mosquito cell line results in extraordinarily high copy numbers of the plasmid.

PubMed Central

Monroe, T J; Muhlmann-Diaz, M C; Kovach, M J; Carlson, J O; Bedford, J S; Beaty, B J

1992-01-01

Stable incorporation of high copy numbers (greater than 10,000 per cell) of a plasmid vector containing a gene conferring resistance to the antibiotic hygromycin was achieved in a cell line derived from the Aedes albopictus mosquito. Plasmid sequences were readily observed by ethidium bromide staining of cellular DNA after restriction endonuclease digestion and agarose gel electrophoresis. The plasmid was demonstrated by in situ hybridization to be present in large arrays integrated in metaphase chromosomes and in minute and double-minute replicating elements. In one subclone, approximately 60,000 copies of the plasmid were organized in a large array that resembles a chromosome, morphologically and in the segregation of its chromatids during anaphase. The original as well as modified versions of the plasmid were rescued by transformation of Escherichia coli using total cellular DNA. Southern blot analyses of recovered plasmids indicate the presence of mosquito-derived sequences. Images PMID:1631052

Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

PubMed Central

MacAlpine, Heather K.; Lubelsky, Yoav; Hartemink, Alexander J.

2015-01-01

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2–7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we “footprinted” nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2–7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS). PMID:25593310

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

PubMed Central

Gai, Dahai; Wang, Damian; Li, Shu-Xing; Chen, Xiaojiang S

2016-01-01

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001 PMID:27921994

Analysis of model replication origins in Drosophila reveals new aspects of the chromatin landscape and its relationship to origin activity and the prereplicative complex

PubMed Central

Liu, Jun; McConnell, Kristopher; Dixon, Michael; Calvi, Brian R.

2012-01-01

Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development. PMID:22049023

CLB5-dependent activation of late replication origins in S. cerevisiae.

PubMed

Donaldson, A D; Raghuraman, M K; Friedman, K L; Cross, F R; Brewer, B J; Fangman, W L

1998-08-01

Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.

Lack of evolvability in self-sustaining autocatalytic networks constraints metabolism-first scenarios for the origin of life.

PubMed

Vasas, Vera; Szathmáry, Eörs; Santos, Mauro

2010-01-26

A basic property of life is its capacity to experience Darwinian evolution. The replicator concept is at the core of genetics-first theories of the origin of life, which suggest that self-replicating oligonucleotides or their similar ancestors may have been the first "living" systems and may have led to the evolution of an RNA world. But problems with the nonenzymatic synthesis of biopolymers and the origin of template replication have spurred the alternative metabolism-first scenario, where self-reproducing and evolving proto-metabolic networks are assumed to have predated self-replicating genes. Recent theoretical work shows that "compositional genomes" (i.e., the counts of different molecular species in an assembly) are able to propagate compositional information and can provide a setup on which natural selection acts. Accordingly, if we stick to the notion of replicator as an entity that passes on its structure largely intact in successive replications, those macromolecular aggregates could be dubbed "ensemble replicators" (composomes) and quite different from the more familiar genes and memes. In sharp contrast with template-dependent replication dynamics, we demonstrate here that replication of compositional information is so inaccurate that fitter compositional genomes cannot be maintained by selection and, therefore, the system lacks evolvability (i.e., it cannot substantially depart from the asymptotic steady-state solution already built-in in the dynamical equations). We conclude that this fundamental limitation of ensemble replicators cautions against metabolism-first theories of the origin of life, although ancient metabolic systems could have provided a stable habitat within which polymer replicators later evolved.

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage

PubMed Central

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F. Peter; Zhang, Huidong

2017-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, E. coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. PMID:27234563

Mechanisms of mutagenesis: DNA replication in the presence of DNA damage.

PubMed

Liu, Binyan; Xue, Qizhen; Tang, Yong; Cao, Jia; Guengerich, F Peter; Zhang, Huidong

2016-01-01

Environmental mutagens cause DNA damage that disturbs replication and produces mutations, leading to cancer and other diseases. We discuss mechanisms of mutagenesis resulting from DNA damage, from the level of DNA replication by a single polymerase to the complex DNA replisome of some typical model organisms (including bacteriophage T7, T4, Sulfolobus solfataricus, Escherichia coli, yeast and human). For a single DNA polymerase, DNA damage can affect replication in three major ways: reducing replication fidelity, causing frameshift mutations, and blocking replication. For the DNA replisome, protein interactions and the functions of accessory proteins can yield rather different results even with a single DNA polymerase. The mechanism of mutation during replication performed by the DNA replisome is a long-standing question. Using new methods and techniques, the replisomes of certain organisms and human cell extracts can now be investigated with regard to the bypass of DNA damage. In this review, we consider the molecular mechanism of mutagenesis resulting from DNA damage in replication at the levels of single DNA polymerases and complex DNA replisomes, including translesion DNA synthesis. Copyright © 2016 Elsevier B.V. All rights reserved.

Antibiotic resistance phenotypes and virulence-associated genes in Escherichia coli isolated from animals and animal food products in Tunisia.

PubMed

Badi, Souhir; Cremonesi, Paola; Abbassi, Mohamed Salah; Ibrahim, Chourouk; Snoussi, Majdi; Bignoli, Giulia; Luini, Mario; Castiglioni, Bianca; Hassen, Abdennaceur

2018-05-01

Livestock and food products of animal origin constitute important reservoirs of intestinal and extraintestinal pathogenic Escherichia coli including antibiotic-resistant E. coli isolates. To assess potential risks to public health related to E. coli strains of animal origin in Tunisia, 65 E. coli isolates recovered from healthy animals and food products of animal origin were studied. Antimicrobial susceptibility was determined according to CLSI guidelines and genes encoding antibiotic resistance as well as virulence factors were investigated by PCR. High rates of antibiotic resistance were observed to kanamycin (78.4%), gentamicin (75.3%) and streptomycin (75.3%, encoded by strA-strB (7 isolates)), amoxicillin (64.6%), amoxicillin/clavulanic acid (60%), tetracycline (44.6%; tetA (8 isolates) and tetB (7 isolates)), nalidixic acid (27.6%, qnrS (3 isolates), qnrB (2 isolates) and qnrA (one isolate)) and sulfonamides (36.9%; sul1 (1 isolate), sul2 (4 isolates), and sul3 (1 isolate)). Virulotypes classified some isolates as STEC (3%), MNEC (1.5%) and atypical EPEC (1.5%). This study demonstrated high rates of antimicrobial resistance and the presence of some pathogenic pathovars from animal origins that are a cause of concern for public health.

Replication of the Chicken β-Globin Locus: Early-Firing Origins at the 5′ HS4 Insulator and the ρ- and βA-Globin Genes Show Opposite Epigenetic Modifications

PubMed Central

Prioleau, Marie-Noëlle; Gendron, Marie-Claude; Hyrien, Olivier

2003-01-01

Chromatin structure is believed to exert a strong effect on replication origin function. We have studied the replication of the chicken β-globin locus, whose chromatin structure has been extensively characterized. This locus is delimited by hypersensitive sites (HSs) that mark the position of insulator elements. A stretch of condensed chromatin and another HS separate the β-globin domain from an adjacent folate receptor (FR) gene. We demonstrate here that in erythroid cells that express the FR but not the globin genes, replication initiates at four sites within the β-globin domain, one at the 5′ HS4 insulator and the other three near the ρ- and βA-globin genes. Three origins consist of G+C-rich sequences enriched in CpG dinucleotides. The fourth origin is A+T rich. Together with previous work, these data reveal that the insulator origin has unmethylated CpGs, hyperacetylated histones H3 and H4, and lysine 4-methylated histone H3. In contrast, opposite modifications are observed at the other G+C-rich origins. We also show that the whole region, including the stretch of condensed chromatin, replicates early in S phase in these cells. Therefore, different early-firing origins within the same locus may have opposite patterns of epigenetic modifications. The role of insulator elements in DNA replication is discussed. PMID:12724412

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT

PubMed Central

Noguchi, Yasunori; Katayama, Tsutomu

2016-01-01

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions. PMID:26973617

The Escherichia coli Cryptic Prophage Protein YfdR Binds to DnaA and Initiation of Chromosomal Replication Is Inhibited by Overexpression of the Gene Cluster yfdQ-yfdR-yfdS-yfdT.

PubMed

Noguchi, Yasunori; Katayama, Tsutomu

2016-01-01

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation. One of the suppressors comprised the yfdQ-yfdR-yfdS-yfdT gene cluster carried on a cryptic prophage. Increased copy numbers of yfdQRT or yfdQRS inhibited not only hda-185-dependent overinitiation, but also replication overinitiation in a hyperactive dnaA mutant, and in a mutant lacking an oriC-binding initiation-inhibitor SeqA. In addition, increasing the copy number of the gene set inhibited the growth of cells bearing specific, initiation-impairing dnaA mutations. In wild-type cells, multicopy supply of yfdQRT or yfdQRS also inhibited replication initiation and increased hydroxyurea (HU)-resistance, as seen in cells lacking DiaA, a stimulator of DnaA assembly on oriC. Deletion of the yfdQ-yfdR-yfdS-yfdT genes did not affect either HU resistance or initiation regulation. Furthermore, we found that DnaA bound specifically to YfdR in soluble protein extracts oversupplied with YfdQRST. Purified YfdR also bound to DnaA, and DnaA Phe46, an amino acid residue crucial for DnaA interactions with DiaA and DnaB replicative helicase was important for this interaction. Consistently, YfdR moderately inhibited DiaA-DnaA and DnaB-DnaA interactions. In addition, protein extracts oversupplied with YfdQRST inhibited replication initiation in vitro. Given the roles of yfdQ and yfdS in cell tolerance to specific environmental stresses, the yfdQ-yfdR-yfdS-yfdT genes might downregulate the initiator DnaA-oriC complex under specific growth conditions.

Concerted activities of Mcm4, Sld3, and Dbf4 in control of origin activation and DNA replication fork progression

PubMed Central

Sheu, Yi-Jun; Kinney, Justin B.; Stillman, Bruce

2016-01-01

Eukaryotic chromosomes initiate DNA synthesis from multiple replication origins in a temporally specific manner during S phase. The replicative helicase Mcm2-7 functions in both initiation and fork progression and thus is an important target of regulation. Mcm4, a helicase subunit, possesses an unstructured regulatory domain that mediates control from multiple kinase signaling pathways, including the Dbf4-dependent Cdc7 kinase (DDK). Following replication stress in S phase, Dbf4 and Sld3, an initiation factor and essential target of Cyclin-Dependent Kinase (CDK), are targets of the checkpoint kinase Rad53 for inhibition of initiation from origins that have yet to be activated, so-called late origins. Here, whole-genome DNA replication profile analysis is used to access under various conditions the effect of mutations that alter the Mcm4 regulatory domain and the Rad53 targets, Sld3 and Dbf4. Late origin firing occurs under genotoxic stress when the controls on Mcm4, Sld3, and Dbf4 are simultaneously eliminated. The regulatory domain of Mcm4 plays an important role in the timing of late origin firing, both in an unperturbed S phase and in dNTP limitation. Furthermore, checkpoint control of Sld3 impacts fork progression under replication stress. This effect is parallel to the role of the Mcm4 regulatory domain in monitoring fork progression. Hypomorph mutations in sld3 are suppressed by a mcm4 regulatory domain mutation. Thus, in response to cellular conditions, the functions executed by Sld3, Dbf4, and the regulatory domain of Mcm4 intersect to control origin firing and replication fork progression, thereby ensuring genome stability. PMID:26733669

Initiation of DNA replication: functional and evolutionary aspects

PubMed Central

Bryant, John A.; Aves, Stephen J.

2011-01-01

Background The initiation of DNA replication is a very important and highly regulated step in the cell division cycle. It is of interest to compare different groups of eukaryotic organisms (a) to identify the essential molecular events that occur in all eukaryotes, (b) to start to identify higher-level regulatory mechanisms that are specific to particular groups and (c) to gain insights into the evolution of initiation mechanisms. Scope This review features a wide-ranging literature survey covering replication origins, origin recognition and usage, modification of origin usage (especially in response to plant hormones), assembly of the pre-replication complex, loading of the replisome, genomics, and the likely origin of these mechanisms and proteins in Archaea. Conclusions In all eukaryotes, chromatin is organized for DNA replication as multiple replicons. In each replicon, replication is initiated at an origin. With the exception of those in budding yeast, replication origins, including the only one to be isolated so far from a plant, do not appear to embody a specific sequence; rather, they are AT-rich, with short tracts of locally bent DNA. The proteins involved in initiation are remarkably similar across the range of eukaryotes. Nevertheless, their activity may be modified by plant-specific mechanisms, including regulation by plant hormones. The molecular features of initiation are seen in a much simpler form in the Archaea. In particular, where eukaryotes possess a number of closely related proteins that form ‘hetero-complexes’ (such as the origin recognition complex and the MCM complex), archaeans typically possess one type of protein (e.g. one MCM) that forms a homo-complex. This suggests that several eukaryotic initiation proteins have evolved from archaeal ancestors by gene duplication and divergence. PMID:21508040

A 4-study replication of the moderating effects of greed on socioeconomic status and unethical behaviour.

PubMed

Balakrishnan, Anjana; Palma, Paolo A; Patenaude, Joshua; Campbell, Lorne

2017-01-31

Four replications of Piff and colleagues' study examined the moderating effects of greed attitudes on the relationship between socio-economic status (SES) and unethical behaviour (Study 7). In the original study, the researchers found that both greed and SES predicted increased propensity to engage in unethical behavior. Furthermore, this association was moderated such that the effects of SES on unethical behaviour were no longer present in the greed prime condition versus the neutral condition. In replication 1 of the original study main effects of greed attitudes and SES were found, but no interaction was found. Main effects for greed emerged in replications 3 and 4. However no main effects for SES or interactions emerged for replications 2-4. A meta-analysis was conducted with all replications and the original study, and found no moderating effect of greed on the relationship between SES and unethical behavior.

A 4-study replication of the moderating effects of greed on socioeconomic status and unethical behaviour

PubMed Central

Balakrishnan, Anjana; Palma, Paolo A.; Patenaude, Joshua; Campbell, Lorne

2017-01-01

Four replications of Piff and colleagues’ study examined the moderating effects of greed attitudes on the relationship between socio-economic status (SES) and unethical behaviour (Study 7). In the original study, the researchers found that both greed and SES predicted increased propensity to engage in unethical behavior. Furthermore, this association was moderated such that the effects of SES on unethical behaviour were no longer present in the greed prime condition versus the neutral condition. In replication 1 of the original study main effects of greed attitudes and SES were found, but no interaction was found. Main effects for greed emerged in replications 3 and 4. However no main effects for SES or interactions emerged for replications 2–4. A meta-analysis was conducted with all replications and the original study, and found no moderating effect of greed on the relationship between SES and unethical behavior. PMID:28140396

Interplay between CedA, rpoB and double stranded DNA: A step towards understanding CedA mediated cell division in E. coli.

PubMed

Sharma, Pankaj; Tomar, Anil Kumar; Kundu, Bishwajit

2018-02-01

Cell division is compromised in DnaAcos mutant E. coli cells due to chromosome over-replication. In these cells, CedA acts as a regulatory protein and initiates cell division by a hitherto unknown mechanism. CedA, a double stranded DNA binding protein, interacts with various subunits of RNA polymerase complex, including rpoB. To reveal how this concert between CedA, rpoB and DNA brings about cell division in E. coli, we performed biophysical and in silico analysis and obtained mechanistic insights. Interaction between CedA and rpoB was shown by circular dichroism spectrometry and in silico docking experiments. Further, CedA and rpoB were allowed to interact individually to a selected DNA and their binding was monitored by fluorescence spectroscopy. The binding constants of these interactions as determined by BioLayer Interferometry clearly show that rpoB binds to DNA with higher affinity (K D2 =<1.0E-12M) as compared to CedA (K D2 =9.58E-09M). These findings were supported by docking analysis where 12 intermolecular H-bonds were formed in rpoB-DNA complex as compared to 4 in CedA-DNA complex. Based on our data we propose that in E. coli cells chromosome over-replication signals CedA to recruit rpoB to specific DNA site(s), which initiates transcription of cell division regulatory elements. Copyright © 2017 Elsevier B.V. All rights reserved.

Solution structure of the N-terminal domain of a replication restart primosome factor, PriC, in Escherichia coli

PubMed Central

Aramaki, Takahiko; Abe, Yoshito; Katayama, Tsutomu; Ueda, Tadashi

2013-01-01

In eubacterial organisms, the oriC-independent primosome plays an essential role in replication restart after the dissociation of the replication DNA-protein complex by DNA damage. PriC is a key protein component in the replication restart primosome. Our recent study suggested that PriC is divided into two domains: an N-terminal and a C-terminal domain. In the present study, we determined the solution structure of the N-terminal domain, whose structure and function have remained unknown until now. The revealed structure was composed of three helices and one extended loop. We also observed chemical shift changes in the heteronuclear NMR spectrum and oligomerization in the presence of ssDNA. These abilities may contribute to the PriC-ssDNA complex, which is important for the replication restart primosome. PMID:23868391

New insights into replication origin characteristics in metazoans

PubMed Central

Puy, Aurore; Rialle, Stéphanie; Kaplan, Noam; Segal, Eran

2012-01-01

We recently reported the identification and characterization of DNA replication origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA replication origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of replication origins. PMID:22373526

Duplication of the genome in normal and cancer cell cycles.

PubMed

Bandura, Jennifer L; Calvi, Brian R

2002-01-01

It is critical to discover the mechanisms of normal cell cycle regulation if we are to fully understand what goes awry in cancer cells. The normal eukaryotic cell tightly regulates the activity of origins of DNA replication so that the genome is duplicated exactly once per cell cycle. Over the last ten years much has been learned concerning the cell cycle regulation of origin activity. It is now clear that the proteins and cell cycle mechanisms that control origin activity are largely conserved from yeast to humans. Despite this conservation, the composition of origins of DNA replication in higher eukaryotes remains ill defined. A DNA consensus for predicting origins has yet to emerge, and it is of some debate whether primary DNA sequence determines where replication initiates. In this review we outline what is known about origin structure and the mechanism of once per cell cycle DNA replication with an emphasis on recent advances in mammalian cells. We discuss the possible relevance of these regulatory pathways for cancer biology and therapy.

Near-atomic structural model for bacterial DNA replication initiation complex and its functional insights.

PubMed

Shimizu, Masahiro; Noguchi, Yasunori; Sakiyama, Yukari; Kawakami, Hironori; Katayama, Tsutomu; Takada, Shoji

2016-12-13

Upon DNA replication initiation in Escherichia coli, the initiator protein DnaA forms higher-order complexes with the chromosomal origin oriC and a DNA-bending protein IHF. Although tertiary structures of DnaA and IHF have previously been elucidated, dynamic structures of oriC-DnaA-IHF complexes remain unknown. Here, combining computer simulations with biochemical assays, we obtained models at almost-atomic resolution for the central part of the oriC-DnaA-IHF complex. This complex can be divided into three subcomplexes; the left and right subcomplexes include pentameric DnaA bound in a head-to-tail manner and the middle subcomplex contains only a single DnaA. In the left and right subcomplexes, DnaA ATPases associated with various cellular activities (AAA+) domain III formed helices with specific structural differences in interdomain orientations, provoking a bend in the bound DNA. In the left subcomplex a continuous DnaA chain exists, including insertion of IHF into the DNA looping, consistent with the DNA unwinding function of the complex. The intervening spaces in those subcomplexes are crucial for DNA unwinding and loading of DnaB helicases. Taken together, this model provides a reasonable near-atomic level structural solution of the initiation complex, including the dynamic conformations and spatial arrangements of DnaA subcomplexes.

How MCM loading and spreading specify eukaryotic DNA replication initiation sites

PubMed Central

Hyrien, Olivier

2016-01-01

DNA replication origins strikingly differ between eukaryotic species and cell types. Origins are localized and can be highly efficient in budding yeast, are randomly located in early fly and frog embryos, which do not transcribe their genomes, and are clustered in broad (10-100 kb) non-transcribed zones, frequently abutting transcribed genes, in mammalian cells. Nonetheless, in all cases, origins are established during the G1-phase of the cell cycle by the loading of double hexamers of the Mcm 2-7 proteins (MCM DHs), the core of the replicative helicase. MCM DH activation in S-phase leads to origin unwinding, polymerase recruitment, and initiation of bidirectional DNA synthesis. Although MCM DHs are initially loaded at sites defined by the binding of the origin recognition complex (ORC), they ultimately bind chromatin in much greater numbers than ORC and only a fraction are activated in any one S-phase. Data suggest that the multiplicity and functional redundancy of MCM DHs provide robustness to the replication process and affect replication time and that MCM DHs can slide along the DNA and spread over large distances around the ORC. Recent studies further show that MCM DHs are displaced along the DNA by collision with transcription complexes but remain functional for initiation after displacement. Therefore, eukaryotic DNA replication relies on intrinsically mobile and flexible origins, a strategy fundamentally different from bacteria but conserved from yeast to human. These properties of MCM DHs likely contribute to the establishment of broad, intergenic replication initiation zones in higher eukaryotes. PMID:27635237

OriDB, the DNA replication origin database updated and extended.

PubMed

Siow, Cheuk C; Nieduszynska, Sian R; Müller, Carolin A; Nieduszynski, Conrad A

2012-01-01

OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted replication origin sites. The original database collated and curated Saccharomyces cerevisiae origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated origin mapping studies, and to include the collation of replication origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/.

DNA Replication Profiling Using Deep Sequencing.

PubMed

Saayman, Xanita; Ramos-Pérez, Cristina; Brown, Grant W

2018-01-01

Profiling of DNA replication during progression through S phase allows a quantitative snap-shot of replication origin usage and DNA replication fork progression. We present a method for using deep sequencing data to profile DNA replication in S. cerevisiae.

DNA replication and cancer: From dysfunctional replication origin activities to therapeutic opportunities.

PubMed

Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard

2016-06-01

A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Termination of DNA replication forks: "Breaking up is hard to do".

PubMed

Bailey, Rachael; Priego Moreno, Sara; Gambus, Agnieszka

2015-01-01

To ensure duplication of the entire genome, eukaryotic DNA replication initiates from thousands of replication origins. The replication forks move through the chromatin until they encounter forks from neighboring origins. During replication fork termination forks converge, the replisomes disassemble and topoisomerase II resolves the daughter DNA molecules. If not resolved efficiently, terminating forks result in genomic instability through the formation of pathogenic structures. Our recent findings shed light onto the mechanism of replisome disassembly upon replication fork termination. We have shown that termination-specific polyubiquitylation of the replicative helicase component - Mcm7, leads to dissolution of the active helicase in a process dependent on the p97/VCP/Cdc48 segregase. The inhibition of terminating helicase disassembly resulted in a replication termination defect. In this extended view we present hypothetical models of replication fork termination and discuss remaining and emerging questions in the DNA replication termination field.

Conserved Sequences at the Origin of Adenovirus DNA Replication

PubMed Central

Stillman, Bruce W.; Topp, William C.; Engler, Jeffrey A.

1982-01-01

The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis. Images PMID:7143575

Different cellular origins and functions of extracellular proteins from Escherichia coli O157:H7 and O104:H4 as determined by comparative proteomic analysis

USDA-ARS?s Scientific Manuscript database

Escherichia coli is a diverse species of bacteria, including several pathotypes that cause a variety of diseases in humans. Enterohemorrhagic E. coli (EHEC) and recently emerged shigatoxingenic enteroaggregative E. coli (EAEC) produce Shigatoxins and are major foodborne pathogens that can cause hem...

A Link between ORC-Origin Binding Mechanisms and Origin Activation Time Revealed in Budding Yeast

PubMed Central

Hoggard, Timothy; Shor, Erika; Müller, Carolin A.; Nieduszynski, Conrad A.; Fox, Catherine A.

2013-01-01

Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal positions and triggers molecular events culminating in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome uses multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern relevant to differentiation and genome stability. It is unclear whether ORC-origin interactions are relevant to origin activation time. We applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. These two origin classes differed in terms of nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Finally, the ‘chromatin-dependent’ class was enriched for origins that fire early in S-phase, while the DNA-dependent class was enriched for later firing origins. Conversely, the latest firing origins showed a positive association with the ORC-origin DNA paradigm for normal levels of ORC binding, whereas the earliest firing origins did not. These data reveal a novel association between ORC-origin binding mechanisms and the regulation of origin activation time. PMID:24068963

Synthesis of bacteriophage phiC DNA in dna mutants of Esherichia coli.

PubMed

Kodaira, K I; Taketo, A

1978-06-01

Host dna functions involved in the replication of microvirid phage phiC DNA were investigated in vivo. Although growth of this phage was markedly inhibited even at 35-37 degrees C even in dna+ host, conversion of the infecting single-stranded DNA into the double-stranded parental replicative form (stage I synthesis) occurred normally at 43 degrees C in dna+, dnaA, dnaB, dnaC(D), and dnaE cells. In dnaG mutant, the stage I synthesis was severely inhibited at 43 degrees C but not at 30 degrees C. The stage I replication of phiC DNA was clearly thermosensitive in dnaZ cells incubated in nutrient broth. In Tris-casamino acids-glucose medium, however, the dnaZ mutant sufficiently supported synthesis of the parental replicative form. At 43 degrees C, synthesis of the progeny replicative form DNA (stage II replication) was significantly inhibited even in dna+ cells and was nearly completely blocked in dnaB or dnaC(D) mutant. At 37 degrees C, the stage II replication proceeded normally in dna+ bacteria.

Clonal distribution and associated characteristics of Escherichia coli clinical and surveillance isolates from a military medical center.

PubMed

Manges, Amee R; Mende, Katrin; Murray, Clinton K; Johnston, Brian D; Sokurenko, Evgeni V; Tchesnokova, Veronika; Johnson, James R

2017-04-01

Antimicrobial-resistant Escherichia coli are a concern for military health services. We studied 100 extended-spectrum beta-lactamase (ESBL)-producing and non-producing E. coli clinical and surveillance isolates from military personnel and civilians at Brooke Army Medical Center (2007-2011). Major E. coli lineages, most prominently ST10 (24%), ST131 (16%), and ST648 (8%), were distributed much as reported for other North American populations. ST131, represented mainly by its resistance-associated ST131-H30 clonal subset, was uniquely associated with a clinical origin, regardless of ESBL status. Thus, clonal background predicted resistance phenotype and clinical versus surveillance origin, and these findings could assist military clinicians and epidemiologists. Published by Elsevier Inc.

Escherichia coli sampling reliability at a frequently closed Chicago beach: monitoring and management implications

USGS Publications Warehouse

Whitman, Richard L.; Nevers, Meredith B.

2004-01-01

Monitoring beaches for recreational water quality is becoming more common, but few sampling designs or policy approaches have evaluated the efficacy of monitoring programs. The authors intensively sampled water for E. coli (N=1770) at 63rd Street Beach, Chicago for 6 months in 2000 in order to (1) characterize spatial-temporal trends, (2) determine between and within transect variation, and (3) estimate sample size requirements and determine sampling reliability.E. coli counts were highly variable within and between sampling sites but spatially and diurnally autocorrelated. Variation in counts decreased with water depth and time of day. Required number of samples was high for 70% precision around the critical closure level (i.e., 6 within or 24 between transect replicates). Since spatial replication may be cost prohibitive, composite sampling is an alternative once sources of error have been well defined. The results suggest that beach monitoring programs may be requiring too few samples to fulfill management objectives desired. As the recreational water quality national database is developed, it is important that sampling strategies are empirically derived from a thorough understanding of the sources of variation and the reliability of collected data. Greater monitoring efficacy will yield better policy decisions, risk assessments, programmatic goals, and future usefulness of the information.

Quantifying Impact of Chromosome Copy Number on Recombination in Escherichia coli.

PubMed

Reynolds, T Steele; Gill, Ryan T

2015-07-17

The ability to precisely and efficiently recombineer synthetic DNA into organisms of interest in a quantitative manner is a key requirement in genome engineering. Even though considerable effort has gone into the characterization of recombination in Escherichia coli, there is still substantial variability in reported recombination efficiencies. We hypothesized that this observed variability could, in part, be explained by the variability in chromosome copy number as well as the location of the replication forks relative to the recombination site. During rapid growth, E. coli cells may contain several pairs of open replication forks. While recombineered forks are resolving and segregating within the population, changes in apparent recombineering efficiency should be observed. In the case of dominant phenotypes, we predicted and then experimentally confirmed that the apparent recombination efficiency declined during recovery until complete segregation of recombineered and wild-type genomes had occurred. We observed the reverse trend for recessive phenotypes. The observed changes in apparent recombination efficiency were found to be in agreement with mathematical calculations based on our proposed mechanism. We also provide a model that can be used to estimate the total segregated recombination efficiency based on an initial efficiency and growth rate. These results emphasize the importance of employing quantitative strategies in the design of genome-scale engineering efforts.

Links between genome replication and chromatin landscapes.

PubMed

Sequeira-Mendes, Joana; Gutierrez, Crisanto

2015-07-01

Post-embryonic organogenesis in plants requires the continuous production of cells in the organ primordia, their expansion and a coordinated exit to differentiation. Genome replication is one of the most important processes that occur during the cell cycle, as the maintenance of genomic integrity is of primary relevance for development. As it is chromatin that must be duplicated, a strict coordination occurs between DNA replication, the deposition of new histones, and the introduction of histone modifications and variants. In turn, the chromatin landscape affects several stages during genome replication. Thus, chromatin accessibility is crucial for the initial stages and to specify the location of DNA replication origins with different chromatin signatures. The chromatin landscape also determines the timing of activation during the S phase. Genome replication must occur fully, but only once during each cell cycle. The re-replication avoidance mechanisms rely primarily on restricting the availability of certain replication factors; however, the presence of specific histone modifications are also revealed as contributing to the mechanisms that avoid re-replication, in particular for heterochromatin replication. We provide here an update of genome replication mostly focused on data from Arabidopsis, and the advances that genomic approaches are likely to provide in the coming years. The data available, both in plants and animals, point to the relevance of the chromatin landscape in genome replication, and require a critical evaluation of the existing views about the nature of replication origins, the mechanisms of origin specification and the relevance of epigenetic modifications for genome replication. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Epstein-Barr virus origin of lytic replication mediates association of replicating episomes with promyelocytic leukaemia protein nuclear bodies and replication compartments.

PubMed

Amon, Wolfgang; White, Robert E; Farrell, Paul J

2006-05-01

Epstein-Barr virus (EBV) establishes a latent persistence from which it can be reactivated to undergo lytic replication. Late lytic-cycle gene expression is linked to lytic DNA replication, as it is sensitive to the same inhibitors that block lytic replication, and it has recently been shown that the viral origin of lytic replication (ori lyt) is required in cis for late-gene expression. During the lytic cycle, the viral genome forms replication compartments, which are usually adjacent to promyelocytic leukaemia protein (PML) nuclear bodies. A tetracycline repressor DNA-binding domain-enhanced green fluorescent protein fusion was used to visualize replicating plasmids carrying a tetracycline operator sequence array. ori lyt mediated the production of plasmid replication compartments that were associated with PML nuclear bodies. Plasmids carrying ori lyt and EBV itself were visualized in the same cells and replicated in similar regions of the nucleus, further supporting the validity of the plasmids for studying late-gene regulation.

Tissue- and age-specific DNA replication patterns at the CTG/CAG-expanded human myotonic dystrophy type 1 locus.

PubMed

Cleary, John D; Tomé, Stéphanie; López Castel, Arturo; Panigrahi, Gagan B; Foiry, Laurent; Hagerman, Katharine A; Sroka, Hana; Chitayat, David; Gourdon, Geneviève; Pearson, Christopher E

2010-09-01

Myotonic dystrophy, caused by DM1 CTG/CAG repeat expansions, shows varying instability levels between tissues and across ages within patients. We determined DNA replication profiles at the DM1 locus in patient fibroblasts and tissues from DM1 transgenic mice of various ages showing different instability. In patient cells, the repeat is flanked by two replication origins demarcated by CTCF sites, with replication diminished at the expansion. In mice, the expansion replicated from only the downstream origin (CAG as lagging template). In testes from mice of three different ages, replication toward the repeat paused at the earliest age and was relieved at later ages-coinciding with increased instability. Brain, pancreas and thymus replication varied with CpG methylation at DM1 CTCF sites. CTCF sites between progressing forks and repeats reduced replication depending on chromatin. Thus, varying replication progression may affect tissue- and age-specific repeat instability.

Ribosome biogenesis in replicating cells: Integration of experiment and theory.

PubMed

Earnest, Tyler M; Cole, John A; Peterson, Joseph R; Hallock, Michael J; Kuhlman, Thomas E; Luthey-Schulten, Zaida

2016-10-01

Ribosomes-the primary macromolecular machines responsible for translating the genetic code into proteins-are complexes of precisely folded RNA and proteins. The ways in which their production and assembly are managed by the living cell is of deep biological importance. Here we extend a recent spatially resolved whole-cell model of ribosome biogenesis in a fixed volume [Earnest et al., Biophys J 2015, 109, 1117-1135] to include the effects of growth, DNA replication, and cell division. All biological processes are described in terms of reaction-diffusion master equations and solved stochastically using the Lattice Microbes simulation software. In order to determine the replication parameters, we construct and analyze a series of Escherichia coli strains with fluorescently labeled genes distributed evenly throughout their chromosomes. By measuring these cells' lengths and number of gene copies at the single-cell level, we could fit a statistical model of the initiation and duration of chromosome replication. We found that for our slow-growing (120 min doubling time) E. coli cells, replication was initiated 42 min into the cell cycle and completed after an additional 42 min. While simulations of the biogenesis model produce the correct ribosome and mRNA counts over the cell cycle, the kinetic parameters for transcription and degradation are lower than anticipated from a recent analytical time dependent model of in vivo mRNA production. Describing expression in terms of a simple chemical master equation, we show that the discrepancies are due to the lack of nonribosomal genes in the extended biogenesis model which effects the competition of mRNA for ribosome binding, and suggest corrections to parameters to be used in the whole-cell model when modeling expression of the entire transcriptome. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 735-751, 2016. © 2016 Wiley Periodicals, Inc.

DNA Replication Origin Function Is Promoted by H3K4 Di-methylation in Saccharomyces cerevisiae

PubMed Central

Rizzardi, Lindsay F.; Dorn, Elizabeth S.; Strahl, Brian D.; Cook, Jeanette Gowen

2012-01-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication. PMID:22851644

DNA replication origin function is promoted by H3K4 di-methylation in Saccharomyces cerevisiae.

PubMed

Rizzardi, Lindsay F; Dorn, Elizabeth S; Strahl, Brian D; Cook, Jeanette Gowen

2012-10-01

DNA replication is a highly regulated process that is initiated from replication origins, but the elements of chromatin structure that contribute to origin activity have not been fully elucidated. To identify histone post-translational modifications important for DNA replication, we initiated a genetic screen to identify interactions between genes encoding chromatin-modifying enzymes and those encoding proteins required for origin function in the budding yeast Saccharomyces cerevisiae. We found that enzymes required for histone H3K4 methylation, both the histone methyltransferase Set1 and the E3 ubiquitin ligase Bre1, are required for robust growth of several hypomorphic replication mutants, including cdc6-1. Consistent with a role for these enzymes in DNA replication, we found that both Set1 and Bre1 are required for efficient minichromosome maintenance. These phenotypes are recapitulated in yeast strains bearing mutations in the histone substrates (H3K4 and H2BK123). Set1 functions as part of the COMPASS complex to mono-, di-, and tri-methylate H3K4. By analyzing strains lacking specific COMPASS complex members or containing H2B mutations that differentially affect H3K4 methylation states, we determined that these replication defects were due to loss of H3K4 di-methylation. Furthermore, histone H3K4 di-methylation is enriched at chromosomal origins. These data suggest that H3K4 di-methylation is necessary and sufficient for normal origin function. We propose that histone H3K4 di-methylation functions in concert with other histone post-translational modifications to support robust genome duplication.

Monocyte-derived macrophages from Crohn's disease patients are impaired in the ability to control intracellular adherent-invasive Escherichia coli and exhibit disordered cytokine secretion profile.

PubMed

Vazeille, Emilie; Buisson, Anthony; Bringer, Marie-Agnès; Goutte, Marion; Ouchchane, Lemlih; Hugot, Jean-Pierre; de Vallée, Amélie; Barnich, Nicolas; Bommelaer, Gilles; Darfeuille-Michaud, Arlette

2015-05-01

Ileal lesions of Crohn's disease [CD] patients are colonised by adherent-invasive Escherichia coli [AIEC] able to survive in macrophage cell lines. We analysed the ability of monocyte-derived macrophages [MDM] from CD patients to control AIEC intracellular replication and the pro-inflammatory cytokine response of the infected-MDM. Peripheral blood MDM were obtained from 24 CD genotyped for NOD2 and ATG16L1 mutations, 5 ulcerative colitis [UC] patients and 12 healthy controls [HC]. The numbers of intracellular bacteria were determined using gentamicin assay. Cytokine secretion was quantified by ELISA assay. We observed that higher levels of bacteria were internalised within MDM from CD patients than MDM from HC or UC patients. MDM from CD patients were unable to restrict AIEC intracellular replication. Infection of MDM from CD patients with AIEC resulted in significantly increased secretion of IL-6 and tumour necrosis factor alpha [TNF α] than did infection with non-pathogenic E. coli. AIEC-infected MDM from CD patients exhibited a disordered cytokines secretion compared with MDM from UC patients and HC. AIEC-infected MDM from patients with quiescent CD released significantly higher amounts of IL-6 and TNF-alpha than those with active disease or those from HC. The level of secreted TNF-alpha was correlated to the number of intracellular AIEC in MDM from CD patients. Treatment of MDM with infliximab did not change the MDM behaviour. MDM from CD patients are unable to restrict intracellular AIEC replication, leading to disordered inflammatory response influenced by disease activity. Copyright © 2015 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Mutagenic and cytotoxic properties of 6-thioguanine, S6-methylthioguanine, and guanine-S6-sulfonic acid.

PubMed

Yuan, Bifeng; Wang, Yinsheng

2008-08-29

Thiopurine drugs, including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine, are widely employed anticancer agents and immunosuppressants. The formation of (S)G nucleotides from the thiopurine prodrugs and their subsequent incorporation into nucleic acids are important for the drugs to exert their cytotoxic effects. (S)G in DNA can be methylated by S-adenosyl-l-methionine to give S(6)-methylthioguanine (S(6)mG) and oxidized by UVA light to render guanine-S(6)-sulfonic acid ((SO3H)G). Here, we constructed single-stranded M13 shuttle vectors carrying a (S)G, S(6)mG, or (SO3H)G at a unique site and allowed the vectors to propagate in wild-type and bypass polymerase-deficient Escherichia coli cells. Analysis of the replication products by using the competitive replication and adduct bypass and a slightly modified restriction enzyme digestion and post-labeling assays revealed that, although none of the three thionucleosides considerably blocked DNA replication in all transfected E. coli cells, both S(6)mG and (SO3H)G were highly mutagenic, which resulted in G-->A mutation at frequencies of 94 and 77%, respectively, in wild-type E. coli cells. Deficiency in bypass polymerases does not result in alteration of mutation frequencies of these two lesions. In contrast to what was found from previous steady-state kinetic analysis, our data demonstrated that 6-thioguanine is mutagenic, with G-->A transition occurring at a frequency of approximately 10%. The mutagenic properties of 6-thioguanine and its derivatives revealed in the present study offered important knowledge about the biological implications of these thionucleosides.

Ku Protein Levels, Localization and Association to Replication Origins in Different Stages of Breast Tumor Progression

PubMed Central

Abdelbaqi, Khalil; Di Paola, Domenic; Rampakakis, Emmanouil; Zannis-Hadjopoulos, Maria

2013-01-01

Human origins of DNA replication are specific sequences within the genome whereby DNA replication is initiated. A select group of proteins, known as the pre-replication (pre-RC) complex, in whose formation the Ku protein (Ku70/Ku86) was shown to play a role, bind to replication origins to initiate DNA replication. In this study, we have examined the involvement of Ku in breast tumorigenesis and tumor progression and found that the Ku protein expression levels in human breast metastatic (MCF10AC1a) cells were higher in the chromatin fraction compared to hyperplastic (MCF10AT) and normal (MCF10A) human breast cells, but remained constant in both the nuclear and cytoplasmic fractions. In contrast, in human intestinal cells, the Ku expression level was relatively constant for all cell fractions. Nascent DNA abundance and chromatin association of Ku70/86 revealed that the c-myc origin activity in MCF10AC1a is 2.5 to 5-fold higher than in MCF10AT and MCF10A, respectively, and Ku was bound to the c-myc origin more abundantly in MCF10AC1a, by approximately 1.5 to 4.2-fold higher than in MCF10AT and MCF10A, respectively. In contrast, similar nascent DNA abundance and chromatin association was found for all cell lines for the lamin B2 origin, associated with the constitutively active housekeeping lamin B2 gene. Electrophoretic mobility shift assays (EMSAs) performed on the nuclear extracts (NEs) of the three cell types revealed the presence of protein-DNA replication complexes on both the c-myc and lamin B2 origins, but an increase in binding activity was observed from normal, to transformed, to cancer cells for the c-myc origin, whereas no such difference was seen for the lamin B2 origin. Overall, the results suggest that increased Ku chromatin association, beyond wild type levels, alters cellular processes, which have been implicated in tumorigenesis. PMID:23781282

Genome-wide Analysis Reveals Extensive Functional Interaction between DNA Replication Initiation and Transcription in the Genome of Trypanosoma brucei

PubMed Central

Tiengwe, Calvin; Marcello, Lucio; Farr, Helen; Dickens, Nicholas; Kelly, Steven; Swiderski, Michal; Vaughan, Diane; Gull, Keith; Barry, J. David; Bell, Stephen D.; McCulloch, Richard

2012-01-01

Summary Identification of replication initiation sites, termed origins, is a crucial step in understanding genome transmission in any organism. Transcription of the Trypanosoma brucei genome is highly unusual, with each chromosome comprising a few discrete transcription units. To understand how DNA replication occurs in the context of such organization, we have performed genome-wide mapping of the binding sites of the replication initiator ORC1/CDC6 and have identified replication origins, revealing that both localize to the boundaries of the transcription units. A remarkably small number of active origins is seen, whose spacing is greater than in any other eukaryote. We show that replication and transcription in T. brucei have a profound functional overlap, as reducing ORC1/CDC6 levels leads to genome-wide increases in mRNA levels arising from the boundaries of the transcription units. In addition, ORC1/CDC6 loss causes derepression of silent Variant Surface Glycoprotein genes, which are critical for host immune evasion. PMID:22840408

THE E1 PROTEINS

PubMed Central

Bergvall, Monika; Melendy, Thomas; Archambault, Jacques

2013-01-01

E1, an ATP-dependent DNA helicase, is the only enzyme encoded by papillomaviruses (PVs). It is essential for replication and amplification of the viral episome in the nucleus of infected cells. To do so, E1 assembles into a double-hexamer at the viral origin, unwinds DNA at the origin and ahead of the replication fork and interacts with cellular DNA replication factors. Biochemical and structural studies have revealed the assembly pathway of E1 at the origin and how the enzyme unwinds DNA using a spiral escalator mechanism. E1 is tightly regulated in vivo, in particular by post-translational modifications that restrict its accumulation in the nucleus. Here we review how different functional domains of E1 orchestrate viral DNA replication, with an emphasis on their interactions with substrate DNA, host DNA replication factors and modifying enzymes. These studies have made E1 one of the best characterized helicases and provided unique insights on how PVs usurp different host-cell machineries to replicate and amplify their genome in a tightly controlled manner. PMID:24029589

Active role of a human genomic insert in replication of a yeast artificial chromosome.

PubMed

van Brabant, A J; Fangman, W L; Brewer, B J

1999-06-01

Yeast artificial chromosomes (YACs) are a common tool for cloning eukaryotic DNA. The manner by which large pieces of foreign DNA are assimilated by yeast cells into a functional chromosome is poorly understood, as is the reason why some of them are stably maintained and some are not. We examined the replication of a stable YAC containing a 240-kb insert of DNA from the human T-cell receptor beta locus. The human insert contains multiple sites that serve as origins of replication. The activity of these origins appears to require the yeast ARS consensus sequence and, as with yeast origins, additional flanking sequences. In addition, the origins in the human insert exhibit a spacing, a range of activation efficiencies, and a variation in times of activation during S phase similar to those found for normal yeast chromosomes. We propose that an appropriate combination of replication origin density, activation times, and initiation efficiencies is necessary for the successful maintenance of YAC inserts.

Interaction of the Retinoblastoma Protein with Orc1 and Its Recruitment to Human Origins of DNA Replication

PubMed Central

Mendoza-Maldonado, Ramiro; Paolinelli, Roberta; Galbiati, Laura; Giadrossi, Sara; Giacca, Mauro

2010-01-01

Background The retinoblastoma protein (Rb) is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. Methodology/Principal Findings Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1) specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. Conclusions/Significance The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication. PMID:21085491

Survival characteristics of diarrheagenic Escherichia coli pathotypes and Helicobacter pylori during passage through the free-living ciliate, Tetrahymena sp.

PubMed

Smith, Charlotte D; Berk, Sharon G; Brandl, Maria T; Riley, Lee W

2012-12-01

Free-living protozoa have been implicated in the survival and transport of pathogens in the environment, but the relationship between non-Shiga toxin-producing Escherichia coli or Helicobacter pylori and ciliates has not been characterized. Six diarrheagenic pathotypes of E. coli and an isolate of H. pylori were evaluated for their susceptibility to digestion by Tetrahymena, an aquatic ciliate. Tetrahymena strain MB125 was fed E. coli or H. pylori, and the ciliate's egested products examined for viable bacterial pathogens by the BacLight(™) LIVE/DEAD (™) assay, a cell elongation method, and by colony counts. All six diarrheagenic E. coli pathotypes survived digestion, whereas H. pylori was digested. Growth of E. coli on agar plates indicated that the bacteria were able to replicate after passage through the ciliate. Transmission electron micrographs of E. coli cells as intact rods vs. degraded H. pylori cells corroborated these results. Scanning electron microscopy revealed a net-like matrix around intact E. coli cells in fecal pellets. These results suggest a possible role for Tetrahymena and its egested fecal pellets in the dissemination of diarrheagenic E. coli in the environment. This bacterial-protozoan interaction may increase opportunities for transmission of diarrheagenic E. coli to mammalian hosts including humans. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Megabase replication domains along the human genome: relation to chromatin structure and genome organisation.

PubMed

Audit, Benjamin; Zaghloul, Lamia; Baker, Antoine; Arneodo, Alain; Chen, Chun-Long; d'Aubenton-Carafa, Yves; Thermes, Claude

2013-01-01

In higher eukaryotes, the absence of specific sequence motifs, marking the origins of replication has been a serious hindrance to the understanding of (i) the mechanisms that regulate the spatio-temporal replication program, and (ii) the links between origins activation, chromatin structure and transcription. In this chapter, we review the partitioning of the human genome into megabased-size replication domains delineated as N-shaped motifs in the strand compositional asymmetry profiles. They collectively span 28.3% of the genome and are bordered by more than 1,000 putative replication origins. We recapitulate the comparison of this partition of the human genome with high-resolution experimental data that confirms that replication domain borders are likely to be preferential replication initiation zones in the germline. In addition, we highlight the specific distribution of experimental and numerical chromatin marks along replication domains. Domain borders correspond to particular open chromatin regions, possibly encoded in the DNA sequence, and around which replication and transcription are highly coordinated. These regions also present a high evolutionary breakpoint density, suggesting that susceptibility to breakage might be linked to local open chromatin fiber state. Altogether, this chapter presents a compartmentalization of the human genome into replication domains that are landmarks of the human genome organization and are likely to play a key role in genome dynamics during evolution and in pathological situations.

Visualization and Enumeration of Bacteria Carrying a Specific Gene Sequence by In Situ Rolling Circle Amplification

PubMed Central

Maruyama, Fumito; Kenzaka, Takehiko; Yamaguchi, Nobuyasu; Tani, Katsuji; Nasu, Masao

2005-01-01

Rolling circle amplification (RCA) generates large single-stranded and tandem repeats of target DNA as amplicons. This technique was applied to in situ nucleic acid amplification (in situ RCA) to visualize and count single Escherichia coli cells carrying a specific gene sequence. The method features (i) one short target sequence (35 to 39 bp) that allows specific detection; (ii) maintaining constant fluorescent intensity of positive cells permeabilized extensively after amplicon detection by fluorescence in situ hybridization, which facilitates the detection of target bacteria in various physiological states; and (iii) reliable enumeration of target bacteria by concentration on a gelatin-coated membrane filter. To test our approach, the presence of the following genes were visualized by in situ RCA: green fluorescent protein gene, the ampicillin resistance gene and the replication origin region on multicopy pUC19 plasmid, as well as the single-copy Shiga-like toxin gene on chromosomes inside E. coli cells. Fluorescent antibody staining after in situ RCA also simultaneously identified cells harboring target genes and determined the specificity of in situ RCA. E. coli cells in a nonculturable state from a prolonged incubation were periodically sampled and used for plasmid uptake study. The numbers of cells taking up plasmids determined by in situ RCA was up to 106-fold higher than that measured by selective plating. In addition, in situ RCA allowed the detection of cells taking up plasmids even when colony-forming cells were not detected during the incubation period. By optimizing the cell permeabilization condition for in situ RCA, this method can become a valuable tool for studying free DNA uptake, especially in nonculturable bacteria. PMID:16332770

A novel approach for Escherichia coli genome editing combining in vivo cloning and targeted long-length chromosomal insertion.

PubMed

Hook, Ch D; Samsonov, V V; Ublinskaya, A A; Kuvaeva, T M; Andreeva, E V; Gorbacheva, L Yu; Stoynova, N V

2016-11-01

Despite the abundance of genetic manipulation approaches, particularly for Escherichia coli, new techniques and increased flexibility in the application of existing techniques are required to address novel aims. The most widely used approaches for chromosome editing are based on bacteriophage site-specific and λRed/RecET-mediated homologous recombination. In the present study, these techniques were combined to develop a novel approach for in vivo cloning and targeted long-length chromosomal insertion. This approach permits direct λRed-mediated cloning of DNA fragment with lengths of 10kb or greater from the E. coli chromosome into the plasmid vector pGL2, which carries the ori of pSC101, the ϕ80-attP site of ϕ80 phage, and an excisable Cm R marker bracketed by λ-attL/attR sites. In pGL2-based recombinant plasmids, the origin of replication can be eliminated in vitro via hydrolysis by SceI endonuclease and recircularization by DNA ligase. The resulting ori-less circular recombinant DNA can be used for targeted insertion of the cloned sequence into the chromosome at a selected site via ϕ80 phage-specific integrase-mediated recombination using the Dual-In/Out approach (Minaeva et al., 2008). At the final stage of chromosomal editing, the Cm R -marker can be excised from the chromosome due to expression of the λint/xis genes. Notably, the desired fragment can be inserted as multiple copies in the chromosome by combining insertions at different sites in one strain using the P1 general transduction technique (Moore, 2011). The developed approach is useful for the construction of plasmidless, markerless recombinant strains for fundamental and industrial purposes. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Biology and Genomics of an Historic Therapeutic Escherichia coli Bacteriophage Collection.

PubMed

Baig, Abiyad; Colom, Joan; Barrow, Paul; Schouler, Catherine; Moodley, Arshnee; Lavigne, Rob; Atterbury, Robert

2017-01-01

We have performed microbiological and genomic characterization of an historic collection of nine bacteriophages, specifically infecting a K1 E. coli O18:K1:H7 ColV + strain. These phages were isolated from sewage and tested for their efficacy in vivo for the treatment of systemic E. coli infection in a mouse infection model by Smith and Huggins (1982). The aim of the study was to identify common microbiological and genomic characteristics, which co-relate to the performance of these phages in in vivo study. These features will allow an informed selection of phages for use as therapeutic agents. Transmission electron microscopy showed that six of the nine phages were Podoviridae and the remaining three were Siphoviridae . The four best performing phages in vivo belonged to the Podoviridae family. In vitro , these phages exhibited very short latent and rise periods in our study. In agreement with their microbiological profiles, characterization by genome sequencing showed that all six podoviruses belong to the Autographivirinae subfamily. Of these, four were isolates of the same species (99% identity), whereas two had divergent genomes compared to other podoviruses. The Siphoviridae phages, which were moderate to poor performers in vivo , exhibited longer latent and rise periods in vitro . Two of the three siphoviruses were closely related to each other (99% identity), but all can be associated with the Guernseyvirinae subfamily. Genome sequence comparison of both types of phages showed that a gene encoding for DNA-dependent RNA polymerase was only present in phages with faster replication cycle, which may account for their better performance in vivo . These data define a combination of microbiological, genomic and in vivo characteristics which allow a more rational evaluation of the original in vivo data and pave the way for the selection of phages for future phage therapy trails.

Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

NASA Astrophysics Data System (ADS)

Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

2016-03-01

We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

Adaptive laboratory evolution resolves energy depletion to maintain high aromatic metabolite phenotypes in Escherichia coli strains lacking the Phosphotransferase System.

PubMed

McCloskey, Douglas; Xu, Sibei; Sandberg, Troy E; Brunk, Elizabeth; Hefner, Ying; Szubin, Richard; Feist, Adam M; Palsson, Bernhard O

2018-06-15

Aromatic metabolites provide the backbone for numerous industrial and pharmaceutical compounds of high value. The Phosphotransferase System (PTS) is common to many bacteria, and is the primary mechanism for glucose uptake by Escherichia coli. The PTS was removed to conserve phosphoenolpyruvate (pep), which is a precursor for aromatic metabolites and consumed by the PTS, for aromatic metabolite production. Replicate adaptive laboratory evolution (ALE) of PTS and detailed omics data sets collected revealed that the PTS bridged the gap between respiration and fermentation, leading to distinct high fermentative and high respiratory rate phenotypes. It was also found that while all strains retained high levels of aromatic amino acid (AAA) biosynthetic precursors, only one replicate from the high glycolytic clade retained high levels of intracellular AAAs. The fast growth and high AAA precursor phenotypes could provide a starting host for cell factories targeting the overproduction aromatic metabolites. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Modular structural elements in the replication origin region of Tetrahymena rDNA.

PubMed Central

Du, C; Sanzgiri, R P; Shaiu, W L; Choi, J K; Hou, Z; Benbow, R M; Dobbs, D L

1995-01-01

Computer analyses of the DNA replication origin region in the amplified rRNA genes of Tetrahymena thermophila identified a potential initiation zone in the 5'NTS [Dobbs, Shaiu and Benbow (1994), Nucleic Acids Res. 22, 2479-2489]. This region consists of a putative DNA unwinding element (DUE) aligned with predicted bent DNA segments, nuclear matrix or scaffold associated region (MAR/SAR) consensus sequences, and other common modular sequence elements previously shown to be clustered in eukaryotic chromosomal origin regions. In this study, two mung bean nuclease-hypersensitive sites in super-coiled plasmid DNA were localized within the major DUE-like element predicted by thermodynamic analyses. Three restriction fragments of the 5'NTS region predicted to contain bent DNA segments exhibited anomalous migration characteristic of bent DNA during electrophoresis on polyacrylamide gels. Restriction fragments containing the 5'NTS region bound Tetrahymena nuclear matrices in an in vitro binding assay, consistent with an association of the replication origin region with the nuclear matrix in vivo. The direct demonstration in a protozoan origin region of elements previously identified in Drosophila, chick and mammalian origin regions suggests that clusters of modular structural elements may be a conserved feature of eukaryotic chromosomal origins of replication. Images PMID:7784181

Analyzing the dynamics of DNA replication in Mammalian cells using DNA combing.

PubMed

Bialic, Marta; Coulon, Vincent; Drac, Marjorie; Gostan, Thierry; Schwob, Etienne

2015-01-01

How cells duplicate their chromosomes is a key determinant of cell identity and genome stability. DNA replication can initiate from more than 100,000 sites distributed along mammalian chromosomes, yet a given cell uses only a subset of these origins due to inefficient origin activation and regulation by developmental or environmental cues. An impractical consequence of cell-to-cell variations in origin firing is that population-based techniques do not accurately describe how chromosomes are replicated in single cells. DNA combing is a biophysical DNA fiber stretching method which permits visualization of ongoing DNA synthesis along Mb-sized single-DNA molecules purified from cells that were previously pulse-labeled with thymidine analogues. This allows quantitative measurements of several salient features of chromosome replication dynamics, such as fork velocity, fork asymmetry, inter-origin distances, and global instant fork density. In this chapter we describe how to obtain this information from asynchronous cultures of mammalian cells.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase.

PubMed

Xia, Jun; Chen, Li-Tzu; Mei, Qian; Ma, Chien-Hui; Halliday, Jennifer A; Lin, Hsin-Yu; Magnan, David; Pribis, John P; Fitzgerald, Devon M; Hamilton, Holly M; Richters, Megan; Nehring, Ralf B; Shen, Xi; Li, Lei; Bates, David; Hastings, P J; Herman, Christophe; Jayaram, Makkuni; Rosenberg, Susan M

2016-11-01

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli . We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR-HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells.

The aminoglycoside antibiotic kanamycin damages DNA bases in Escherichia coli: caffeine potentiates the DNA-damaging effects of kanamycin while suppressing cell killing by ciprofloxacin in Escherichia coli and Bacillus anthracis.

PubMed

Kang, Tina Manzhu; Yuan, Jessica; Nguyen, Angelyn; Becket, Elinne; Yang, Hanjing; Miller, Jeffrey H

2012-06-01

The distribution of mutants in the Keio collection of Escherichia coli gene knockout mutants that display increased sensitivity to the aminoglycosides kanamycin and neomycin indicates that damaged bases resulting from antibiotic action can lead to cell death. Strains lacking one of a number of glycosylases (e.g., AlkA, YzaB, Ogt, KsgA) or other specific repair proteins (AlkB, PhrB, SmbC) are more sensitive to these antibiotics. Mutants lacking AlkB display the strongest sensitivity among the glycosylase- or direct lesion removal-deficient strains. This perhaps suggests the involvement of ethenoadenine adducts, resulting from reactive oxygen species and lipid peroxidation, since AlkB removes this lesion. Other sensitivities displayed by mutants lacking UvrA, polymerase V (Pol V), or components of double-strand break repair indicate that kanamycin results in damaged base pairs that need to be removed or replicated past in order to avoid double-strand breaks that saturate the cellular repair capacity. Caffeine enhances the sensitivities of these repair-deficient strains to kanamycin and neomycin. The gene knockout mutants that display increased sensitivity to caffeine (dnaQ, holC, holD, and priA knockout mutants) indicate that caffeine blocks DNA replication, ultimately leading to double-strand breaks that require recombinational repair by functions encoded by recA, recB, and recC, among others. Additionally, caffeine partially protects cells of both Escherichia coli and Bacillus anthracis from killing by the widely used fluoroquinolone antibiotic ciprofloxacin.

Holliday junction trap shows how cells use recombination and a junction-guardian role of RecQ helicase

PubMed Central

Xia, Jun; Chen, Li-Tzu; Mei, Qian; Ma, Chien-Hui; Halliday, Jennifer A.; Lin, Hsin-Yu; Magnan, David; Pribis, John P.; Fitzgerald, Devon M.; Hamilton, Holly M.; Richters, Megan; Nehring, Ralf B.; Shen, Xi; Li, Lei; Bates, David; Hastings, P. J.; Herman, Christophe; Jayaram, Makkuni; Rosenberg, Susan M.

2016-01-01

DNA repair by homologous recombination (HR) underpins cell survival and fuels genome instability, cancer, and evolution. However, the main kinds and sources of DNA damage repaired by HR in somatic cells and the roles of important HR proteins remain elusive. We present engineered proteins that trap, map, and quantify Holliday junctions (HJs), a central DNA intermediate in HR, based on catalytically deficient mutant RuvC protein of Escherichia coli. We use RuvCDefGFP (RDG) to map genomic footprints of HR at defined DNA breaks in E. coli and demonstrate genome-scale directionality of double-strand break (DSB) repair along the chromosome. Unexpectedly, most spontaneous HR-HJ foci are instigated, not by DSBs, but rather by single-stranded DNA damage generated by replication. We show that RecQ, the E. coli ortholog of five human cancer proteins, nonredundantly promotes HR-HJ formation in single cells and, in a novel junction-guardian role, also prevents apparent non-HR–HJs promoted by RecA overproduction. We propose that one or more human RecQ orthologs may act similarly in human cancers overexpressing the RecA ortholog RAD51 and find that cancer genome expression data implicate the orthologs BLM and RECQL4 in conjunction with EME1 and GEN1 as probable HJ reducers in such cancers. Our results support RecA-overproducing E. coli as a model of the many human tumors with up-regulated RAD51 and provide the first glimpses of important, previously elusive reaction intermediates in DNA replication and repair in single living cells. PMID:28090586

Structures of minute virus of mice replication initiator protein N-terminal domain: Insights into DNA nicking and origin binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tewary, Sunil K.; Liang, Lingfei; Lin, Zihan

Members of the Parvoviridae family all encode a non-structural protein 1 (NS1) that directs replication of single-stranded viral DNA, packages viral DNA into capsid, and serves as a potent transcriptional activator. Here we report the X-ray structure of the minute virus of mice (MVM) NS1 N-terminal domain at 1.45 Å resolution, showing that sites for dsDNA binding, ssDNA binding and cleavage, nuclear localization, and other functions are integrated on a canonical fold of the histidine-hydrophobic-histidine superfamily of nucleases, including elements specific for this Protoparvovirus but distinct from its Bocaparvovirus or Dependoparvovirus orthologs. High resolution structural analysis reveals a nickase activemore » site with an architecture that allows highly versatile metal ligand binding. The structures support a unified mechanism of replication origin recognition for homotelomeric and heterotelomeric parvoviruses, mediated by a basic-residue-rich hairpin and an adjacent helix in the initiator proteins and by tandem tetranucleotide motifs in the replication origins. - Highlights: • The structure of a parvovirus replication initiator protein has been determined; • The structure sheds light on mechanisms of ssDNA binding and cleavage; • The nickase active site is preconfigured for versatile metal ligand binding; • The binding site for the double-stranded replication origin DNA is identified; • A single domain integrates multiple functions in virus replication.« less

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

Isolation and characterization of novel mutations in the pSC101 origin that increase copy number

DOE PAGES

Thompson, Mitchell G.; Sedaghatian, Nima; Barajas, Jesus F.; ...

2018-01-25

pSC101 is a narrow host range, low-copy plasmid commonly used for genetically manipulating Escherichia coli. As a byproduct of a genetic screen for a more sensitive lactam biosensor, we identified multiple novel mutations that increase the copy number of plasmids with the pSC101 origin. All mutations identified in this study occurred on plasmids which also contained at least one mutation localized to the RepA protein encoded within the origin. Homology modelling predicts that many of these mutations occur within the dimerization interface of RepA. Mutant RepA resulted in plasmid copy numbers between ~31 and ~113 copies/cell, relative to ~5 copies/cellmore » in wild-type pSC101 plasmids. Combining the mutations that were predicted to disrupt multiple contacts on the dimerization interface resulted in copy numbers of ~500 copies/cell, while also attenuating growth in host strains. Fluorescent protein production expressed from an arabinose-inducible promoter on mutant origin derived plasmids did correlate with copy number. Plasmids harboring RepA with one of two mutations, E83K and N99D, resulted in fluorescent protein production similar to that from p15a- (~20 copies/cell) and ColE1- (~31 copies/cell) based plasmids, respectively. The mutant copy number variants retained compatibility with p15a, pBBR, and ColE1 origins of replication. Thus, these pSC101 variants may be useful in future metabolic engineering efforts that require medium or high-copy vectors compatible with p15a- and ColE1-based plasmids.« less

A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs

PubMed Central

Lee, Dong-Hoon; Liu, Yinling; Lee, Hyun-Wook; Xia, Bo; Brice, Allyn R.; Park, Sung-Hyun; Balduf, Hunter; Dominy, Brian N.; Cao, Weiguo

2015-01-01

The uracil DNA glycosylase superfamily consists of several distinct families. Family 2 mismatch-specific uracil DNA glycosylase (MUG) from Escherichia coli is known to exhibit glycosylase activity on three mismatched base pairs, T/U, G/U and C/U. Family 1 uracil N-glycosylase (UNG) from E. coli is an extremely efficient enzyme that can remove uracil from any uracil-containing base pairs including the A/U base pair. Here, we report the identification of an important structural determinant that underlies the functional difference between MUG and UNG. Substitution of a Lys residue at position 68 with Asn in MUG not only accelerates the removal of uracil from mismatched base pairs but also enables the enzyme to gain catalytic activity on A/U base pairs. Binding and kinetic analysis demonstrate that the MUG-K68N substitution results in enhanced ground state binding and transition state interactions. Molecular modeling reveals that MUG-K68N, UNG-N123 and family 5 Thermus thermophiles UDGb-A111N can form bidentate hydrogen bonds with the N3 and O4 moieties of the uracil base. Genetic analysis indicates the gain of function for A/U base pairs allows the MUG-K68N mutant to remove uracil incorporated into the genome during DNA replication. The implications of this study in the origin of life are discussed. PMID:25550433

Design of an Escherichia coli system for whole cell mediated steroid synthesis and molecular evolution of steroid hydroxylases.

PubMed

Hannemann, Frank; Virus, Cornelia; Bernhardt, Rita

2006-06-25

The 15beta-hydroxylase (CYP106A2) from Bacillus megaterium, one of the few bacterial steroid hydroxylases, which has been isolated and characterized so far, catalyses the 15beta-hydroxylation of a variety of steroids. The enzyme can be supported in its activity with adrenodoxin (Adx) and adrenodoxin reductase (AdR) from bovine adrenals, supplying this enzyme with the reducing equivalents necessary for steroid hydroxylation activity. This three-component electron transfer chain was implemented in Escherichia coli by coexpression of the corresponding coding sequences from two plasmids, containing different selection markers and compatible origins of replication. The cDNAs of AdR and Adx on the first plasmid were separated by a ribosome binding sequence, with the reductase preceding the ferredoxin. The second plasmid for CYP106A2 expression was constructed with all features necessary for a molecular evolution approach. The transformed bacteria show the inducible ability to efficiently convert 11-deoxycorticosterone (DOC) to 15beta-DOC at an average rate of 1 mM/d in culture volumes of 300 ml. The steroid conversion system was downscaled to the microtiter plate format and a robot set-up was developed for a fluorescence-based conversion assay as well as a CO difference spectroscopy assay, which enables the screening for enzyme variants with higher activity and stability.

Features of genomic organization in a nucleotide-resolution molecular model of the Escherichia coli chromosome.

PubMed

Hacker, William C; Li, Shuxiang; Elcock, Adrian H

2017-07-27

We describe structural models of the Escherichia coli chromosome in which the positions of all 4.6 million nucleotides of each DNA strand are resolved. Models consistent with two basic chromosomal orientations, differing in their positioning of the origin of replication, have been constructed. In both types of model, the chromosome is partitioned into plectoneme-abundant and plectoneme-free regions, with plectoneme lengths and branching patterns matching experimental distributions, and with spatial distributions of highly-transcribed chromosomal regions matching recent experimental measurements of the distribution of RNA polymerases. Physical analysis of the models indicates that the effective persistence length of the DNA and relative contributions of twist and writhe to the chromosome's negative supercoiling are in good correspondence with experimental estimates. The models exhibit characteristics similar to those of 'fractal globules,' and even the most genomically-distant parts of the chromosome can be physically connected, through paths combining linear diffusion and inter-segmental transfer, by an average of only ∼10 000 bp. Finally, macrodomain structures and the spatial distributions of co-expressed genes are analyzed: the latter are shown to depend strongly on the overall orientation of the chromosome. We anticipate that the models will prove useful in exploring other static and dynamic features of the bacterial chromosome. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Digital replication of chest radiographs without altering diagnostic observer performance

NASA Astrophysics Data System (ADS)

Flynn, Michael J.; Davies, Eric; Spizarny, David; Beute, Gordon H.; Peterson, Edward; Eyler, William R.; Gross, Barry; Chen, Ji

1991-05-01

A study to test the ability of a high-fidelity system to digitize chest radiographs, store the data in a computer, and reprint the film without altering diagnostic observer performance is reported. Two hundred and fifty-two (252) chest films with subtle image features indicative of interstitial disease, pulmonary nodule, or pneumothorax, along with 36 normal chest films were used in the study. Films were selected from a key word search on a computerized report archive and were graded by two experienced radiologists. Each film was digitized with 86 micron pixels and stored in 4000 X 5000 arrays using a research instrument. Replicates were printed using a commercial laser film printer (Eastman Kodak Company) having 80 micron pixels. Originals and replicates were observed separately by two different experienced radiologists. Each indicated a graded response for the three possible pathologies. The agreement of observers between responses for replicates and originals was described by the kappa statistic and compared to the agreement when rereading the original film. The final result of this study supports a hypothesis that the replicate is indistinguishable from the original.

Chromatin Constrains the Initiation and Elongation of DNA Replication.

PubMed

Devbhandari, Sujan; Jiang, Jieqing; Kumar, Charanya; Whitehouse, Iestyn; Remus, Dirk

2017-01-05

Eukaryotic chromosomal DNA is faithfully replicated in a complex series of cell-cycle-regulated events that are incompletely understood. Here we report the reconstitution of DNA replication free in solution with purified proteins from the budding yeast Saccharomyces cerevisiae. The system recapitulates regulated bidirectional origin activation; synthesis of leading and lagging strands by the three replicative DNA polymerases Pol α, Pol δ, and Pol ε; and canonical maturation of Okazaki fragments into continuous daughter strands. We uncover a dual regulatory role for chromatin during DNA replication: promoting origin dependence and determining Okazaki fragment length by restricting Pol δ progression. This system thus provides a functional platform for the detailed mechanistic analysis of eukaryotic chromosome replication. Copyright © 2017 Elsevier Inc. All rights reserved.

Geographic variability of Escherichia coli ribotypes from animals in Idaho and Georgia.

PubMed

Hartel, Peter G; Summer, Jacob D; Hill, Jennifer L; Collins, J Victoria; Entry, James A; Segars, William I

2002-01-01

Several genotypic methods have been developed for determining the host origin of fecal bacteria in contaminated waters. Some of these methods rely on a host origin database to identify environmental isolates. It is not well understood to what degree these host origin isolates are geographically variable (i.e., cosmopolitan or endemic). This is important because a geographically limited host origin database may or may not be universally applicable. The objective of our study was to use one genotypic method, ribotyping, to determine the geographic variability of the fecal bacterium, Escherichia coli, from one location in Idaho and three locations in Georgia for cattle (Bos taurus), horse (Equus caballus), swine (Sus scrofa), and chicken (Gallus gallus domesticus). A total of 568 fecal E. coli isolates from Kimberly, ID (125 isolates), Athens, GA (210 isolates), Brunswick, GA (102 isolates), and Tifton, GA (131 isolates), yielded 213 ribotypes. The percentage of ribotype sharing within an animal species increased with decreased distance between geographic locations for cattle and horses, but not for swine and chicken. When the E. coli ribotypes among the four host species were compared at one location, the percent of unshared ribotypes was 86, 89, 81, and 79% for Kimberly, Athens, Brunswick, and Tifton, respectively. These data suggest that there is good ribotype separation among host animal species at each location. The ability to match environmental isolates to a host origin database may depend on a large number of environmental and host origin isolates that ideally are not geographically separated.

Effect of Sand Bed Depth and Medium Age on Escherichia coli and Turbidity Removal in Biosand Filters.

PubMed

Napotnik, Julie A; Baker, Derek; Jellison, Kristen L

2017-03-21

The main objective of this study was to build several full-scale biosand filters (BSFs) and assess the long-term (9 month) efficacy for particulate and Escherichia coli removal under simulated real-world usage. Four replicates of three different filter designs were built: the traditional concrete BSF and two scaled-down versions that use a 5 or 2 gal bucket as the casing material. The smaller sand bed depths in the bucket-sized filters did not impact filter performance with respect to (i) turbidity and E. coli removal or (ii) effluent levels of turbidity and E. coli. All filters produced effluents with a mean turbidity of <0.6 nephelometric turbidity unit. In addition, 78, 74, and 72% of effluent samples for the concrete, 5 gal, and 2 gal filters, respectively, had E. coli concentrations of <1 colony-forming unit/100 mL. The bucket-sized filters were found to be a potential alternative to the concrete BSFs for the removal of E. coli and turbidity from drinking water. Because smaller BSFs must be filled more frequently than larger BSFs to produce comparable water volumes, the effect of shorter pause periods on BSF performance should be investigated.

Life Origination and Development Hydrate theory (LOH-Theory): new approaches to the problems of the optimal nutrition and life prolongation

NASA Astrophysics Data System (ADS)

Kadyshevich, E. A.; Ostrovskii, V. E.

2014-04-01

Life Origination Hydrate Theory (LOH-Theory) and Mitosis and Replication Hydrate Theory (MRHTheory), both grounded on the notion of honeycomb gas-hydrate structures formation/destruction as the physicochemical phenomenon governing the DNA origination and replication, allow new approaches to the optimal nutrition and life prolongation problems.

Replicating the Conditions on Mars: the Possibility of Sustaining Life

NASA Astrophysics Data System (ADS)

Abdul-Masih, Michael; Guinan, E. F.

2013-01-01

We describe a laboratory experiment that replicates environmental conditions on Mars’s surface to determine the viability of Earth-based life under conditions on Mars. Of all the organisms currently known, the ones that are best suited for the harsh environments found throughout our solar system are the extremophiles found on Earth that tolerate extreme environmental conditions. The environmental conditions on Mars are the least extreme of all the planets in our solar system excluding Earth. The average temperature near the equator varies from +10 C to -20 C and the surface pressure is P ~ 0.01 Atm. The thin atmosphere means that the UV radiation from the Sun is relatively unshielded. Carbon Dioxide (CO2) makes up approximately 95% of Mars’ atmosphere and the soil is composed primarily of iron oxides. This being said, water can exist in liquid form under the surface and because of this, so can life. To test this, we constructed an air-tight chamber out of Plexiglas that replicates the environmental conditions on Mars. As a first step, two different organisms were tested for survival: E-coli, which was the control, and Hypsibius (Tardigrades), a member of the Tardigradas phylum. We hypothesized that the E-coli would perish while the more hardy Tardigrades would survive. The trials were two days each and the chosen temperature was ~4°C. Many trials were conducted. After the first compilation of the results, the hypothesis did not hold. The E-coli surprisingly survived while the Tardigrades did not survive. The E-coli did not grow while in the chamber, but after being removed they grew showing that they were still viable and that the conditions were not harsh enough to kill them. After these initial results, the experiment was altered slightly to ensure that the Tardigrades were able to successfully enter their hibernation like state that allows them to survive extreme conditions. These experiments are continuing and we will discuss the results under controlled differing environmental conditions. This research is supported by a grant from Villanova University Center for Undergraduate Research and Fellowships & also supported by NSF/RUI Grant AST-1009903.

Rif1 is a global regulator of timing of replication origin firing in fission yeast

PubMed Central

Hayano, Motoshi; Kanoh, Yutaka; Matsumoto, Seiji; Renard-Guillet, Claire; Shirahige, Katsuhiko; Masai, Hisao

2012-01-01

One of the long-standing questions in eukaryotic DNA replication is the mechanisms that determine where and when a particular segment of the genome is replicated. Cdc7/Hsk1 is a conserved kinase required for initiation of DNA replication and may affect the site selection and timing of origin firing. We identified rif1Δ, a null mutant of rif1+, a conserved telomere-binding factor, as an efficient bypass mutant of fission yeast hsk1. Extensive deregulation of dormant origins over a wide range of the chromosomes occurs in rif1Δ in the presence or absence of hydroxyurea (HU). At the same time, many early-firing, efficient origins are suppressed or delayed in firing timing in rif1Δ. Rif1 binds not only to telomeres, but also to many specific locations on the arm segments that only partially overlap with the prereplicative complex assembly sites, although Rif1 tends to bind in the vicinity of the late/dormant origins activated in rif1Δ. The binding to the arm segments occurs through M to G1 phase in a manner independent of Taz1 and appears to be essential for the replication timing program during the normal cell cycle. Our data demonstrate that Rif1 is a critical determinant of the origin activation program on the fission yeast chromosomes. PMID:22279046

Escherichia coli challenge and one type of smectite alter intestinal barrier of pigs

PubMed Central

2013-01-01

An experiment was conducted to determine how an E. coli challenge and dietary clays affect the intestinal barrier of pigs. Two groups of 32 pigs (initial BW: 6.9 ± 1.0 kg) were distributed in a 2 × 4 factorial arrangement of a randomized complete block design (2 challenge treatments: sham or E. coli, and 4 dietary treatments: control, 0.3% smectite A, 0.3% smectite B and 0.3% zeolite), with 8 replicates total. Diarrhea score, growth performance, goblet cell size and number, bacterial translocation from intestinal lumen to lymph nodes, intestinal morphology, and relative amounts of sulfo and sialo mucins were measured. The E. coli challenge reduced performance, increased goblet cell size and number in the ileum, increased bacterial translocation from the intestinal lumen to the lymph nodes, and increased ileal crypt depth. One of the clays (smectite A) tended to increase goblet cell size in ileum, which may indicate enhanced protection. In conclusion, E. coli infection degrades intestinal barrier integrity but smectite A may enhance it. PMID:24359581

Escherichia coli challenge and one type of smectite alter intestinal barrier of pigs.

PubMed

Almeida, Juliana Abranches Soares; Liu, Yanhong; Song, Minho; Lee, Jeong Jae; Gaskins, H Rex; Maddox, Carol Wolfgang; Osuna, Orlando; Pettigrew, James Eugene

2013-12-20

An experiment was conducted to determine how an E. coli challenge and dietary clays affect the intestinal barrier of pigs. Two groups of 32 pigs (initial BW: 6.9 ± 1.0 kg) were distributed in a 2 × 4 factorial arrangement of a randomized complete block design (2 challenge treatments: sham or E. coli, and 4 dietary treatments: control, 0.3% smectite A, 0.3% smectite B and 0.3% zeolite), with 8 replicates total. Diarrhea score, growth performance, goblet cell size and number, bacterial translocation from intestinal lumen to lymph nodes, intestinal morphology, and relative amounts of sulfo and sialo mucins were measured. The E. coli challenge reduced performance, increased goblet cell size and number in the ileum, increased bacterial translocation from the intestinal lumen to the lymph nodes, and increased ileal crypt depth. One of the clays (smectite A) tended to increase goblet cell size in ileum, which may indicate enhanced protection. In conclusion, E. coli infection degrades intestinal barrier integrity but smectite A may enhance it.

Persistence and microbial source tracking of Escherichia coli at a swimming beach at Lake of the Ozarks State Park, Missouri

USGS Publications Warehouse

Wilson, Jordan L.; Schumacher, John G.; Burken, Joel G.

2016-01-01

The Missouri Department of Natural Resources (MDNR) has closed or posted advisories at public beaches at Lake of the Ozarks State Park in Missouri because of Escherichia coli (E. coli) concentration exceedances in recent years. Spatial and temporal patterns of E. coliconcentrations, microbial source tracking, novel sampling techniques, and beach-use patterns were studied during the 2012 recreational season to identify possible sources, origins, and occurrence of E. coli contamination at Grand Glaize Beach (GGB). Results indicate an important source of E. coli contamination at GGB was E. coli released into the water column by bathers resuspending avian-contaminated sediments, especially during high-use days early in the recreational season. Escherichia coli concentrations in water, sediment, and resuspended sediment samples all decreased throughout the recreational season likely because of decreasing lake levels resulting in sampling locations receding away from the initial spring shoreline as well as natural decay and physical transport out of the cove. Weekly MDNR beach monitoring, based solely on E. coli concentrations, at GGB during this study inaccurately predicted E. coli exceedances, especially on weekends and holidays. Interestingly, E. coli of human origin were measured at concentrations indicative of raw sewage in runoff from an excavation of a nearby abandoned septic tank that had not been used for nearly two years.

Molecular typing of avian pathogenic Escherichia coli colonies originating from outbreaks of E. coli peritonitis syndrome in chicken flocks.

PubMed

Landman, W J M; Buter, G J; Dijkman, R; van Eck, J H H

2014-01-01

Escherichia coli colonies isolated from the bone marrow of fresh dead hens of laying flocks with the E. coli peritonitis syndrome (EPS) were genotyped using pulsed-field gel electrophoresis (PFGE). Typing is important from an epidemiological point of view and also if the use of autogenous (auto)vaccines is considered. Birds with EPS originated from one house of each of three layer farms and one broiler breeder farm. Farms were considered as separate epidemiological units. In total, six flocks were examined including two successive flocks of one layer farm and the broiler breeder farm. E. coli colonies (one per bird) from nine to 16 hens of each flock were genotyped. The clonality of E. coli within birds was studied using five colonies of each of nine to 14 birds per flock. E. coli genotypes, which totalled 15, differed between farms and flocks except for two successive layer flocks that shared three genotypes. One to five genotypes were found per flock with one or two genotypes dominating each outbreak. Within hens, E. coli bacteria were always clonal. Colonies of the same PFGE type always had the same multilocus sequence type. However, four PFGE types shared sequence type 95. Neither PFGE types nor multilocus sequence types were unambiguously related to avian pathogenic E. coli from EPS. In cases where persistence of E. coli strains associated with EPS is found to occur frequently, routine genotyping to select strains for autovaccines should be considered.

The rolling-circle melting-pot model for porcine circovirus DNA replication

USDA-ARS?s Scientific Manuscript database

A stem-loop structure, formed by a pair of inverted repeats during DNA replication, is a conserved feature at the origin of DNA replication (Ori) among plant and animal viruses, bacteriophages and plasmids that replicate their genomes via the rolling-circle replication (RCR) mechanism. Porcine circo...

Diverged composition and regulation of the Trypanosoma brucei origin recognition complex that mediates DNA replication initiation

PubMed Central

Marques, Catarina A.; Tiengwe, Calvin; Lemgruber, Leandro; Damasceno, Jeziel D.; Scott, Alan; Paape, Daniel; Marcello, Lucio; McCulloch, Richard

2016-01-01

Abstract Initiation of DNA replication depends upon recognition of genomic sites, termed origins, by AAA+ ATPases. In prokaryotes a single factor binds each origin, whereas in eukaryotes this role is played by a six-protein origin recognition complex (ORC). Why eukaryotes evolved a multisubunit initiator, and the roles of each component, remains unclear. In Trypanosoma brucei, an ancient unicellular eukaryote, only one ORC-related initiator, TbORC1/CDC6, has been identified by sequence homology. Here we show that three TbORC1/CDC6-interacting factors also act in T. brucei nuclear DNA replication and demonstrate that TbORC1/CDC6 interacts in a high molecular complex in which a diverged Orc4 homologue and one replicative helicase subunit can also be found. Analysing the subcellular localization of four TbORC1/CDC6-interacting factors during the cell cycle reveals that one factor, TbORC1B, is not a static constituent of ORC but displays S-phase restricted nuclear localization and expression, suggesting it positively regulates replication. This work shows that ORC architecture and regulation are diverged features of DNA replication initiation in T. brucei, providing new insight into this key stage of eukaryotic genome copying. PMID:26951375

Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex.

PubMed

Jaiswal, Rahul; Singh, Samarendra K; Bastia, Deepak; Escalante, Carlos R

2015-04-01

The Reb1 protein from Schizosaccharomyces pombe is a member of a family of proteins that control programmed replication termination and/or transcription termination in eukaryotic cells. These events occur at naturally occurring replication fork barriers (RFBs), where Reb1 binds to termination (Ter) DNA sites and coordinates the polar arrest of replication forks and transcription approaching in opposite directions. The Reb1 DNA-binding and replication-termination domain was expressed in Escherichia coli, purified and crystallized in complex with a 26-mer DNA Ter site. Batch crystallization under oil was required to produce crystals of good quality for data collection. Crystals grew in space group P2₁, with unit-cell parameters a = 68.9, b = 162.9, c = 71.1 Å, β = 94.7°. The crystals diffracted to a resolution of 3.0 Å. The crystals were mosaic and required two or three cycles of annealing. This study is the first to yield structural information about this important family of proteins and will provide insights into the mechanism of replication and transcription termination.

Transfer of Herb-Resistance Plasmid From Escherichia coli to Staphylococcus aureus Residing in the Human Urinary Tract

PubMed Central

Tong, Yan Qing; Xin, Bing; Zhu, Li

2014-01-01

Background: Plasmid transfer among bacteria provides a means for dissemination of resistance. Plasmid Analysis has made it possible to track plasmids that induce resistance in bacterial population. Objectives: To screen the presence of herb-resistance plasmid in Escherichia coli strains and determine the transferability of this resistance plasmid directly from E. coli to the Gram-positive, Staphylococcus aureus. Materials and Methods: The donor strain E. coli CP9 and recipient strain S. aureus RN450RF were isolated from UTI patients. E. coli CP9 was highly resistant to herbal concoction. Isolates of S. aureus RN450RF were fully susceptible. Total plasmid DNA was prepared and transferred into E. coli DH5α. Transconjugants were selected on agar plates containing serial dilutions of herbal concoction. Resistance plasmid was transferred to susceptible S. aureus RN450RFin triple replicas. The mating experiments were repeated twice. Results: The identified 45 kb herb-resistance plasmid could be transferred from E. coli CP9 isolates to E. coli DH5α. As a consequence E. coli DH5α transconjugant MIC increased from 0.0125 g/mL to 0.25 g/mL. The plasmid was easily transferred from E. coli CP9 strain to S. aureus RN450RF with a mean transfer rate of 1×10-2 transconjugants/recipient. The E. coli donor and the S. aureus RN450RF transconjugant contained a plasmid of the same size, which was absent in the recipient before mating. Susceptibility testing showed that the S. aureus RN450RF transconjugant was resistant to herbal concoction. Conclusions: E. coli herb-resistance plasmid can replicate and be expressed in S. aureus. PMID:25147679

Novel Chromosome Organization Pattern in Actinomycetales-Overlapping Replication Cycles Combined with Diploidy.

PubMed

Böhm, Kati; Meyer, Fabian; Rhomberg, Agata; Kalinowski, Jörn; Donovan, Catriona; Bramkamp, Marc

2017-06-06

Bacteria regulate chromosome replication and segregation tightly with cell division to ensure faithful segregation of DNA to daughter generations. The underlying mechanisms have been addressed in several model species. It became apparent that bacteria have evolved quite different strategies to regulate DNA segregation and chromosomal organization. We have investigated here how the actinobacterium Corynebacterium glutamicum organizes chromosome segregation and DNA replication. Unexpectedly, we found that C. glutamicum cells are at least diploid under all of the conditions tested and that these organisms have overlapping C periods during replication, with both origins initiating replication simultaneously. On the basis of experimental data, we propose growth rate-dependent cell cycle models for C. glutamicum IMPORTANCE Bacterial cell cycles are known for few model organisms and can vary significantly between species. Here, we studied the cell cycle of Corynebacterium glutamicum , an emerging cell biological model organism for mycolic acid-containing bacteria, including mycobacteria. Our data suggest that C. glutamicum carries two pole-attached chromosomes that replicate with overlapping C periods, thus initiating a new round of DNA replication before the previous one is terminated. The newly replicated origins segregate to midcell positions, where cell division occurs between the two new origins. Even after long starvation or under extremely slow-growth conditions, C. glutamicum cells are at least diploid, likely as an adaptation to environmental stress that may cause DNA damage. The cell cycle of C. glutamicum combines features of slow-growing organisms, such as polar origin localization, and fast-growing organisms, such as overlapping C periods. Copyright © 2017 Böhm et al.

Molecular mechanism of DNA replication-coupled inactivation of the initiator protein in Escherichia coli: interaction of DnaA with the sliding clamp-loaded DNA and the sliding clamp-Hda complex.

PubMed

Su'etsugu, Masayuki; Takata, Makoto; Kubota, Toshio; Matsuda, Yusaku; Katayama, Tsutomu

2004-06-01

In Escherichia coli, the ATP-DnaA protein initiates chromosomal replication. After the DNA polymerase III holoenzyme is loaded on to DNA, DnaA-bound ATP is hydrolysed in a manner depending on Hda protein and the DNA-loaded form of the DNA polymerase III sliding clamp subunit, which yields ADP-DnaA, an inactivated form for initiation. This regulatory DnaA-inactivation represses extra initiation events. In this study, in vitro replication intermediates and structured DNA mimicking replicational intermediates were first used to identify structural prerequisites in the process of DnaA-ATP hydrolysis. Unlike duplex DNA loaded with sliding clamps, primer RNA-DNA heteroduplexes loaded with clamps were not associated with DnaA-ATP hydrolysis, and duplex DNA provided in trans did not rescue this defect. At least 40-bp duplex DNA is competent for the DnaA-ATP hydrolysis when a single clamp was loaded. The DnaA-ATP hydrolysis was inhibited when ATP-DnaA was tightly bound to a DnaA box-bearing oligonucleotide. These results imply that the DnaA-ATP hydrolysis involves the direct interaction of ATP-DnaA with duplex DNA flanking the sliding clamp. Furthermore, Hda protein formed a stable complex with the sliding clamp. Based on these, we suggest a mechanical basis in the DnaA-inactivation that ATP-DnaA interacts with the Hda-clamp complex with the aid of DNA binding. Copyright Blackwell Publishing Limited

Chromosome demise in the wake of ligase-deficient replication.

PubMed

Kouzminova, Elena A; Kuzminov, Andrei

2012-06-01

Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. © 2012 Blackwell Publishing Ltd.

Role for a region of helically unstable DNA within the Epstein-Barr virus latent cycle origin of DNA replication oriP in origin function

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polonskaya, Zhanna; Benham, Craig J.; Hearing, Janet

The minimal replicator of the Epstein-Barr virus (EBV) latent cycle origin of DNA replication oriP is composed of two binding sites for the Epstein-Barr virus nuclear antigen-1 (EBNA-1) and flanking inverted repeats that bind the telomere repeat binding factor TRF2. Although not required for minimal replicator activity, additional binding sites for EBNA-1 and TRF2 and one or more auxiliary elements located to the right of the EBNA-1/TRF2 sites are required for the efficient replication of oriP plasmids. Another region of oriP that is predicted to be destabilized by DNA supercoiling is shown here to be an important functional component ofmore » oriP. The ability of DNA fragments of unrelated sequence and possessing supercoiled-induced DNA duplex destabilized (SIDD) structures, but not fragments characterized by helically stable DNA, to substitute for this component of oriP demonstrates a role for the SIDD region in the initiation of oriP-plasmid DNA replication.« less

Differential Chromatin Structure Encompassing Replication Origins in Transformed and Normal Cells

PubMed Central

Di Paola, Domenic; Rampakakis, Emmanouil; Chan, Man Kid

2012-01-01

This study examines the chromatin structure encompassing replication origins in transformed and normal cells. Analysis of the global levels of histone H3 acetylated at K9&14 (open chromatin) and histone H3 trimethylated at K9 (closed chromatin) revealed a higher ratio of open to closed chromatin in the transformed cells. Also, the trithorax and polycomb group proteins, Brg-1 and Bmi-1, respectively, were overexpressed and more abundantly bound to chromatin in the transformed cells. Quantitative comparative analyses of episomal and in situ chromosomal replication origin activity as well as chromatin immunoprecipitation (ChIP) assays, using specific antibodies targeting members of the pre-replication complex (pre-RC) as well as open/closed chromatin markers encompassing both episomal and chromosomal origins, revealed that episomal origins had similar levels of in vivo activity, nascent DNA abundance, pre-RC protein association, and elevated open chromatin structure at the origin in both cell types. In contrast, the chromosomal origins corresponding to 20mer1, 20mer2, and c-myc displayed a 2- to 3-fold higher activity and pre-RC protein abundance as well as higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in the transformed cells, whereas the origin associated with the housekeeping lamin B2 gene exhibited similar levels of activity, pre-RC protein abundance, and higher ratios of open to closed chromatin and of Brg-1 to Bmi-1 in both cell types. Nucleosomal positioning analysis, using an MNase-Southern blot assay, showed that all the origin regions examined were situated within regions of inconsistently positioned nucleosomes, with the nucleosomes being spaced farther apart from each other prior to the onset of S phase in both cell types. Overall, the results indicate that cellular transformation is associated with differential epigenetic regulation, whereby chromatin structure is more open, rendering replication origins more accessible to initiator proteins, thus allowing increased origin activity. PMID:23050047

Construction of a novel gene bank of Bacillus subtilis using a low copy number vector in Escherichia coli.

PubMed

Hasnain, S; Thomas, C M

1986-07-01

Low copy number vector plasmid pCT571 was constructed to clone Bacillus subtilis genomic fragments in Escherichia coli. pCT571 confers KmR, TcR and CmR in E. coli and CmR in B. subtilis. It has unique restriction sites within the KmR and TcR markers to allow screening for recombinant plasmids by insertional inactivation of these genes. It contains the pSC101 replicon and replicates normally at six to eight copies per chromosome equivalent in E. coli. It also contains oriVRK2, which when supplied with the product of the trfA gene of RK2 in trans, allows pCT571 to replicate at 35-40 copies per chromosome equivalent. A B. subtilis gene bank was created by cloning partially Sau3A-digested and size-fractionated fragments of B. subtilis chromosomal DNA into the BamHI site of pCT571. DNA from 1097 KmR TcS transformants was extracted and analysed electrophoretically as supercoiled DNA and after digesting with EcoRI or EcoRI and SalI. Approximately 1000 hybrid plasmids were found with reasonably sized B. subtilis fragments. The mean size of the inserts in pCT571 is 8 kb, ranging from 4 to 20 kb in different plasmids. The gene bank covers most of the B. subtilis chromosome, as demonstrated by the results of screening the gene bank for selectable nutritional markers in E. coli and B. subtilis. Hybrid plasmids which complement E. coli mutants for arg, his, lys, met, pdx, pyr and thr markers were identified from the gene bank. In B. subtilis the presence of argC, cysA, dal, hisA, ilvA, leuA, lys, metB, metC, phe, purA, purB, thr and trpC was established by transformation experiments. The effects of copy number on cloning and long-term maintenance in the bacterial strains were also investigated. At high copy number some hybrid plasmids cannot be maintained at all, while others show an increased rate of structural deletions and rearrangements.

Development of a new fluorescent reporter:operator system: location of AraC regulated genes in Escherichia coli K-12.

PubMed

Sellars, Laura E; Bryant, Jack A; Sánchez-Romero, María-Antonia; Sánchez-Morán, Eugenio; Busby, Stephen J W; Lee, David J

2017-08-03

In bacteria, many transcription activator and repressor proteins regulate multiple transcription units that are often distally distributed on the bacterial genome. To investigate the subcellular location of DNA bound proteins in the folded bacterial nucleoid, fluorescent reporters have been developed which can be targeted to specific DNA operator sites. Such Fluorescent Reporter-Operator System (FROS) probes consist of a fluorescent protein fused to a DNA binding protein, which binds to an array of DNA operator sites located within the genome. Here we have developed a new FROS probe using the Escherichia coli MalI transcription factor, fused to mCherry fluorescent protein. We have used this in combination with a LacI repressor::GFP protein based FROS probe to assess the cellular location of commonly regulated transcription units that are distal on the Escherichia coli genome. We developed a new DNA binding fluorescent reporter, consisting of the Escherichia coli MalI protein fused to the mCherry fluorescent protein. This was used in combination with a Lac repressor:green fluorescent protein fusion to examine the spatial positioning and possible co-localisation of target genes, regulated by the Escherichia coli AraC protein. We report that induction of gene expression with arabinose does not result in co-localisation of AraC-regulated transcription units. However, measurable repositioning was observed when gene expression was induced at the AraC-regulated promoter controlling expression of the araFGH genes, located close to the DNA replication terminus on the chromosome. Moreover, in dividing cells, arabinose-induced expression at the araFGH locus enhanced chromosome segregation after replication. Regions of the chromosome regulated by AraC do not colocalise, but transcription events can induce movement of chromosome loci in bacteria and our observations suggest a role for gene expression in chromosome segregation.

A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae.

PubMed Central

Ehrenhofer-Murray, A E; Kamakaka, R T; Rine, J

1999-01-01

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed. PMID:10545450

Fragile genomic sites are associated with origins of replication.

PubMed

Di Rienzi, Sara C; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-09-09

Genome rearrangements are mediators of evolution and disease. Such rearrangements are frequently bounded by transfer RNAs (tRNAs), transposable elements, and other repeated elements, suggesting a functional role for these elements in creating or repairing breakpoints. Though not well explored, there is evidence that origins of replication also colocalize with breakpoints. To investigate a potential correlation between breakpoints and origins, we analyzed evolutionary breakpoints defined between Saccharomyces cerevisiae and Kluyveromyces waltii and S. cerevisiae and a hypothetical ancestor of both yeasts, as well as breakpoints reported in the experimental literature. We find that origins correlate strongly with both evolutionary breakpoints and those described in the literature. Specifically, we find that origins firing earlier in S phase are more strongly correlated with breakpoints than are later-firing origins. Despite origins being located in genomic regions also bearing tRNAs and Ty elements, the correlation we observe between origins and breakpoints appears to be independent of these genomic features. This study lays the groundwork for understanding the mechanisms by which origins of replication may impact genome architecture and disease.

Outsider to insider: resetting the natural host niche of commensal E. coli K-12.

PubMed

Sahu, Upasana; Kar, Sudeshna

2012-01-01

The status of E. coli K-12 as an exclusively non-invasive, non-pathogenic bacterium has almost been incontrovertible. Our recent finding that a mutation in one of its main architectural protein, HU, converts E. coli K-12 to an actively invasive form suggests that gaining host cell entry might be an expedient survival tactic for traditional commensals during certain altered host conditions. The mutant E. coli (SK3842) exhibits properties usually associated with pathogenic bacteria: host cell invasion, phagosomal disruption and intracellular replication. However, unlike the situation with some pathogens, internalized SK3842 imparts anti-apoptotic and cyto-protective effects rather than lethality on the host cell, both in vitro and in vivo. Here, we show that SK3842 also provides colonization resistance against other invasive pathogens--a trait not shared by the parental commensal strain. Thus, the altered lifestyle of SK3842 encompasses characteristics both from traditional pathogens as well as beneficial probiotic strains.

Asymmetric Assembly of Merkel Cell Polyomavirus Large T-Antigen Origin Binding Domains at the Viral Origin

DOE Office of Scientific and Technical Information (OSTI.GOV)

C Harrison; G Meinke; H Kwun

2011-12-31

The double-stranded DNA polyomavirus Merkel cell polyomavirus (MCV) causes Merkel cell carcinoma, an aggressive but rare human skin cancer that most often affects immunosuppressed and elderly persons. As in other polyomaviruses, the large T-antigen of MCV recognizes the viral origin of replication by binding repeating G(A/G)GGC pentamers. The spacing, number, orientation, and necessity of repeats for viral replication differ, however, from other family members such as SV40 and murine polyomavirus. We report here the 2.9 {angstrom} crystal structure of the MCV large T-antigen origin binding domain (OBD) in complex with a DNA fragment from the MCV origin of replication. Consistentmore » with replication data showing that three of the G(A/G)GGC-like binding sites near the center of the origin are required for replication, the crystal structure contains three copies of the OBD. This stoichiometry was verified using isothermal titration calorimetry. The affinity for G(A/G)GGC-containing double-stranded DNA was found to be {approx} 740 nM, approximately 8-fold weaker than the equivalent domain in SV40 for the analogous region of the SV40 origin. The difference in affinity is partially attributable to DNA-binding residue Lys331 (Arg154 in SV40). In contrast to SV40, a small protein-protein interface is observed between MCV OBDs when bound to the central region of the origin. This protein-protein interface is reminiscent of that seen in bovine papilloma virus E1 protein. Mutational analysis indicates, however, that this interface contributes little to DNA binding energy.« less

Prokaryotic and eukaryotic DNA helicases. Essential molecular motor proteins for cellular machinery.

PubMed

Tuteja, Narendra; Tuteja, Renu

2004-05-01

DNA helicases are ubiquitous molecular motor proteins which harness the chemical free energy of ATP hydrolysis to catalyze the unwinding of energetically stable duplex DNA, and thus play important roles in nearly all aspects of nucleic acid metabolism, including replication, repair, recombination, and transcription. They break the hydrogen bonds between the duplex helix and move unidirectionally along the bound strand. All helicases are also translocases and DNA-dependent ATPases. Most contain conserved helicase motifs that act as an engine to power DNA unwinding. All DNA helicases share some common properties, including nucleic acid binding, NTP binding and hydrolysis, and unwinding of duplex DNA in the 3' to 5' or 5' to 3' direction. The minichromosome maintenance (Mcm) protein complex (Mcm4/6/7) provides a DNA-unwinding function at the origin of replication in all eukaryotes and may act as a licensing factor for DNA replication. The RecQ family of helicases is highly conserved from bacteria to humans and is required for the maintenance of genome integrity. They have also been implicated in a variety of human genetic disorders. Since the discovery of the first DNA helicase in Escherichia coli in 1976, and the first eukaryotic one in the lily in 1978, a large number of these enzymes have been isolated from both prokaryotic and eukaryotic systems, and the number is still growing. In this review we cover the historical background of DNA helicases, helicase assays, biochemical properties, prokaryotic and eukaryotic DNA helicases including Mcm proteins and the RecQ family of helicases. The properties of most of the known DNA helicases from prokaryotic and eukaryotic systems, including viruses and bacteriophages, are summarized in tables.

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

PubMed

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

PubMed Central

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Molecular homogeneity of heat-stable enterotoxins produced by bovine enterotoxigenic Escherichia coli.

PubMed Central

Saeed, A M; Magnuson, N S; Sriranganathan, N; Burger, D; Cosand, W

1984-01-01

Heat-stable enterotoxins (STs) from four strains of bovine enterotoxigenic Escherichia coli representing four serogroups were purified to homogeneity by utilizing previously published purification schemata. Biochemical characterization of the purified STs showed that they met the basic criteria for the heat-stable enterotoxins of E. coli. Amino acid analysis of the purified STs revealed that they were peptides of identical amino acid composition. This composition consisted of 18 residues of 10 different amino acids, 6 of which were cysteine. The amino acid composition of the four ST peptides was identical to that reported for the STs of human and porcine E. coli. In addition, complete sequence analysis of two of the ST peptides and partial sequencing of several others revealed strong homology to the sequences of STs from human and porcine E. coli and to the sequence predicted from the last 18 codons of the transposon Tn1681. There was also substantial homology to the sequence predicted from the ST-coding genetic element of human E. coli, which may indicate the existence of identical bioactive configuration among ST peptides of E. coli strains of various host origins. These data support the hypothesis that STs produced by human, bovine, and porcine E. coli are coded by a closely related genetic element which may have originated from a single, widely disseminated transposon. Images PMID:6376355

Interpreting the Dependence of Mutation Rates on Age and Time

PubMed Central

Gao, Ziyue; Wyman, Minyoung J.; Sella, Guy; Przeworski, Molly

2016-01-01

Mutations can originate from the chance misincorporation of nucleotides during DNA replication or from DNA lesions that arise between replication cycles and are not repaired correctly. We introduce a model that relates the source of mutations to their accumulation with cell divisions, providing a framework for understanding how mutation rates depend on sex, age, and cell division rate. We show that the accrual of mutations should track cell divisions not only when mutations are replicative in origin but also when they are non-replicative and repaired efficiently. One implication is that observations from diverse fields that to date have been interpreted as pointing to a replicative origin of most mutations could instead reflect the accumulation of mutations arising from endogenous reactions or exogenous mutagens. We further find that only mutations that arise from inefficiently repaired lesions will accrue according to absolute time; thus, unless life history traits co-vary, the phylogenetic “molecular clock” should not be expected to run steadily across species. PMID:26761240

The Alleged Crisis and the Illusion of Exact Replication.

PubMed

Stroebe, Wolfgang; Strack, Fritz

2014-01-01

There has been increasing criticism of the way psychologists conduct and analyze studies. These critiques as well as failures to replicate several high-profile studies have been used as justification to proclaim a "replication crisis" in psychology. Psychologists are encouraged to conduct more "exact" replications of published studies to assess the reproducibility of psychological research. This article argues that the alleged "crisis of replicability" is primarily due to an epistemological misunderstanding that emphasizes the phenomenon instead of its underlying mechanisms. As a consequence, a replicated phenomenon may not serve as a rigorous test of a theoretical hypothesis because identical operationalizations of variables in studies conducted at different times and with different subject populations might test different theoretical constructs. Therefore, we propose that for meaningful replications, attempts at reinstating the original circumstances are not sufficient. Instead, replicators must ascertain that conditions are realized that reflect the theoretical variable(s) manipulated (and/or measured) in the original study. © The Author(s) 2013.

Controlled progressive innate immune stimulation regimen prevents the induction of sickness behavior in the open field test

PubMed Central

Chen, Qun; Tarr, Andrew J; Liu, Xiaoyu; Wang, Yufen; Reed, Nathaniel S; DeMarsh, Cameron P; Sheridan, John F; Quan, Ning

2013-01-01

Peripheral immune activation by bacterial mimics or live replicating pathogens is well known to induce central nervous system activation. Sickness behavior alterations are often associated with inflammation-induced increases in peripheral proinflammatory cytokines (eg, interleukin [IL]-1β and IL-6). However, most researchers have used acute high dose endotoxin/bacterial challenges to observe these outcomes. Using this methodology may pose inherent risks in the translational interpretation of the experimental data in these studies. Studies using Escherichia coli have yet to establish the full kinetics of repeated E. coli peripheral injections. Therefore, we sought to examine the effects of repeated low dose E. coli on sickness behavior and local peripheral inflammation in the open field test. Results from the current experiments showed a behavioral dose response, where increased amounts of E. coli resulted in correspondingly increased sickness behavior. Furthermore, animals that received a subthreshold dose (ie, one that did not cause sickness behavior) of E. coli 24 hours prior were able to withstand a larger dose of E. coli on the second day (a dose that would normally cause sickness behavior in mice without prior exposure) without inducing sickness behavior. In addition, animals that received escalating subthreshold doses of E. coli on days 1 and 2 behaviorally tolerated a dose of E. coli 25 times higher than what would normally cause sickness behavior if given acutely. Lastly, increased levels of E. coli caused increased IL-6 and IL-1β protein expression in the peritoneal cavity, and this increase was blocked by administering a subthreshold dose of E. coli 24 hours prior. These data show that progressive challenges with subthreshold levels of E. coli may obviate the induction of sickness behavior and proinflammatory cytokine expression. PMID:23950656

Controlled progressive innate immune stimulation regimen prevents the induction of sickness behavior in the open field test.

PubMed

Chen, Qun; Tarr, Andrew J; Liu, Xiaoyu; Wang, Yufen; Reed, Nathaniel S; Demarsh, Cameron P; Sheridan, John F; Quan, Ning

2013-01-01

Peripheral immune activation by bacterial mimics or live replicating pathogens is well known to induce central nervous system activation. Sickness behavior alterations are often associated with inflammation-induced increases in peripheral proinflammatory cytokines (eg, interleukin [IL]-1β and IL-6). However, most researchers have used acute high dose endotoxin/bacterial challenges to observe these outcomes. Using this methodology may pose inherent risks in the translational interpretation of the experimental data in these studies. Studies using Escherichia coli have yet to establish the full kinetics of repeated E. coli peripheral injections. Therefore, we sought to examine the effects of repeated low dose E. coli on sickness behavior and local peripheral inflammation in the open field test. Results from the current experiments showed a behavioral dose response, where increased amounts of E. coli resulted in correspondingly increased sickness behavior. Furthermore, animals that received a subthreshold dose (ie, one that did not cause sickness behavior) of E. coli 24 hours prior were able to withstand a larger dose of E. coli on the second day (a dose that would normally cause sickness behavior in mice without prior exposure) without inducing sickness behavior. In addition, animals that received escalating subthreshold doses of E. coli on days 1 and 2 behaviorally tolerated a dose of E. coli 25 times higher than what would normally cause sickness behavior if given acutely. Lastly, increased levels of E. coli caused increased IL-6 and IL-1β protein expression in the peritoneal cavity, and this increase was blocked by administering a subthreshold dose of E. coli 24 hours prior. These data show that progressive challenges with subthreshold levels of E. coli may obviate the induction of sickness behavior and proinflammatory cytokine expression.

Life in the Clouds of Venus? An Experimental Synthetic Biology Approach

NASA Technical Reports Server (NTRS)

Rothschild, L. J.; Paulino-Lima, I. G.; Amatya, D.; Bajar, B.; Geilich, B.; Hu, J.; Jackson, C. J.

2015-01-01

The surface of Venus constitutes the most hellish and biologically inhospitable planetary surface in our solar system, boasting a pH of 0, blistering winds that can melt lead, and pressures of 60 atm. However, during the earlier years of the solar system, without the runaway greenhouse effect that has plagued the planet, Venus potentially housed oceans and perhaps even life. There is a possibility that microbes could have retreated into hospitable niches in the atmosphere, as suggested by Carl Sagan as early as 1967 [1]. For example, 50 km above the raging hell of the Venusian surface, exists a relatively temperate environment that might serve as reservoir for life. This astrobiology project seeks to explore life at the extremes and to theorize whether microbial communities could not only survive but also reproduce in the Venusian atmosphere. Specifically, we ask: are aerosols viable microbial environments? But before we can test for life in the clouds, we have to develop a proper reporter to visualize cell growth in situ. For this purpose, we aimed to develop cell-growth dependent reporters to serve as remote biosensors for cell growth. We developed two using the polA promoter, a DNA-replication dependent promoter, and nrd operon promoter, a cell-cycle dependent promoter. Using these cell-growth reporters, the next step is to aerosolize microbes expressing these reporters in a suspension chamber adapted from a Millikan Drop Apparatus to assay reproduction in an aerosolized environment. Better yet is to test the reproduction of microbes in a microgravity regime such as on ISS.Approach: We engineered two cell-cycle dependent genetic reporters. One was the polA promoter which codes for DNA Polymerase I, a gene active in DNA replication [2]. The other was the nrdP. The activation of ribonucleotide reductase reduces ribonucleotides into deoxyribonucleotides and is involved in the bacterial cell cycle [3]. This promoter began activation during the initiation of DNA replication and is cell-cycle dependent [4]. These promoters were fused to a GFP reporter, transformed into E. coli. The constructs were deposited in the iGEM registry as K847210: Escherichia coli DNA-replication dependent polA promoter K847211: Escherichia coli cell-division dependent nrd promoter Results: Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrdP-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polAP-E0840 construct (which uses mut3b GFP) exhibited low expression; while fluorescence was visible under the microscope, the signal was too weak for the camera to recognize. The polA promoter was therefore digest-ed with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness [5] Fluoresence time course data demonsrated that the genes were induced in a cell cycle dependant manner [6]. Our assays via microscopy and the bulk assay shows that our promoters are functional as cell cycle reporters.Conclusions: The application of such tools are widespread and not limited to astrobiology; nrdP could be used to determine doubling times empirically and could possibly extrapolate DNA content from intensity of signals expressed by polAP. However, we are pri-marily interested in its use in astrobiology.

DNA sequence divergence among derivatives of Escherichia coli K-12 detected by arbitrary primer PCR (random amplified polymorphic DNA) fingerprinting.

PubMed Central

Brikun, I; Suziedelis, K; Berg, D E

1994-01-01

Derivatives of Escherichia coli K-12 of known ancestry were characterized by random amplified polymorphic DNA (RAPD) fingerprinting to better understand genome evolution in this family of closely related strains. This sensitive method entails PCR amplification with arbitrary primers at low stringency and yields arrays of anonymous DNA fragments that are strain specific. Among 150 fragments scored, eight were polymorphic in that they were produced from some but not all strains. Seven polymorphic bands were chromosomal, and one was from the F-factor plasmid. Five of the six mapped polymorphic chromosomal bands came from just 7% of the genome, a 340-kb segment that includes the terminus of replication. Two of these were from the cryptic Rac prophage, and the inability to amplify them from strains was attributable to deletion (excision) or to rearrangement of Rac. Two other terminus-region segments that resulted in polymorphic bands appeared to have sustained point mutations that affected the ability to amplify them. Control experiments showed that RAPD bands from the 340-kb terminus-region segment and also from two plasmids (P1 and F) were represented in approximate proportion to their size. Optimization experiments showed that the concentration of thermostable polymerase strongly affected the arrays of RAPD products obtained. Comparison of RAPD polymorphisms and positions of strains exhibiting them in the pedigree suggests that many sequence changes occurred in these historic E. coli strains during their storage. We propose that the clustering of such mutations near the terminus reflects errors during completion of chromosome replication, possibly during slow growth in the stab cultures that were often used to store E. coli strains in the early years of bacterial genetics. Images PMID:8132463

More than just a metabolic regulator - elucidation and validation of new targets of PdhR in Escherichia coli

PubMed Central

2011-01-01

Background The pyruvate dehydrogenase regulator protein (PdhR) of Escherichia coli acts as a transcriptional regulator in a pyruvate dependent manner to control central metabolic fluxes. However, the complete PdhR regulon has not yet been uncovered. To achieve an extended understanding of its gene regulatory network, we combined large-scale network inference and experimental verification of results obtained by a systems biology approach. Results 22 new genes contained in two operons controlled by PdhR (previously only 20 regulatory targets in eight operons were known) were identified by analysing a large-scale dataset of E. coli from the Many Microbes Microarray Database and novel expression data from a pdhR knockout strain, as well as a PdhR overproducing strain. We identified a regulation of the glycolate utilization operon glcDEFGBA using chromatin immunoprecipitation and gel shift assays. We show that this regulation could be part of a cross-induction between genes necessary for acetate and pyruvate utilisation controlled through PdhR. Moreover, a link of PdhR regulation to the replication machinery of the cell via control of the transcription of the dcw-cluster was verified in experiments. This augments our knowledge of the functions of the PdhR-regulon and demonstrates its central importance for further cellular processes in E. coli. Conclusions We extended the PdhR regulon by 22 new genes contained in two operons and validated the regulation of the glcDEFGBA operon for glycolate utilisation and the dcw-cluster for cell division proteins experimentally. Our results provide, for the first time, a plausible regulatory link between the nutritional status of the cell and cell replication mediated by PdhR. PMID:22168595

Analysis of re-replication from deregulated origin licensing by DNA fiber spreading

PubMed Central

Dorn, Elizabeth S.; Chastain, Paul D.; Hall, Jonathan R.; Cook, Jeanette Gowen

2009-01-01

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability. PMID:19010964

Intrinsically bent DNA in replication origins and gene promoters.

PubMed

Gimenes, F; Takeda, K I; Fiorini, A; Gouveia, F S; Fernandez, M A

2008-06-24

Intrinsically bent DNA is an alternative conformation of the DNA molecule caused by the presence of dA/dT tracts, 2 to 6 bp long, in a helical turn phase DNA or with multiple intervals of 10 to 11 bp. Other than flexibility, intrinsic bending sites induce DNA curvature in particular chromosome regions such as replication origins and promoters. Intrinsically bent DNA sites are important in initiating DNA replication, and are sometimes found near to regions associated with the nuclear matrix. Many methods have been developed to localize bent sites, for example, circular permutation, computational analysis, and atomic force microscopy. This review discusses intrinsically bent DNA sites associated with replication origins and gene promoter regions in prokaryote and eukaryote cells. We also describe methods for identifying bent DNA sites for circular permutation and computational analysis.

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

PubMed Central

Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

2013-01-01

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity. PMID:23564203

DNA replication checkpoint signaling depends on a Rad53-Dbf4 N-terminal interaction in Saccharomyces cerevisiae.

PubMed

Chen, Ying-Chou; Kenworthy, Jessica; Gabrielse, Carrie; Hänni, Christine; Zegerman, Philip; Weinreich, Michael

2013-06-01

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53-Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4-Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53-Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.

Novel features of ARS selection in budding yeast Lachancea kluyveri

PubMed Central

2011-01-01

Background The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. Results In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. Conclusions Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs. PMID:22204614

Novel features of ARS selection in budding yeast Lachancea kluyveri.

PubMed

Liachko, Ivan; Tanaka, Emi; Cox, Katherine; Chung, Shau Chee Claire; Yang, Lu; Seher, Arael; Hallas, Lindsay; Cha, Eugene; Kang, Gina; Pace, Heather; Barrow, Jasmine; Inada, Maki; Tye, Bik-Kwoon; Keich, Uri

2011-12-28

The characterization of DNA replication origins in yeast has shed much light on the mechanisms of initiation of DNA replication. However, very little is known about the evolution of origins or the evolution of mechanisms through which origins are recognized by the initiation machinery. This lack of understanding is largely due to the vast evolutionary distances between model organisms in which origins have been examined. In this study we have isolated and characterized autonomously replicating sequences (ARSs) in Lachancea kluyveri - a pre-whole genome duplication (WGD) budding yeast. Through a combination of experimental work and rigorous computational analysis, we show that L. kluyveri ARSs require a sequence that is similar but much longer than the ARS Consensus Sequence well defined in Saccharomyces cerevisiae. Moreover, compared with S. cerevisiae and K. lactis, the replication licensing machinery in L. kluyveri seems more tolerant to variations in the ARS sequence composition. It is able to initiate replication from almost all S. cerevisiae ARSs tested and most Kluyveromyces lactis ARSs. In contrast, only about half of the L. kluyveri ARSs function in S. cerevisiae and less than 10% function in K. lactis. Our findings demonstrate a replication initiation system with novel features and underscore the functional diversity within the budding yeasts. Furthermore, we have developed new approaches for analyzing biologically functional DNA sequences with ill-defined motifs.

MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

PubMed

Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

2016-01-01

The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes.

PubMed

Lo, Andy; Weiner, Joel H; Li, Liang

2013-09-17

Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed. Copyright © 2013 Elsevier B.V. All rights reserved.

Resistance of Pseudomonas aeruginosa PAO to nalidixic acid and low levels of beta-lactam antibiotics: mapping of chromosomal genes.

PubMed Central

Rella, M; Haas, D

1982-01-01

Resistance to high concentrations of nalidixic acid in Pseudomonas aeruginosa PAO was due to mutations in one locus designated nalA, which was mapped by transduction between hex-9001 and leu-10. The nalA mutants were cross-resistant to pipemidic acid, a nalidixic acid analog, at relatively low concentrations. Replicative DNA synthesis was resistant to both drugs in permeabilized cells of nalA mutants. A locus coding for low-level resistance to nalidixic acid, nalB, was cotransducible with pyrB, proC, and met-28. The nalB mutants were also resistant to low levels of pipemidic acid, novobiocin, and beta-lactam antibiotics (e.g., carbenicillin, azlocillin, and cefsulodin), but not to other drugs, such as gentamicin, rifampin, kanamycin, or tetracycline. In nalB mutants, DNA replication showed wild-type sensitivity to nalidixic acid, whereas carbenicillin-induced filamentation required higher drug levels than in the wild-type strain. Thus, nalB mutations appear to decrease cell permeability to some antibiotics. The sensitivity of replicative DNA synthesis to nalidixic acid and novobiocin was very similar in P. aeruginosa and Escherichia coli; by contrast, the concentrations of these drugs needed to inhibit growth of P. aeruginosa were higher than those reported for E. coli by one or two orders of magnitude. PMID:6821455

Characterization of a Theta-Type Plasmid from Lactobacillus sakei: a Potential Basis for Low-Copy-Number Vectors in Lactobacilli

PubMed Central

Alpert, Carl-Alfred; Crutz-Le Coq, Anne-Marie; Malleret, Christine; Zagorec, Monique

2003-01-01

The complete nucleotide sequence of the 13-kb plasmid pRV500, isolated from Lactobacillus sakei RV332, was determined. Sequence analysis enabled the identification of genes coding for a putative type I restriction-modification system, two genes coding for putative recombinases of the integrase family, and a region likely involved in replication. The structural features of this region, comprising a putative ori segment containing 11- and 22-bp repeats and a repA gene coding for a putative initiator protein, indicated that pRV500 belongs to the pUCL287 subfamily of theta-type replicons. A 3.7-kb fragment encompassing this region was fused to an Escherichia coli replicon to produce the shuttle vector pRV566 and was observed to be functional in L. sakei for plasmid replication. The L. sakei replicon alone could not support replication in E. coli. Plasmid pRV500 and its derivative pRV566 were determined to be at very low copy numbers in L. sakei. pRV566 was maintained at a reasonable rate over 20 generations in several lactobacilli, such as Lactobacillus curvatus, Lactobacillus casei, and Lactobacillus plantarum, in addition to L. sakei, making it an interesting basis for developing vectors. Sequence relationships with other plasmids are described and discussed. PMID:12957947

[Single-molecule detection and characterization of DNA replication based on DNA origami].

PubMed

Wang, Qi; Fan, Youjie; Li, Bin

2014-08-01

To investigate single-molecule detection and characterization of DNA replication. Single-stranded DNA (ssDNA) as the template of DNA replication was attached to DNA origami by a hybridization reaction based on the complementary base-pairing principle. DNA replication catalyzed by E.coli DNA polymerase I Klenow Fragment (KF) was detected using atomic force microscopy (AFM). The height variations between the ssDNA and the double-stranded DNA (dsDNA), the distribution of KF during DNA replication and biotin-streptavidin (BA) complexes on the DNA strand after replication were detected. Agarose gel electrophoresis was employed to analyze the changes in the DNA after replication. The designed ssDNA could be anchored on the target positions of over 50% of the DNA origami. The KF was capable of binding to the ssDNA fixed on DNA origami and performing its catalytic activities, and was finally dissociated from the DNA after replication. The height of DNA strand increased by about 0.7 nm after replication. The addition of streptavidin also resulted in an DNA height increase to about 4.9 nm due to the formation of BA complexes on the biotinylated dsDNA. The resulting dsDNA and BA complex were subsequently confirmed by agarose gel electrophoresis. The combination of AFM and DNA origami allows detection and characterization of DNA replication at the single molecule level, and this approach provides better insights into the mechanism of DNA polymerase and the factors affecting DNA replication.

Household Microbial Water Quality Testing in a Peruvian Demographic and Health Survey: Evaluation of the Compartment Bag Test for Escherichia coli.

PubMed

Wang, Alice; McMahan, Lanakila; Rutstein, Shea; Stauber, Christine; Reyes, Jorge; Sobsey, Mark D

2017-04-01

AbstractThe Joint Monitoring Program relies on household surveys to classify access to improved water sources instead of measuring microbiological quality. The aim of this research was to pilot a novel test for Escherichia coli quantification of household drinking water in the 2011 Demographic and Health Survey (DHS) in Peru. In the Compartment Bag Test (CBT), a 100-mL water sample is supplemented with chromogenic medium to support the growth of E. coli , poured into a bag with compartments, and incubated. A color change indicates E. coli growth, and the concentration of E. coli /100 mL is estimated as a most probable number. Triplicate water samples from 704 households were collected; one sample was analyzed in the field using the CBT, another replicate sample using the CBT was analyzed by reference laboratories, and one sample using membrane filtration (MF) was analyzed by reference laboratories. There were no statistically significant differences in E. coli concentrations between the field and laboratory CBT results, or when compared with MF results. These results suggest that the CBT for E. coli is an effective method to quantify fecal bacteria in household drinking water. The CBT can be incorporated into DHS and other national household surveys as a direct measure of drinking water safety based on microbial quality to better document access to safe drinking water.

Household Microbial Water Quality Testing in a Peruvian Demographic and Health Survey: Evaluation of the Compartment Bag Test for Escherichia coli

PubMed Central

Wang, Alice; McMahan, Lanakila; Rutstein, Shea; Stauber, Christine; Reyes, Jorge; Sobsey, Mark D.

2017-01-01

The Joint Monitoring Program relies on household surveys to classify access to improved water sources instead of measuring microbiological quality. The aim of this research was to pilot a novel test for Escherichia coli quantification of household drinking water in the 2011 Demographic and Health Survey (DHS) in Peru. In the Compartment Bag Test (CBT), a 100-mL water sample is supplemented with chromogenic medium to support the growth of E. coli, poured into a bag with compartments, and incubated. A color change indicates E. coli growth, and the concentration of E. coli/100 mL is estimated as a most probable number. Triplicate water samples from 704 households were collected; one sample was analyzed in the field using the CBT, another replicate sample using the CBT was analyzed by reference laboratories, and one sample using membrane filtration (MF) was analyzed by reference laboratories. There were no statistically significant differences in E. coli concentrations between the field and laboratory CBT results, or when compared with MF results. These results suggest that the CBT for E. coli is an effective method to quantify fecal bacteria in household drinking water. The CBT can be incorporated into DHS and other national household surveys as a direct measure of drinking water safety based on microbial quality to better document access to safe drinking water. PMID:28500818

PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

PubMed

Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

2013-06-14

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

Chromatin Controls DNA Replication Origin Selection, Lagging-Strand Synthesis, and Replication Fork Rates.

PubMed

Kurat, Christoph F; Yeeles, Joseph T P; Patel, Harshil; Early, Anne; Diffley, John F X

2017-01-05

The integrity of eukaryotic genomes requires rapid and regulated chromatin replication. How this is accomplished is still poorly understood. Using purified yeast replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA replication origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at replication forks. Finally, nucleosomes disrupted during replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin replication in vitro and shows how multiple chromatin factors might modulate replication fork rates in vivo. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Metabolism and Fitness of Urinary Tract Pathogens.

PubMed

Alteri, Christopher J; Mobley, Harry L T

2015-06-01

Among common infections, urinary tract infections (UTI) are the most frequently diagnosed urologic disease. The majority of UTIs are caused by uropathogenic Escherichia coli. The primary niche occupied by E. coli is the lower intestinal tract of mammals, where it resides as a beneficial component of the commensal microbiota. Although it is well-known that E. coli resides in the human intestine as a harmless commensal, specific strains or pathotypes have the potential to cause a wide spectrum of intestinal and diarrheal diseases. In contrast, extraintestinal E. coli pathotypes reside harmlessly in the human intestinal microenvironment but, upon access to sites outside of the intestine, become a major cause of human morbidity and mortality as a consequence of invasive UTI (pyelonephritis, bacteremia, or septicemia). Thus, extraintestinal pathotypes like uropathogenic E. coli (UPEC) possess an enhanced ability to cause infection outside of the intestinal tract and colonize the urinary tract, the bloodstream, or cerebrospinal fluid of human hosts. Due to the requirement for these E. coli to replicate in and colonize both the intestine and extraintestinal environments, we posit that physiology and metabolism of UPEC strains is paramount. Here we discuss that the ability to survive in the urinary tract depends as much on bacterial physiology and metabolism as it does on the well-considered virulence determinants.

Effect of storage temperatures on growth and survival of Escherichia coli O157: H7 inoculated in foods from a neotropical environment.

PubMed

Arias, M L; Monge-Rojas, R; Chaves, C; Antillón, F

2001-06-01

Escherichia coli O157: H7 has emerged as a new pathogen and is found worldwide. We studied the effect of several storage temperatures on the survival of this bacterium in common foods from a neotropical environment (Costa Rica) because at least seven clinical cases have been reported from the country, and no epidemiological link or probable food association has been described. High (10(6)-10(8) CFU/ml) and low (10(4)-10(6) CFU/ml) populations of E. coli were inoculated (three replications) in ground meat, chopped cabbage, chicken giblets and pasteurized milk and incubated at 0, 6 and 12 degrees C for 24, 48 and 72 h. Vegetables and milk were also stored at 22 degrees C for the same periods. The E. coli O157: H7 enumeration was done according to the methodology described in the Bacteriological Analytical Manual. Populations of E. coli O157: H7 showed either an increasing or decreasing trend, according to temperature, time or food base. Our data indicate that E. coli O157: H7 is capable of surviving and growing in meat, cabbage, milk and chicken giblets; food items commonly consumed by Costa Ricans.

Dormant origins as a built-in safeguard in eukaryotic DNA replication against genome instability and disease development.

PubMed

Shima, Naoko; Pederson, Kayla D

2017-08-01

DNA replication is a prerequisite for cell proliferation, yet it can be increasingly challenging for a eukaryotic cell to faithfully duplicate its genome as its size and complexity expands. Dormant origins now emerge as a key component for cells to successfully accomplish such a demanding but essential task. In this perspective, we will first provide an overview of the fundamental processes eukaryotic cells have developed to regulate origin licensing and firing. With a special focus on mammalian systems, we will then highlight the role of dormant origins in preventing replication-associated genome instability and their functional interplay with proteins involved in the DNA damage repair response for tumor suppression. Lastly, deficiencies in the origin licensing machinery will be discussed in relation to their influence on stem cell maintenance and human diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli.

PubMed

Suzuki, E; Kondo, T; Makise, M; Mima, S; Sakamoto, K; Tsuchiya, T; Mizushima, T

1998-04-01

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli, has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42 degrees C (non-permissive temperature) and at 37 degrees C (semi-permissive temperature), but not at 28 degrees C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which over-initiation of DNA replication occurs at low temperature (28 degrees C), showed a higher level of unsaturation of fatty acids at 28 degrees C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.

Replication in hydroxyurea: it's a matter of time.

PubMed

Alvino, Gina M; Collingwood, David; Murphy, John M; Delrow, Jeffrey; Brewer, Bonita J; Raghuraman, M K

2007-09-01

Hydroxyurea (HU) is a DNA replication inhibitor that negatively affects both the elongation and initiation phases of replication and triggers the "intra-S phase checkpoint." Previous work with budding yeast has shown that, during a short exposure to HU, MEC1/RAD53 prevent initiation at some late S phase origins. In this study, we have performed microarray experiments to follow the fate of all origins over an extended exposure to HU. We show that the genome-wide progression of DNA synthesis, including origin activation, follows the same pattern in the presence of HU as in its absence, although the time frames are very different. We find no evidence for a specific effect that excludes initiation from late origins. Rather, HU causes S phase to proceed in slow motion; all temporal classes of origins are affected, but the order in which they become active is maintained. We propose a revised model for the checkpoint response to HU that accounts for the continued but slowed pace of the temporal program of origin activation.

The plant cell cycle: Pre-Replication complex formation and controls

PubMed Central

Brasil, Juliana Nogueira; Costa, Carinne N. Monteiro; Cabral, Luiz Mors; Ferreira, Paulo C. G.; Hemerly, Adriana S.

2017-01-01

Abstract The multiplication of cells in all living organisms requires a tight regulation of DNA replication. Several mechanisms take place to ensure that the DNA is replicated faithfully and just once per cell cycle in order to originate through mitoses two new daughter cells that contain exactly the same information from the previous one. A key control mechanism that occurs before cells enter S phase is the formation of a pre-replication complex (pre-RC) that is assembled at replication origins by the sequential association of the origin recognition complex, followed by Cdt1, Cdc6 and finally MCMs, licensing DNA to start replication. The identification of pre-RC members in all animal and plant species shows that this complex is conserved in eukaryotes and, more importantly, the differences between kingdoms might reflect their divergence in strategies on cell cycle regulation, as it must be integrated and adapted to the niche, ecosystem, and the organism peculiarities. Here, we provide an overview of the knowledge generated so far on the formation and the developmental controls of the pre-RC mechanism in plants, analyzing some particular aspects in comparison to other eukaryotes. PMID:28304073

Open chromatin structures regulate the efficiencies of pre-RC formation and replication initiation in Epstein-Barr virus

PubMed Central

Papior, Peer; Arteaga-Salas, José M.; Günther, Thomas; Grundhoff, Adam

2012-01-01

Whether or not metazoan replication initiates at random or specific but flexible sites is an unsolved question. The lack of sequence specificity in origin recognition complex (ORC) DNA binding complicates genome-scale chromatin immunoprecipitation (ChIP)-based studies. Epstein-Barr virus (EBV) persists as chromatinized minichromosomes that are replicated by the host replication machinery. We used EBV to investigate the link between zones of pre-replication complex (pre-RC) assembly, replication initiation, and micrococcal nuclease (MNase) sensitivity at different cell cycle stages in a genome-wide fashion. The dyad symmetry element (DS) of EBV’s latent origin, a well-established and very efficient pre-RC assembly region, served as an internal control. We identified 64 pre-RC zones that correlate spatially with 57 short nascent strand (SNS) zones. MNase experiments revealed that pre-RC and SNS zones were linked to regions of increased MNase sensitivity, which is a marker of origin strength. Interestingly, although spatially correlated, pre-RC and SNS zones were characterized by different features. We propose that pre-RCs are formed at flexible but distinct sites, from which only a few are activated per single genome and cell cycle. PMID:22891264

The hunt for origins of DNA replication in multicellular eukaryotes

PubMed Central

Urban, John M.; Foulk, Michael S.; Casella, Cinzia

2015-01-01

Origins of DNA replication (ORIs) occur at defined regions in the genome. Although DNA sequence defines the position of ORIs in budding yeast, the factors for ORI specification remain elusive in metazoa. Several methods have been used recently to map ORIs in metazoan genomes with the hope that features for ORI specification might emerge. These methods are reviewed here with analysis of their advantages and shortcomings. The various factors that may influence ORI selection for initiation of DNA replication are discussed. PMID:25926981

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication

PubMed Central

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-01

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. PMID:27679476

Adherent and Invasive Escherichia coli Is Associated with Granulomatous Colitis in Boxer Dogs

PubMed Central

Simpson, Kenneth W.; Dogan, Belgin; Rishniw, Mark; Goldstein, Richard E.; Klaessig, Suzanne; McDonough, Patrick L.; German, Alex J.; Yates, Robin M.; Russell, David G.; Johnson, Susan E.; Berg, Douglas E.; Harel, Josee; Bruant, Guillaume; McDonough, Sean P.; Schukken, Ynte H.

2006-01-01

The mucosa-associated microflora is increasingly considered to play a pivotal role in the pathogenesis of inflammatory bowel disease. This study explored the possibility that an abnormal mucosal flora is involved in the etiopathogenesis of granulomatous colitis of Boxer dogs (GCB). Colonic biopsy samples from affected dogs (n = 13) and controls (n = 38) were examined by fluorescent in situ hybridization (FISH) with a eubacterial 16S rRNA probe. Culture, 16S ribosomal DNA sequencing, and histochemistry were used to guide subsequent FISH. GCB-associated Escherichia coli isolates were evaluated for their ability to invade and persist in cultured epithelial cells and macrophages as well as for serotype, phylogenetic group, genome size, overall genotype, and presence of virulence genes. Intramucosal gram-negative coccobacilli were present in 100% of GCB samples but not controls. Invasive bacteria hybridized with FISH probes to E. coli. Three of four GCB-associated E. coli isolates adhered to, invaded, and replicated within cultured epithelial cells. Invasion triggered a “splash”-type response, was decreased by cytochalasin D, genistein, colchicine, and wortmannin, and paralleled the behavior of the Crohn's disease-associated strain E. coli LF 82. GCB E. coli and LF 82 were diverse in serotype and overall genotype but similar in phylogeny (B2 and D), in virulence gene profiles (fyuA, irp1, irp2, chuA, fepC, ibeA, kpsMII, iss), in having a larger genome size than commensal E. coli, and in the presence of novel multilocus sequence types. We conclude that GCB is associated with selective intramucosal colonization by E. coli. E. coli strains associated with GCB and Crohn's disease have an adherent and invasive phenotype and novel multilocus sequence types and resemble E. coli associated with extraintestinal disease in phylogeny and virulence gene profile. PMID:16861666

Role of Escherichia coli dnaG function in coliphage M13 DNA synthesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dasgupta, S.; Mitra, S.

Examination of the role of Escherichia coli dnaG function in different stages of M13 phage DNA synthesis by ultracentrifugal analysis of intracellular phage DNA in a thermosensitive dnaG mutant shows that: (a) the formation of parental double-strand replicative-form DNA (rfDNA) from the infecting virus is independent of dnaG function; (b) the synthesis of progeny rfDNA requires dnaG product; (c) after a pool of rfDNA is made up, dnaG function is not required for the progeny single-strand DNA (ssDNA) synthesis. The ssDNAs produced under nonpermissive condition are mostly circular and biologically functional.

Herpesvirus papio contains a plasmid origin of replication that acts in cis interspecies with an Epstein-Barr virus trans-acting function.

PubMed Central

Pesano, R L; Pagano, J S

1986-01-01

Herpesvirus papio (HVP) and Epstein-Barr virus (EBV) are closely related biologically and biochemically; lymphoblastoid cells infected with either virus contain episomal viral DNA. The putative origin of replication for EBV plasmids (oriP) has been assigned to a 1,790-base-pair fragment (cis) in the short unique region of the genome which requires a viral function supplied in trans from elsewhere in the genome (J. Yates, N. Warren, D. Reisman, and B. Sugden, Proc. Natl. Acad. Sci. USA 81:3806-3810, 1984). We report here the identification of the putative origin of replication (cis) in HVP; we assigned it to the HVP EcoRI K fragment. The results indicate that the HVP replication process requires both a cis and a trans-acting function, analogous to that found in EBV. Images PMID:3023667

Editorial Decisions May Perpetuate Belief in Invalid Research Findings

PubMed Central

Eriksson, Kimmo; Simpson, Brent

2013-01-01

Social psychology and related disciplines are seeing a resurgence of interest in replication, as well as actual replication efforts. But prior work suggests that even a clear demonstration that a finding is invalid often fails to shake acceptance of the finding. This threatens the full impact of these replication efforts. Here we show that the actions of two key players – journal editors and the authors of original (invalidated) research findings – are critical to the broader public’s continued belief in an invalidated research conclusion. Across three experiments, we show that belief in an invalidated finding falls sharply when a critical failed replication is published in the same – versus different – journal as the original finding, and when the authors of the original finding acknowledge that the new findings invalidate their conclusions. We conclude by discussing policy implications of our key findings. PMID:24023863

Multiple-locus variable-nucleotide tandem repeat subtype analysis implicates European starlings as biological vectors for Escherichia coli O157:H7 in Ohio, USA.

PubMed

Williams, M L; Pearl, D L; Lejeune, J T

2011-10-01

To provide molecular epidemiological evidence of avian transmission of Escherichia coli O157:H7 between dairy farms in Ohio, this study was designed to identify genetic relatedness between isolates originating from bovine faecal samples and intestinal contents of European starlings captured on these farms. During a three-year period (2007-2009), cattle (n = 9000) and starlings (n = 430) on 150 different dairy farms in northern Ohio were sampled for the presence of E. coli O157:H7. Isolates were subjected to multiple-locus variable-nucleotide tandem repeat analysis (MLVA). Distinct allelic groups were identified on most farms; however, isolates clustering into three MLVA groups originated from both cattle and birds on different farms. Sharing of indistinguishable epidemiologically linked E. coli O157 MLVA subtypes between starlings and cattle on different farms supports the hypothesis that these birds contribute to the transmission of E. coli O157:H7 between dairy farms. A continued need exists to identify and to improve preharvest measures for controlling E. coli O157:H7. Controlling wildlife intrusion, particularly European starlings, on livestock operations, may be an important strategy for reducing dissemination of E. coli O157:H7 between farms and thereby potentially decreasing the on-farm prevalence of E. coli O157:H7 and enhancing the safety of the food supply. © 2011 The Authors. Journal of Applied Microbiology © 2011 The Society for Applied Microbiology.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Cloning-independent plasmid construction for genetic studies in streptococci

PubMed Central

Xie, Zhoujie; Qi, Fengxia; Merritt, Justin

2013-01-01

Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in E. coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5×103 – 2×105 CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli – Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts. PMID:23673081

Cloning-independent plasmid construction for genetic studies in streptococci.

PubMed

Xie, Zhoujie; Qi, Fengxia; Merritt, Justin

2013-08-01

Shuttle plasmids are among the few routinely utilized tools in the Streptococcus mutans genetic system that still require the use of classical cloning methodologies and intermediate hosts for genetic manipulation. Accordingly, it typically requires considerably less time and effort to introduce mutations onto the S. mutans chromosome than it does to construct shuttle vectors for expressing genes in trans. Occasionally, shuttle vector constructs also exhibit toxicity in Escherichia coli, which prevents their proper assembly. To circumvent these limitations, we modified a prolonged overlap extension PCR (POE-PCR) protocol to facilitate direct plasmid assembly in S. mutans. Using solely PCR, we created the reporter vector pZX7, which contains a single minimal streptococcal replication origin and harbors a spectinomycin resistance cassette and the gusA gene encoding β-glucuronidase. We compared the efficiency of pZX7 assembly using multiple strains of S. mutans and were able to obtain from 5 × 10³ to 2 × 10⁵ CFU/μg PCR product. Likewise, we used pZX7 to further demonstrate that Streptococcus sanguinis and Streptococcus gordonii are also excellent hosts for cloning-independent plasmid assembly, which suggests that this system is likely to function in numerous other streptococci. Consequently, it should be possible to completely forgo the use of E. coli-Streptococcus shuttle vectors in many streptococcal species, thereby decreasing the time and effort required to assemble constructs and eliminating any toxicity issues associated with intermediate hosts. Copyright © 2013 Elsevier B.V. All rights reserved.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression.

PubMed

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J; Schmidt, Kristina H

2016-12-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186-212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks.

A Novel Rrm3 Function in Restricting DNA Replication via an Orc5-Binding Domain Is Genetically Separable from Rrm3 Function as an ATPase/Helicase in Facilitating Fork Progression

PubMed Central

Syed, Salahuddin; Desler, Claus; Rasmussen, Lene J.; Schmidt, Kristina H.

2016-01-01

In response to replication stress cells activate the intra-S checkpoint, induce DNA repair pathways, increase nucleotide levels, and inhibit origin firing. Here, we report that Rrm3 associates with a subset of replication origins and controls DNA synthesis during replication stress. The N-terminal domain required for control of DNA synthesis maps to residues 186–212 that are also critical for binding Orc5 of the origin recognition complex. Deletion of this domain is lethal to cells lacking the replication checkpoint mediator Mrc1 and leads to mutations upon exposure to the replication stressor hydroxyurea. This novel Rrm3 function is independent of its established role as an ATPase/helicase in facilitating replication fork progression through polymerase blocking obstacles. Using quantitative mass spectrometry and genetic analyses, we find that the homologous recombination factor Rdh54 and Rad5-dependent error-free DNA damage bypass act as independent mechanisms on DNA lesions that arise when Rrm3 catalytic activity is disrupted whereas these mechanisms are dispensable for DNA damage tolerance when the replication function is disrupted, indicating that the DNA lesions generated by the loss of each Rrm3 function are distinct. Although both lesion types activate the DNA-damage checkpoint, we find that the resultant increase in nucleotide levels is not sufficient for continued DNA synthesis under replication stress. Together, our findings suggest a role of Rrm3, via its Orc5-binding domain, in restricting DNA synthesis that is genetically and physically separable from its established catalytic role in facilitating fork progression through replication blocks. PMID:27923055

DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner.

PubMed

Wiedemann, Eva-Maria; Peycheva, Mihaela; Pavri, Rushad

2016-12-13

Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Identification and characterization of the DNA replication origin recognition complex gene family in the silkworm Bombyx mori.

PubMed

Yang, Hui-Peng; Luo, Su-Juan; Li, Yi-Nü; Zhang, Yao-Zhou; Zhang, Zhi-Fang

2011-10-01

The ORC (origin recognition complex) binds to the DNA replication origin and recruits other replication factors to form the pre-replication complex. The cDNA and genomic sequences of all six subunits of ORC in Bombyx mori (BmORC1-6) were determined by RACE (rapid amplification of cDNA ends) and bioinformatic analysis. The conserved domains were identified in BmOrc1p-6p and the C-terminal of BmOrc6p features a short sequence that may be specific for Lepidoptera. As in other organisms, each of the six BmORC subunits had evolved individually from ancestral genes in early eukaryotes. During embryo development, the six genes were co-regulated, but different ratios of the abundance of mRNAs were observed in 13 tissues of the fifth instar day-6 larvae. Infection by BmNPV (B. mori nucleopolyhedrovirus) initially decreased and then increased the abundance of BmORC. We suggest that some of the BmOrc proteins may have additional functions and that BmOrc proteins participate in the replication of BmNPV.

The Obligate Human Pathogen, Neisseria gonorrhoeae, Is Polyploid

PubMed Central

Tobiason, Deborah M; Seifert, H. Steven

2006-01-01

We show using several methodologies that the Gram-negative, diplococcal-bacterium Neisseria gonorrhoeae has more than one complete genome copy per cell. Gene dosage measurements demonstrated that only a single replication initiation event per chromosome occurs per round of cell division, and that there is a single origin of replication. The region containing the origin does not encode any genes previously associated with bacterial origins of replication. Quantitative PCR results showed that there are on average three genome copies per coccal cell unit. These findings allow a model for gonococcal DNA replication and cell division to be proposed, in which a minimum of two chromosomal copies exist per coccal unit within a monococcal or diplococcal cell, and these chromosomes replicate in unison to produce four chromosomal copies during cell division. Immune evasion via antigenic variation is an important mechanism that allows these organisms to continually infect a high risk population of people. We propose that polyploidy may be necessary for the high frequency gene conversion system that mediates pilin antigenic variation and the propagation of N. gonorrhoeae within its human hosts. PMID:16719561

CDK activity provides temporal and quantitative cues for organizing genome duplication

PubMed Central

Perrot, Anthony; Millington, Christopher Lee; Gómez-Escoda, Blanca; Schausi-Tiffoche, Diane

2018-01-01

In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs), which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome. PMID:29466359

Double silencing of relevant genes suggests the existence of the direct link between DNA replication/repair and central carbon metabolism in human fibroblasts.

PubMed

Wieczorek, Aneta; Fornalewicz, Karolina; Mocarski, Łukasz; Łyżeń, Robert; Węgrzyn, Grzegorz

2018-04-15

Genetic evidence for a link between DNA replication and glycolysis has been demonstrated a decade ago in Bacillus subtilis, where temperature-sensitive mutations in genes coding for replication proteins could be suppressed by mutations in genes of glycolytic enzymes. Then, a strong influence of dysfunctions of particular enzymes from the central carbon metabolism (CCM) on DNA replication and repair in Escherichia coli was reported. Therefore, we asked if such a link occurs only in bacteria or it is a more general phenomenon. Here, we demonstrate that effects of silencing (provoked by siRNA) of expression of genes coding for proteins involved in DNA replication and repair (primase, DNA polymerase ι, ligase IV, and topoisomerase IIIβ) on these processes (less efficient entry into the S phase of the cell cycle and decreased level of DNA synthesis) could be suppressed by silencing of specific genes of enzymes from CMM. Silencing of other pairs of replication/repair and CMM genes resulted in enhancement of the negative effects of lower expression levels of replication/repair genes. We suggest that these results may be proposed as a genetic evidence for the link between DNA replication/repair and CMM in human cells, indicating that it is a common biological phenomenon, occurring from bacteria to humans. Copyright © 2018 Elsevier B.V. All rights reserved.

Genetic variations in the DNA replication origins of human papillomavirus family correlate with their oncogenic potential.

PubMed

Yilmaz, Gulden; Biswas-Fiss, Esther E; Biswas, Subhasis B

2018-04-01

Human papillomaviruses (HPVs) encompass a large family of viruses that range from benign to highly carcinogenic. The crucial differences between benign and carcinogenic types of HPV remain unknown, except that the two HPV types differ in the frequency of DNA replication. We have systematically analyzed the mechanism of HPV DNA replication initiation in low-risk and high-risk HPVs. Our results demonstrate that HPV-encoded E2 initiator protein and its four binding sites in the replication origin play pivotal roles in determining the destiny of the HPV-infected cell. We have identified strain-specific single nucleotide variations in E2 binding sites found only in the high-risk HPVs. We have demonstrated that these variations result in attenuated formation of the E2-DNA complex. E2 binding to these sites is linked to the activation of the DNA replication origin as well as initiation of DNA replication. Both electrophoretic mobility shift assay and atomic force microscopy studies demonstrated that binding of E2 from either low- or high-risk HPVs with variant binding sequences lacked multimeric E2-DNA complex formation in vitro. These results provided a molecular basis of differential DNA replication in the two types of HPVs and pointed to a correlation with the development of cancer. Copyright © 2017. Published by Elsevier B.V.

Isolation of a conjugative F-like plasmid from a multidrug-resistant Escherichia coli strain CM6 using tandem shock wave-mediated transformation.

PubMed

Soto-Alonso, G; Cruz-Medina, J A; Caballero-Pérez, J; Arvizu-Hernández, I; Ávalos-Esparza, L M; Cruz-Hernández, A; Romero-Gómez, S; Rodríguez, A L; Pastrana-Martínez, X; Fernández, F; Loske, A M; Campos-Guillén, J

2015-07-01

Genetic characterization of plasmids from bacterial strains provides insight about multidrug resistance. Ten wild type Escherichia coli (E. coli) strains isolated from cow fecal samples were characterized by their antibiotic resistance profile, plasmid patterns and three different identification methods. From one of the strains, a fertility factor-like plasmid was replicated using tandem shock wave-mediated transformation. Underwater shock waves with a positive pressure peak of up to approximately 40 MPa, followed by a pressure trough of approximately -19 MPa were generated using an experimental piezoelectric shock wave source. Three different shock wave energies and a fixed delay of 750 μs were used to study the relationship between energy and transformation efficiency (TE), as well as the influence of shock wave energy on the integrity of the plasmid. Our results showed that the mean shock wave-mediated TE and the integrity of the large plasmid (~70 kb) were reduced significantly at the energy levels tested. The sequencing analysis of the plasmid revealed a high identity to the pHK17a plasmid, including the replication system, which was similar to the plasmid incompatibility group FII. It also showed that it carried an extended spectrum beta-lactamase gene, ctx-m-14. Furthermore, diverse genes for the conjugative mechanism were identified. Our results may be helpful in improving methodologies for conjugative plasmid transfer and directly selecting the most interesting plasmids from environmental samples. Copyright © 2015 Elsevier B.V. All rights reserved.

Change in the Structure of Escherichia coli Population and the Pattern of Virulence Genes along a Rural Aquatic Continuum

PubMed Central

Petit, Fabienne; Clermont, Olivier; Delannoy, Sabine; Servais, Pierre; Gourmelon, Michèle; Fach, Patrick; Oberlé, Kenny; Fournier, Matthieu; Denamur, Erick; Berthe, Thierry

2017-01-01

The aim of this study was to investigate the diversity of the Escherichia coli population, focusing on the occurrence of pathogenic E. coli, in surface water draining a rural catchment. Two sampling campaigns were carried out in similar hydrological conditions (wet period, low flow) along a river continuum, characterized by two opposite density gradients of animals (cattle and wild animals) and human populations. While the abundance of E. coli slightly increased along the river continuum, the abundance of both human and ruminant-associated Bacteroidales markers, as well as the number of E. coli multi-resistant to antibiotics, evidenced a fecal contamination originating from animals at upstream rural sites, and from humans at downstream urban sites. A strong spatial modification of the structure of the E. coli population was observed. At the upstream site close to a forest, a higher abundance of the B2 phylogroup and Escherichia clade strains were observed. At the pasture upstream site, a greater proportion of both E and B1 phylogroups was detected, therefore suggesting a fecal contamination of mainly bovine origin. Conversely, in downstream urban sites, A, D, and F phylogroups were more abundant. To assess the occurrence of intestinal pathogenic strains, virulence factors [afaD, stx1, stx2, eltB (LT), estA (ST), ipaH, bfpA, eae, aaiC and aatA] were screened among 651 E. coli isolates. Intestinal pathogenic strains STEC O174:H21 (stx2) and EHEC O26:H11 (eae, stx1) were isolated in water and sediments close to the pasture site. In contrast, in the downstream urban site aEPEC/EAEC and DAEC of human origin, as well as extra-intestinal pathogenic E. coli belonging to clonal group A of D phylogroup, were sampled. Even if the estimated input of STEC (Shiga toxin-producing E. coli) – released in water at the upstream pasture site – at the downstream site was low, we show that STEC could persist in sediment. These results show that, the run-off of small cattle farms contributed, as much as the wastewater effluent, in the dissemination of pathogenic E. coli in both water and sediments, even if the microbiological quality of the water was good or to average quality according to the French water index. PMID:28458656

An assessment of fecal indicator and other bacteria from an urbanized coastal lagoon in the City of Los Angeles, California, USA.

PubMed

Dorsey, John H; Carmona-Galindo, Víctor D; Leary, Christopher; Huh, Julie; Valdez, Jennifer

2013-03-01

A study was performed in Del Rey Lagoon, City of Los Angeles, to determine if the lagoon was as a source or sink for fecal indicator bacteria (FIB: total coliforms, Escherichia coli, enterococci) and to screen for the presence of other potentially pathogenic bacteria. The lagoon receives tidal flows from the adjacent Ballona Estuary whose water usually is contaminated with FIB originating from the highly urbanized Ballona Creek Watershed. During 16 sampling events from February 2008 through March 2009, replicate water samples (n = 3) were collected 1 h prior to the high tide and 1 h prior to the following low tide. FIB concentrations were measured by the defined substrate method (IDEXX, Westbrook, Me) followed by culturing of bacterial isolates sampled from positive IDEXX Quanti-Tray wells and were identified using the Vitek 2 Compact (bioMérieux, Durham, NC). Mean concentrations of FIB often differed by an order of magnitude from flood to ebb flow conditions. The lagoon tended to act as a sink for total coliforms based on the ratio of mean flood to ebb densities (R (F/E)) >1.0 during 56 % of the sampling events and during ebb flows, as a source for E. coli and enterococci (R (F/E) <1.69 % of events). Approximately 54 species were identified from 277 isolates cultured from the IDEXX Quanti-Trays. Of these, 54 % were species known to include pathogenic strains that can be naturally occurring, introduced in runoff, or originated from other sources. Diversity and cluster analyses indicated a dynamic assemblage that changes in species composition with day-to-day fluctuations as well as tidal action. The concept of monitoring the lagoon and estuary as a sentinel habitat for pathogenic assemblages is discussed.

Persistence of Escherichia coli and Salmonella in surface soil following application of liquid hog manure for production of pickling cucumbers.

PubMed

Côté, Caroline; Quessy, Sylvain

2005-05-01

Liquid hog manure is routinely applied to farm land as a crop fertilizer. However, this practice raises food safety concerns, especially when manure is used on fruit and vegetable crops. The objectives of this project were to evaluate the persistence of Escherichia coli and Salmonella in surface soil after application of liquid hog manure to fields where pickling cucumbers were grown and to verify the microbiological quality of harvested cucumbers. Mineral fertilizers were replaced by liquid hog manure at various ratios in the production of pickling cucumbers in a 3-year field study. The experimental design was a randomized complete block comprising four replicates in sandy loam (years 1, 2, and 3) and loamy sand (year 3). Soil samples were taken at a depth of 20 cm every 2 weeks after June application of organic and inorganic fertilizers. Vegetable samples were also taken at harvest time. Liquid hog manure, soil, and vegetable (washed and unwashed) samples were analyzed for the presence of Salmonella and E. coli. An exponential decrease of E. coli populations was observed in surface soil after the application of manure. The estimated average time required to reach undetectable concentrations of E. coli in sandy loam varied from 56 to 70 days, whereas the absence of E. coli was estimated at 77 days in loamy sand. The maximal Salmonella persistence in soil was 54 days. E. coli and Salmonella were not detected in any vegetable samples.

Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith-Somerville, H.E.; Huryn, V.B.; Walker, C.

1991-09-01

The processing of phagosomes containing Legionella pneumophila and Escerichia coli were compared in Tetrahymena vorax, a hymenostome ciliated protozoan that prefers lower temperatures. L. pneumophila did not multiply in the ciliate when incubated at 20 to 22C, but vacuoles containing L. pneumophila were retained in the cells for a substantially longer time than vacuoles with E. coli. Electron micrographs showed no evidence of degradation of L. pneumophila cells through 12 h, while E. coli cells in the process of being digested were observed in vacuoles 75 min after the addition of the bacterium T. vorax ingested L. pneumophila normally, butmore » by 10 to 15 min, the vacuolar membrane appeared denser than that surrounding nascent or newly formed phagosomes. In older vacuoles, electron-dense particles lined portions of the membrane. Acidification of the phagosomes indicated by the accumulation of neutral red was similar in T. vorax containing L. pneumophila or E. coli. This ciliate could provide a model for the analysis of virulence-associated intracellular events independent of the replication of L. pneumophila.« less

Postnatal development of the small intestinal mucosa drives age-dependent, regio-selective susceptibility to Escherichia coli K1 infection.

PubMed

Birchenough, George M H; Dalgakiran, Fatma; Witcomb, Luci A; Johansson, Malin E V; McCarthy, Alex J; Hansson, Gunnar C; Taylor, Peter W

2017-03-06

The strong age dependency of neonatal systemic infection with Escherichia coli K1 can be replicated in the neonatal rat. Gastrointestinal (GI) colonization of two-day-old (P2) rats leads to invasion of the blood within 48 h of initiation of colonization; pups become progressively less susceptible to infection over the P2-P9 period. We show that, in animals colonized at P2 but not at P9, E. coli K1 bacteria gain access to the enterocyte surface in the mid-region of the small intestine and translocate through the epithelial cell monolayer by an intracellular pathway to the submucosa. In this region of the GI tract, the protective mucus barrier is poorly developed but matures to full thickness over P2-P9, coincident with the development of resistance to invasion. At P9, E. coli K1 bacteria are physically separated from villi by the mucus layer and their numbers controlled by mucus-embedded antimicrobial peptides, preventing invasion of host tissues.

Evidence supporting a role for TopBP1 and Brd4 in the initiation but not continuation of human papillomavirus 16 E1/E2-mediated DNA replication.

PubMed

Gauson, Elaine J; Donaldson, Mary M; Dornan, Edward S; Wang, Xu; Bristol, Molly; Bodily, Jason M; Morgan, Iain M

2015-05-01

To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Evidence Supporting a Role for TopBP1 and Brd4 in the Initiation but Not Continuation of Human Papillomavirus 16 E1/E2-Mediated DNA Replication

PubMed Central

Gauson, Elaine J.; Donaldson, Mary M.; Dornan, Edward S.; Wang, Xu; Bristol, Molly; Bodily, Jason M.

2015-01-01

ABSTRACT To replicate the double-stranded human papillomavirus 16 (HPV16) DNA genome, viral proteins E1 and E2 associate with the viral origin of replication, and E2 can also regulate transcription from adjacent promoters. E2 interacts with host proteins in order to regulate both transcription and replication; TopBP1 and Brd4 are cellular proteins that interact with HPV16 E2. Previous work with E2 mutants demonstrated the Brd4 requirement for the transactivation properties of E2, while TopBP1 is required for DNA replication induced by E2 from the viral origin of replication in association with E1. More-recent studies have also implicated Brd4 in the regulation of DNA replication by E2 and E1. Here, we demonstrate that both TopBP1 and Brd4 are present at the viral origin of replication and that interaction with E2 is required for optimal initiation of DNA replication. Both cellular proteins are present in E1-E2-containing nuclear foci, and the viral origin of replication is required for the efficient formation of these foci. Short hairpin RNA (shRNA) against either TopBP1 or Brd4 destroys the E1-E2 nuclear bodies but has no effect on E1-E2-mediated levels of DNA replication. An E2 mutation in the context of the complete HPV16 genome that compromises Brd4 interaction fails to efficiently establish episomes in primary human keratinocytes. Overall, the results suggest that interactions between TopBP1 and E2 and between Brd4 and E2 are required to correctly initiate DNA replication but are not required for continuing DNA replication, which may be mediated by alternative processes such as rolling circle amplification and/or homologous recombination. IMPORTANCE Human papillomavirus 16 (HPV16) is causative in many human cancers, including cervical and head and neck cancers, and is responsible for the annual deaths of hundreds of thousands of people worldwide. The current vaccine will save lives in future generations, but antivirals targeting HPV16 are required for the alleviation of disease burden on the current, and future, generations. Targeting viral DNA replication that is mediated by two viral proteins, E1 and E2, in association with cellular proteins such as TopBP1 and Brd4 would have therapeutic benefits. This report suggests a role for these cellular proteins in the initiation of viral DNA replication by HPV16 E1-E2 but not for continuing replication. This is important if viral replication is to be effectively targeted; we need to understand the viral and cellular proteins required at each phase of viral DNA replication so that it can be effectively disrupted. PMID:25694599

An Introduction to Replication Research in Gifted Education: Shiny and New Is Not the Same as Useful

ERIC Educational Resources Information Center

Makel, Matthew C.; Plucker, Jonathan A.

2015-01-01

This methodological brief introduces readers to replication methods and their uses. Broadly defined, replication is the duplication of previously conducted research to verify or expand the original findings. Replication is particularly useful in the gifted education context because so much education theory and research are based on general…

Peripheral re-localization of constitutive heterochromatin advances its replication timing and impairs maintenance of silencing marks.

PubMed

Heinz, Kathrin S; Casas-Delucchi, Corella S; Török, Timea; Cmarko, Dusan; Rapp, Alexander; Raska, Ivan; Cardoso, M Cristina

2018-05-10

The replication of the genome is a highly organized process, both spatially and temporally. Although a lot is known on the composition of the basic replication machinery, how its activity is regulated is mostly unknown. Several chromatin properties have been proposed as regulators, but a potential role of the nuclear DNA position remains unclear. We made use of the prominent structure and well-defined heterochromatic landscape of mouse pericentric chromosome domains as a well-studied example of late replicating constitutive heterochromatin. We established a method to manipulate its nuclear position and evaluated the effect on replication timing, DNA compaction and epigenetic composition. Using time-lapse microscopy, we observed that constitutive heterochromatin, known to replicate during late S-phase, was replicated in mid S-phase when repositioned to the nuclear periphery. Out-of-schedule replication resulted in deficient post-replicative maintenance of chromatin modifications, namely silencing marks. We propose that repositioned constitutive heterochromatin was activated in trans according to the domino model of origin firing by nearby (mid S) firing origins. In summary, our data provide, on the one hand, a novel approach to manipulate nuclear DNA position and, on the other hand, establish nuclear DNA position as a novel mechanism regulating DNA replication timing and epigenetic maintenance.

H3K9me3 demethylase Kdm4d facilitates the formation of pre-initiative complex and regulates DNA replication.

PubMed

Wu, Rentian; Wang, Zhiquan; Zhang, Honglian; Gan, Haiyun; Zhang, Zhiguo

2017-01-09

DNA replication is tightly regulated to occur once and only once per cell cycle. How chromatin, the physiological substrate of DNA replication machinery, regulates DNA replication remains largely unknown. Here we show that histone H3 lysine 9 demethylase Kdm4d regulates DNA replication in eukaryotic cells. Depletion of Kdm4d results in defects in DNA replication, which can be rescued by the expression of H3K9M, a histone H3 mutant transgene that reverses the effect of Kdm4d on H3K9 methylation. Kdm4d interacts with replication proteins, and its recruitment to DNA replication origins depends on the two pre-replicative complex components (origin recognition complex [ORC] and minichromosome maintenance [MCM] complex). Depletion of Kdm4d impairs the recruitment of Cdc45, proliferating cell nuclear antigen (PCNA), and polymerase δ, but not ORC and MCM proteins. These results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate formation of pre-initiative complex. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Cohesin organizes chromatin loops at DNA replication factories

PubMed Central

Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Méndez, Juan

2010-01-01

Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication. PMID:21159821

THE FORK AND THE KINASE: A DNA REPLICATION TALE FROM A CHK1 PERSPECTIVE

PubMed Central

González Besteiro, Marina A.; Gottifredi, Vanesa

2014-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. The checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged-DNA. Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Such findings unveil a puzzling connection between Chk1 and DNA-lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, the multifaceted and versatile functions of Chk1 at ongoing forks and replication origins determine the extent and quality of the cellular response to replication stress. PMID:25795119

A DNA sequence element that advances replication origin activation time in Saccharomyces cerevisiae.

PubMed

Pohl, Thomas J; Kolor, Katherine; Fangman, Walton L; Brewer, Bonita J; Raghuraman, M K

2013-11-06

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.

Does it actually feel right? A replication attempt of the rounded price effect.

PubMed

Harms, Christopher; Genau, Hanna A; Meschede, Carolin; Beauducel, André

2018-04-01

How does the roundedness of prices affect product evaluations? The 'rounded price effect' postulates that depending on the context, rounded or non-rounded prices increase the purchase likelihood of consumers. The study presented here is a replication attempt of these findings and the proposed mediation of the effect through a sense of 'feeling right' when evaluating the product. p -Curve analysis and the R-Index are used to assess the robustness of the originally reported statistics since original data were not available. A pre-registered replication of study 5 from the original article was conducted in a sample of N =588 participants. For both the original product and one alternative product neither an interaction between price roundedness and context, nor a mediation through 'a sense of feeling right' was found. Our results suggest that the effect is either smaller than originally reported or contingent on other, not investigated factors. Further studies might investigate contingencies in larger samples.

Does it actually feel right? A replication attempt of the rounded price effect

PubMed Central

Genau, Hanna A.; Meschede, Carolin; Beauducel, André

2018-01-01

How does the roundedness of prices affect product evaluations? The ‘rounded price effect’ postulates that depending on the context, rounded or non-rounded prices increase the purchase likelihood of consumers. The study presented here is a replication attempt of these findings and the proposed mediation of the effect through a sense of ‘feeling right’ when evaluating the product. p-Curve analysis and the R-Index are used to assess the robustness of the originally reported statistics since original data were not available. A pre-registered replication of study 5 from the original article was conducted in a sample of N=588 participants. For both the original product and one alternative product neither an interaction between price roundedness and context, nor a mediation through ‘a sense of feeling right’ was found. Our results suggest that the effect is either smaller than originally reported or contingent on other, not investigated factors. Further studies might investigate contingencies in larger samples. PMID:29765625

Elements in the transcriptional regulatory region flanking herpes simplex virus type 1 oriS stimulate origin function.

PubMed

Wong, S W; Schaffer, P A

1991-05-01

Like other DNA-containing viruses, the three origins of herpes simplex virus type 1 (HSV-1) DNA replication are flanked by sequences containing transcriptional regulatory elements. In a transient plasmid replication assay, deletion of sequences comprising the transcriptional regulatory elements of ICP4 and ICP22/47, which flank oriS, resulted in a greater than 80-fold decrease in origin function compared with a plasmid, pOS-822, which retains these sequences. In an effort to identify specific cis-acting elements responsible for this effect, we conducted systematic deletion analysis of the flanking region with plasmid pOS-822 and tested the resulting mutant plasmids for origin function. Stimulation by cis-acting elements was shown to be both distance and orientation dependent, as changes in either parameter resulted in a decrease in oriS function. Additional evidence for the stimulatory effect of flanking sequences on origin function was demonstrated by replacement of these sequences with the cytomegalovirus immediate-early promoter, resulting in nearly wild-type levels of oriS function. In competition experiments, cotransfection of cells with the test plasmid, pOS-822, and increasing molar concentrations of a competitor plasmid which contained the ICP4 and ICP22/47 transcriptional regulatory regions but lacked core origin sequences resulted in a significant reduction in the replication efficiency of pOS-822, demonstrating that factors which bind specifically to the oriS-flanking sequences are likely involved as auxiliary proteins in oriS function. Together, these studies demonstrate that trans-acting factors and the sites to which they bind play a critical role in the efficiency of HSV-1 DNA replication from oriS in transient-replication assays.

Identification of DNA gyrase inhibitor (GyrI) in Escherichia coli.

PubMed

Nakanishi, A; Oshida, T; Matsushita, T; Imajoh-Ohmi, S; Ohnuki, T

1998-01-23

DNA gyrase is an essential enzyme in DNA replication in Escherichia coli. It mediates the introduction of negative supercoils near oriC, removal of positive supercoils ahead of the growing DNA fork, and separation of the two daughter duplexes. In the course of purifying DNA gyrase from E. coli KL16, we found an 18-kDa protein that inhibited the supercoiling activity of DNA gyrase, and we coined it DNA gyrase inhibitory protein (GyrI). Its NH2-terminal amino acid sequence of 16 residues was determined to be identical to that of a putative gene product (a polypeptide of 157 amino acids) encoded by yeeB (EMBL accession no. U00009) and sbmC (Baquero, M. R., Bouzon, M., Varea, J., and Moreno, F. (1995) Mol. Microbiol. 18, 301-311) of E. coli. Assuming the identity of the gene (gyrI) encoding GyrI with the previously reported genes yeeB and sbmC, we cloned the gene after amplification by polymerase chain reaction and purified the 18-kDa protein from an E. coli strain overexpressing it. The purified 18-kDa protein was confirmed to inhibit the supercoiling activity of DNA gyrase in vitro. In vivo, both overexpression and antisense expression of the gyrI gene induced filamentous growth of cells and suppressed cell proliferation. GyrI protein is the first identified chromosomally nucleoid-encoded regulatory factor of DNA gyrase in E. coli.

Effect of soil composition, temperature, indigenous microflora, and environmental conditions on the survival of Escherichia coli O157:H7.

PubMed

Vidovic, Sinisa; Block, Hushton C; Korber, Darren R

2007-07-01

The survival of Escherichia coli O157:H7 in replicate soil microcosms was quantified in 2 types of silty clay loam soil (high carbon and low carbon) under either sterile or nonsterile conditions. Microcosms were held at -21, 4, and 22 degrees C under constant soil moisture content. Differences existed (P < 0.05) in survival of E. coli O157:H7 in low- and high-carbon soil at all temperatures, indicating an important role of soil composition on the survival of this pathogen. The highest death rate of E. coli O157:H7 in sterile soil occurred in the low-carbon soil at 4 degrees C, whereas in nonsterile soil the highest death rate was observed in the low-carbon soil at 22 degrees C. These results suggest that the most lethal effects on E. coli O157:H7 in the sterile system occurred via the synergy of nutrient limitation and cold stress, whereas in the nonsterile system lethality was owing to inhibition by indigenous soil microorganisms and starvation. Results obtained from an in situ field survival experiment demonstrated the apparent sensitivity of E. coli O157:H7 cells to dehydration, information that may be used to reduce environmental spread of this pathogen as well as formulate appropriate waste management strategies.

Escherichia coli DnaA forms helical structures along the longitudinal cell axis distinct from MreB filaments.

PubMed

Boeneman, Kelly; Fossum, Solveig; Yang, Yanhua; Fingland, Nicholas; Skarstad, Kirsten; Crooke, Elliott

2009-05-01

DnaA initiates chromosomal replication in Escherichia coli at a well-regulated time in the cell cycle. To determine how the spatial distribution of DnaA is related to the location of chromosomal replication and other cell cycle events, the localization of DnaA in living cells was visualized by confocal fluorescence microscopy. The gfp gene was randomly inserted into a dnaA-bearing plasmid via in vitro transposition to create a library that included internally GFP-tagged DnaA proteins. The library was screened for the ability to rescue dnaA(ts) mutants, and a candidate gfp-dnaA was used to replace the dnaA gene of wild-type cells. The resulting cells produce close to physiological levels of GFP-DnaA from the endogenous promoter as their only source of DnaA and somewhat under-initiate replication with moderate asynchrony. Visualization of GFP-tagged DnaA in living cells revealed that DnaA adopts a helical pattern that spirals along the long axis of the cell, a pattern also seen in wild-type cells by immunofluorescence with affinity purified anti-DnaA antibody. Although the DnaA helices closely resemble the helices of the actin analogue MreB, co-visualization of GFP-tagged DnaA and RFP-tagged MreB demonstrates that DnaA and MreB adopt discrete helical structures along the length of the longitudinal cell axis.

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

PubMed Central

Keyamura, Kenji; Fujikawa, Norie; Ishida, Takuma; Ozaki, Shogo; Su’etsugu, Masayuki; Fujimitsu, Kazuyuki; Kagawa, Wataru; Yokoyama, Shigeyuki; Kurumizaka, Hitoshi; Katayama, Tsutomu

2007-01-01

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP–DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP–DnaA, and stimulated the assembly of inactive ADP–DnaA–oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP–DnaA-specific–oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP–DnaA–oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner. PMID:17699754

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex.

PubMed

Ganaie, Safder S; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve; Qiu, Jianming

2017-05-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases.

Rapid Growth of Uropathogenic Escherichia coli during Human Urinary Tract Infection.

PubMed

Forsyth, Valerie S; Armbruster, Chelsie E; Smith, Sara N; Pirani, Ali; Springman, A Cody; Walters, Matthew S; Nielubowicz, Greta R; Himpsl, Stephanie D; Snitkin, Evan S; Mobley, Harry L T

2018-03-06

Uropathogenic Escherichia coli (UPEC) strains cause most uncomplicated urinary tract infections (UTIs). These strains are a subgroup of extraintestinal pathogenic E. coli (ExPEC) strains that infect extraintestinal sites, including urinary tract, meninges, bloodstream, lungs, and surgical sites. Here, we hypothesize that UPEC isolates adapt to and grow more rapidly within the urinary tract than other E. coli isolates and survive in that niche. To date, there has not been a reliable method available to measure their growth rate in vivo Here we used two methods: segregation of nonreplicating plasmid pGTR902, and peak-to-trough ratio (PTR), a sequencing-based method that enumerates bacterial chromosomal replication forks present during cell division. In the murine model of UTI, UPEC strain growth was robust in vivo , matching or exceeding in vitro growth rates and only slowing after reaching high CFU counts at 24 and 30 h postinoculation (hpi). In contrast, asymptomatic bacteriuria (ABU) strains tended to maintain high growth rates in vivo at 6, 24, and 30 hpi, and population densities did not increase, suggesting that host responses or elimination limited population growth. Fecal strains displayed moderate growth rates at 6 hpi but did not survive to later times. By PTR, E. coli in urine of human patients with UTIs displayed extraordinarily rapid growth during active infection, with a mean doubling time of 22.4 min. Thus, in addition to traditional virulence determinants, including adhesins, toxins, iron acquisition, and motility, very high growth rates in vivo and resistance to the innate immune response appear to be critical phenotypes of UPEC strains. IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains cause most urinary tract infections in otherwise healthy women. While we understand numerous virulence factors are utilized by E. coli to colonize and persist within the urinary tract, these properties are inconsequential unless bacteria can divide rapidly and survive the host immune response. To determine the contribution of growth rate to successful colonization and persistence, we employed two methods: one involving the segregation of a nonreplicating plasmid in bacteria as they divide and the peak-to-trough ratio, a sequencing-based method that enumerates chromosomal replication forks present during cell division. We found that UPEC strains divide extraordinarily rapidly during human UTIs. These techniques will be broadly applicable to measure in vivo growth rates of other bacterial pathogens during host colonization. Copyright © 2018 Forsyth et al.

Manipulation for plasmid elimination by transforming synthetic competitors diversifies lactococcus lactis starters applicable to food products.

PubMed

Kobayashi, Miho; Nomura, Masaru; Kimoto, Hiromi

2007-11-01

This study was designed selectively to eliminate a theta-plasmid from Lactococcus lactis strains by transforming synthetic competitors. A shuttle vector for Escherichia coli and L. lactis, pDB1, was constructed by ligating a partial replicon of pDR1-1B, which is a 7.3 kb theta-plasmid in L. lactis DRC1, with an erythromycin resistance gene into pBluescript II KS(+). This versatile vector was used to construct competitors to common lactococcal theta-plasmids. pDB1 contains the 5' half of the replication origin and the 3' region of repB of pDR1-1B, but lacks the 1.1-kb region normally found between these two segments. A set of primers, Pv3 and Pv4, was designed to amplify the 1.1-kb middle parts of the general theta-replicons of lactococcal plasmids. When the PCR products were cloned into the Nru I and Xho I sites of pDB1, synthetic replicons were constructed and replication activity was restored. A number of theta-plasmids in L. lactis ssp. lactis and cremoris were eliminated selectively by transforming the synthetic competitors. These competitors were easily eliminated by subculture for a short time in the absence of selection. The resulting variants contained no exogenous DNA and are suitable for food products, since part of the phenotype was altered without altering other plasmids indispensable for fermentation.

Construction of new cloning, lacZ reporter and scarless-markerless suicide vectors for genetic studies in Aggregatibacter actinomycetemcomitans

PubMed Central

Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R.

2013-01-01

To elucidate the putative function of a gene, effective tools are required for genetic characterization that facilitate its inactivation, deletion or modification on the bacterial chromosome. In the present study, the nucleotide sequence of the Escherichia coli/Aggregatibacter actinomycetemcomitans shuttle vector pYGK was determined, allowing us to redesign and construct a new shuttle cloning vector, pJT4, and promoterless lacZ transcriptional/translational fusion plasmids, pJT3 and pJT5. Plasmids pJT4 and pJT5 contain the origin of replication necessary to maintain shuttle vector replication. In addition, a new suicide vector, pJT1, was constructed for the generation of scarless and markerless deletion mutations of genes in the oral pathogen A. actinomycetemcomitans. Plasmid pJT1 is a pUC-based suicide vector that is counter-selectable for sucrose sensitivity. This vector does not leave antibiotic markers or scars on the chromosome after gene deletion and thus provides the option to combine several mutations in the same genetic background. The effectiveness of pJT1 was demonstrated by the construction of A. actinomycetemcomitans isogenic qseB single deletion (ΔqseB) mutant and lsrRK double deletion mutants (ΔlsrRK). These new vectors may offer alternatives for genetic studies in A. actinomycetemcomitans and other members of the HACEK (Haemophilus spp., A. actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae) group of Gram-negative bacteria. PMID:23353051

Transformation of Schwanniomyces occidentalis with an ADE2 gene cloned from S. occidentalis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klein, R.D.; Favreau, M.A.

1988-12-01

We have developed an efficient transformation system for the industrial yeast Schwanniomyces occidentalis (formerly Schwanniomyces castellii). The transformation system is based on ade2 mutants of S. occidentalis deficient for phosphoribosylaminoimidazole carboxylase that were generated by mutagenesis. As a selectable marker, we isolated and characterized the S. occidentalis ADE2 gene by complementation in an ade2 strain of Saccharomyces cerevisiae. S. occidentalis was transformed with the recombinant plasmid pADE, consisting of a 4.5-kilobase-pair (kbp) DNA fragment from S. occidentalis containing the ADE2 gene inserted into the S. cerevisiae expression vector pYcDE8 by a modification of the spheroplasting procedure of Beggs. Intact plasmidsmore » were recovered in Escherichia coli from whole-cell lysates of ADE+ transformants, indicating that plasmids were replicating autonomously. High-molecular-mass species of pADE2 were found by Southern hybridization analysis of intact genomic DNA preparations. The shift to higher molecular mass of these plasmids during electrophoresis in the presence ethidium bromide after exposure to shortwave UV suggests that they exist in a supercoiled form in the transformed host. Subclones of the 4.5-kbp insert indicated that ADE2-complementing activity and sequences conferring autonomous replication in S. occidentalis were located within a 2.7-kbp EcoRI-SphI fragment. Plasmids containing this region cloned into the bacterial vector pUC19 complemented ade2 mutants of S. occidentalis with efficiencies identical to those of the original plasmid pADE.« less

Differential decay of Enterococci and Escherichia coli originating from two fecal pollution sources

EPA Science Inventory

Using in situ subtropical aquatic mesocosms, fecal source (cattle manure versus sewage) was shown to be the most important contributor to differential loss in viability of fecal indicator bacteria (FIB), specifically enterococci in freshwater and Escherichia coli in marine habita...

Calcein represses human papillomavirus 16 E1-E2 mediated DNA replication via blocking their binding to the viral origin of replication.

PubMed

Das, Dipon; Smith, Nathan W; Wang, Xu; Richardson, Stacie L; Hartman, Matthew C T; Morgan, Iain M

2017-08-01

Human papillomaviruses are causative agents in several human diseases ranging from genital warts to ano-genital and oropharyngeal cancers. Currently only symptoms of HPV induced disease are treated; there are no antivirals available that directly target the viral life cycle. Previously, we determined that the cellular protein TopBP1 interacts with the HPV16 replication/transcription factor E2. This E2-TopBP1 interaction is essential for optimal E1-E2 DNA replication and for the viral life cycle. The drug calcein disrupts the interaction of TopBP1 with itself and other host proteins to promote cell death. Here we demonstrate that calcein blocks HPV16 E1-E2 DNA replication via blocking the viral replication complex forming at the origin of replication. This occurs at non-toxic levels of calcein and demonstrates specificity as it does not block the ability of E2 to regulate transcription. We propose that calcein or derivatives could be developed as an anti-HPV therapeutic. Copyright © 2017 Elsevier Inc. All rights reserved.

Who Needs Replication?

ERIC Educational Resources Information Center

Porte, Graeme

2013-01-01

In this paper, the editor of a recent Cambridge University Press book on research methods discusses replicating previous key studies to throw more light on their reliability and generalizability. Replication research is presented as an accepted method of validating previous research by providing comparability between the original and replicated…

Primer retention owing to the absence of RNase H1 is catastrophic for mitochondrial DNA replication.

PubMed

Holmes, J Bradley; Akman, Gokhan; Wood, Stuart R; Sakhuja, Kiran; Cerritelli, Susana M; Moss, Chloe; Bowmaker, Mark R; Jacobs, Howard T; Crouch, Robert J; Holt, Ian J

2015-07-28

Encoding ribonuclease H1 (RNase H1) degrades RNA hybridized to DNA, and its function is essential for mitochondrial DNA maintenance in the developing mouse. Here we define the role of RNase H1 in mitochondrial DNA replication. Analysis of replicating mitochondrial DNA in embryonic fibroblasts lacking RNase H1 reveals retention of three primers in the major noncoding region (NCR) and one at the prominent lagging-strand initiation site termed Ori-L. Primer retention does not lead immediately to depletion, as the persistent RNA is fully incorporated in mitochondrial DNA. However, the retained primers present an obstacle to the mitochondrial DNA polymerase γ in subsequent rounds of replication and lead to the catastrophic generation of a double-strand break at the origin when the resulting gapped molecules are copied. Hence, the essential role of RNase H1 in mitochondrial DNA replication is the removal of primers at the origin of replication.

Information-Theoretic Considerations Concerning the Origin of Life

NASA Astrophysics Data System (ADS)

Adami, Christoph

2015-09-01

Research investigating the origins of life usually either focuses on exploring possible life-bearing chemistries in the pre-biotic Earth, or else on synthetic approaches. Comparatively little work has explored fundamental issues concerning the spontaneous emergence of life using only concepts (such as information and evolution) that are divorced from any particular chemistry. Here, I advocate studying the probability of spontaneous molecular self-replication as a function of the information contained in the replicator, and the environmental conditions that might enable this emergence. I show (under certain simplifying assumptions) that the probability to discover a self-replicator by chance depends exponentially on the relative rate of formation of the monomers. If the rate at which monomers are formed is somewhat similar to the rate at which they would occur in a self-replicating polymer, the likelihood to discover such a replicator by chance is increased by many orders of magnitude. I document such an increase in searches for a self-replicator within the digital life system avida.

How and What Can We Learn from Replicating Historical Experiments? A Case Study.

ERIC Educational Resources Information Center

Hottecke, Dietmar

2000-01-01

Argues that historical experiments replicated as closely as possible to the original enable experiences that are intellectual as well as sensual in kind. Demonstrates that learning by replicating makes it possible to learn on different levels of human activity related to mind and body. Introduces a case study of the replication of the kind of…

pUL34 binding near the human cytomegalovirus origin of lytic replication enhances DNA replication and viral growth.

PubMed

Slayton, Mark; Hossain, Tanvir; Biegalke, Bonita J

2018-05-01

The human cytomegalovirus (HCMV) UL34 gene encodes sequence-specific DNA-binding proteins (pUL34) which are required for viral replication. Interactions of pUL34 with DNA binding sites represses transcription of two viral immune evasion genes, US3 and US9. 12 additional predicted pUL34-binding sites are present in the HCMV genome (strain AD169) with three binding sites concentrated near the HCMV origin of lytic replication (oriLyt). We used ChIP-seq analysis of pUL34-DNA interactions to confirm that pUL34 binds to the oriLyt region during infection. Mutagenesis of the UL34-binding sites in an oriLyt-containing plasmid significantly reduced viral-mediated oriLyt-dependent DNA replication. Mutagenesis of these sites in the HCMV genome reduced the replication efficiencies of the resulting viruses. Protein-protein interaction analyses demonstrated that pUL34 interacts with the viral proteins IE2, UL44, and UL84, that are essential for viral DNA replication, suggesting that pUL34-DNA interactions in the oriLyt region are involved in the DNA replication cascade. Copyright © 2018 Elsevier Inc. All rights reserved.

Probable secondary transmission of antimicrobial-resistant Escherichia coli between people living with and without pets

PubMed Central

CHUNG, Yeon Soo; PARK, Young Kyung; PARK, Yong Ho; PARK, Kun Taek

2017-01-01

Companion animals are considered as one of the reservoirs of antimicrobial-resistant (AR) bacteria that can be cross-transmitted to humans. However, limited information is available on the possibility of AR bacteria originating from companion animals being transmitted secondarily from owners to non-owners sharing the same space. To address this issue, the present study investigated clonal relatedness among AR E. coli isolated from dog owners and non-owners in the same college classroom or household. Anal samples (n=48) were obtained from 14 owners and 34 non-owners; 31 E. coli isolates were collected (nine from owners and 22 from non-owners). Of 31 E. coli, 20 isolates (64.5%) were resistant to at least one antimicrobial, and 16 isolates (51.6%) were determined as multi-drug resistant E. coli. Six isolates (19.4%) harbored integrase genes (five harbored class I integrase gene and one harbored class 2 integrase gene, respectively). Pulsed-field gel electrophoretic analysis identified three different E. coli clonal sets among isolates, indicating that cross-transmission of AR E. coli can easily occur between owners and non-owners. The findings emphasize a potential risk of spread of AR bacteria originating from pets within human communities, once they are transferred to humans. Further studies are needed to evaluate the exact risk and identify the risk factors of secondarily transmission by investigating larger numbers of isolates from pets, their owners and non-owners in a community. PMID:28190823

Probable secondary transmission of antimicrobial-resistant Escherichia coli between people living with and without pets.

PubMed

Chung, Yeon Soo; Park, Young Kyung; Park, Yong Ho; Park, Kun Taek

2017-03-18

Companion animals are considered as one of the reservoirs of antimicrobial-resistant (AR) bacteria that can be cross-transmitted to humans. However, limited information is available on the possibility of AR bacteria originating from companion animals being transmitted secondarily from owners to non-owners sharing the same space. To address this issue, the present study investigated clonal relatedness among AR E. coli isolated from dog owners and non-owners in the same college classroom or household. Anal samples (n=48) were obtained from 14 owners and 34 non-owners; 31 E. coli isolates were collected (nine from owners and 22 from non-owners). Of 31 E. coli, 20 isolates (64.5%) were resistant to at least one antimicrobial, and 16 isolates (51.6%) were determined as multi-drug resistant E. coli. Six isolates (19.4%) harbored integrase genes (five harbored class I integrase gene and one harbored class 2 integrase gene, respectively). Pulsed-field gel electrophoretic analysis identified three different E. coli clonal sets among isolates, indicating that cross-transmission of AR E. coli can easily occur between owners and non-owners. The findings emphasize a potential risk of spread of AR bacteria originating from pets within human communities, once they are transferred to humans. Further studies are needed to evaluate the exact risk and identify the risk factors of secondarily transmission by investigating larger numbers of isolates from pets, their owners and non-owners in a community.

Form and function of topologically associating genomic domains in budding yeast.

PubMed

Eser, Umut; Chandler-Brown, Devon; Ay, Ferhat; Straight, Aaron F; Duan, Zhijun; Noble, William Stafford; Skotheim, Jan M

2017-04-11

The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.

The human GINS complex associates with Cdc45 and MCM and is essential for DNA replication

PubMed Central

Aparicio, Tomás; Guillou, Emmanuelle; Coloma, Javier; Montoya, Guillermo; Méndez, Juan

2009-01-01

The GINS complex, originally discovered in Saccharomyces cerevisiae and Xenopus laevis, binds to DNA replication origins shortly before the onset of S phase and travels with the replication forks after initiation. In this study we present a detailed characterization of the human GINS (hGINS) homolog. Using new antibodies that allow the detection of endogenous hGINS in cells and tissues, we have examined its expression, abundance, subcellular localization and association with other DNA replication proteins. Expression of hGINS is restricted to actively proliferating cells. During the S phase, hGINS becomes part of a Cdc45–MCM–GINS (CMG) complex that is assembled on chromatin. Down-regulation of hGINS destabilizes CMG, causes a G1–S arrest and slows down ongoing DNA replication, effectively blocking cell proliferation. Our data support the notion that hGINS is an essential component of the human replisome. PMID:19223333

Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers.

PubMed

Nagy, B; Szmolka, A; Smole Možina, S; Kovač, J; Strauss, A; Schlager, S; Beutlich, J; Appel, B; Lušicky, M; Aprikian, P; Pászti, J; Tóth, I; Kugler, R; Wagner, M

2015-09-16

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe. Copyright © 2015 Elsevier B.V. All rights reserved.

A DNA Sequence Element That Advances Replication Origin Activation Time in Saccharomyces cerevisiae

PubMed Central

Pohl, Thomas J.; Kolor, Katherine; Fangman, Walton L.; Brewer, Bonita J.; Raghuraman, M. K.

2013-01-01

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time. PMID:24022751

MUTATIONAL SPECTRUM AND RECOMBINOGENIC EFFECTS INDUCED BY AMINOFLUORENE ADDUCTS IN BACTERIOPHAGE M13 (JOURNAL VERSION)

EPA Science Inventory

Double stranded replicative form (RFI) DNA of bacteriophage M13mp10 has been modified in vitro to various extents with N-hydroxy-2-aminofluorene (N-OH-AF) and then transfected into E. coli cells. HPLC analysis of the modified DNA shows that only dG-C8-AF adducts are formed. Appro...

Effectiveness of Halogen-Based Disinfectants Against Acinetobacter Baumannii: Wound Care and Environmental Decontamination

DTIC Science & Technology

2006-11-01

EFFECTIVENESS OF HALOGEN-BASED DISINFECTANTS AGAINST Acinetobacter baumannii: WOUND CARE AND ENVIROMENTAL DECONTAMINATION James...a standard E. coli comparator, in a novel bacterial culture system that incorporated a three log range of organic growth media concentrations. We...report the highest dilutions of stock disinfectant able to inhibit replication or kill the bacteria , denoted as the maximum inhibitory dilution

Enhanced detection and identification of Shiga toxin 1 and 2 from pathogenic bacteria by MALDI-TOF-TOF-MS/MS-PSD and top-down proteomic analysis

USDA-ARS?s Scientific Manuscript database

Shiga toxin producing Escherichia coli (STEC) represent a continuing threat to the Nation’s food supply and public health. Shiga toxin genes (stx) are encoded in lambda-like bacteriophages whose genome is inserted into the bacterial DNA. Environmental stress can trigger bacteriophage replication a...

Rifampin-stimulated uv resistance of phage lambda on Escherichia coli K12

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bronner, C.E.; Fluke, D.J.; Pollard, E.C.

1983-01-01

The plaque survival of uv-irradiated phage lambda on excision-proficient E.coli strain AB1157 is greater if the host cells are exposed to rifampin for 10 minutes prior to infection. This repair is accompanied by little or no clear-plaque mutagenesis. Host cells uv-irradiated and incubated for 30 minutes in growth medium prior to treatment with rifampin show some Weigle-reactivation in addition to the repair stimulated by rifampin. Some clear-plaque Weigle-mutagenesis is also observed in the presence of rifampin: however, the amount is less than that seen in the absence of rifampin treatment. In contrast, the uv sensitivity of lambda on strain AB1886,more » an excision-repair deficient mutant, is unchanged by pre-treating the cells with rifampin, and no Weigle-reactivation is observed. These results suggest that repair of lambda on unirradiated cells in the presence of rifampin is an excision-dependent, error-free phenomenon. Since initiation of replication of lambda DNA requires RNA polymerase, and since rifampin blocks transcription by that polymerase, the effect of rifampin on the survival of lambda may be to delay phage replication, thereby allowing more time for excision repair to operate. 6 figures.« less

Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage

PubMed Central

Sattar, Sadia; Bennett, Nicholas J.; Wen, Wesley X.; Guthrie, Jenness M.; Blackwell, Len F.; Conway, James F.; Rakonjac, Jasna

2015-01-01

F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70∘C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative “dipstick” lateral flow diagnostic assay for human plasma fibronectin. PMID:25941520

Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage.

PubMed

Sattar, Sadia; Bennett, Nicholas J; Wen, Wesley X; Guthrie, Jenness M; Blackwell, Len F; Conway, James F; Rakonjac, Jasna

2015-01-01

F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

Versatile plasmid-based expression systems for Gram-negative bacteria--General essentials exemplified with the bacterium Ralstonia eutropha H16.

PubMed

Gruber, Steffen; Schwab, Helmut; Koefinger, Petra

2015-12-25

The Gram-negative bacterium Escherichia coli is currently the most efficient and widely used prokaryotic host for recombinant protein and metabolite production. However, due to some limitations and to various interesting features of other Gram-negative bacteria efficient vector systems applicable to a broad range are desired. Basic building blocks for plasmid-based vectors include besides the need for a suitable selection marker in the first line a proper replication and maintenance system. In addition to these basic requirements, further elements are needed for Gram-negative bacteria beyond E. coli, such as Pseudomonas pudita, Ralstonia eutropha, Burkholderia glumae or Acinetobacter sp.. Established building blocks have to be adapted and new building blocks providing the desired functions need to be identified and exploited. This minireview addresses so far described and used genetic elements for broad host range replication, efficient plasmid maintenance, and conjugative plasmid transfer as well as expression elements and protein secretion signals. The industrially important bacterium R. eutropha H16 was chosen as a model organism to provide specific data on the effectivity and utility of building blocks based on such genetic elements. Copyright © 2015 Elsevier B.V. All rights reserved.

An origin-deficient yeast artificial chromosome triggers a cell cycle checkpoint.

PubMed

van Brabant, A J; Buchanan, C D; Charboneau, E; Fangman, W L; Brewer, B J

2001-04-01

Checkpoint controls coordinate entry into mitosis with the completion of DNA replication. Depletion of nucleotide precursors by treatment with the drug hydroxyurea triggers such a checkpoint response. However, it is not clear whether the signal for this hydroxyurea-induced checkpoint pathway is the presence of unreplicated DNA, or rather the persistence of single-stranded or damaged DNA. In a yeast artificial chromosome (YAC) we have engineered an approximately 170 kb region lacking efficient replication origins that allows us to explore the specific effects of unreplicated DNA on cell cycle progression. Replication of this YAC extends the length of S phase and causes cells to engage an S/M checkpoint. In the absence of Rad9 the YAC becomes unstable, undergoing deletions within the origin-free region.

When is a failure to replicate not a type II error?

PubMed

Vasconcelos, Marco; Urcuioli, Peter J; Lionello-DeNolf, Karen M

2007-05-01

Zentall and Singer (2007) challenge our conclusion that the work-ethic effect reported by Clement, Feltus, Kaiser, and Zentall (2000) may have been a Type I error by arguing that (a) the effect has been extensively replicated and (b) the amount of overtraining our pigeons received may not have been sufficient to produce it. We believe that our conclusion is warranted because (a) the original effect has not been replicated despite multiple attempts to do so and (b) the statement that more extended overtraining may be needed itself suggests that the original effect is not reliable.

Life as a cosmic imperative?

PubMed

de Duve, Christian

2011-02-13

The origin of life on Earth may be divided into two stages separated by the first appearance of replicable molecules, most probably of RNA. The first stage depended exclusively on chemistry. The second stage likewise involved chemistry, but with the additional participation of selection, a necessary concomitant of inevitable replication accidents. Consideration of these two processes suggests that the origin of life may have been close to obligatory under the physical-chemical conditions that prevailed at the site of its birth. Thus, an extrasolar planet in which those conditions were replicated appears as a probable site for the appearance of extra-terrestrial life.

When Is a Failure to Replicate Not a Type II Error?

PubMed Central

Vasconcelos, Marco; Urcuioli, Peter J; Lionello-DeNolf, Karen M

2007-01-01

Zentall and Singer (2007) challenge our conclusion that the work-ethic effect reported by Clement, Feltus, Kaiser, and Zentall (2000) may have been a Type I error by arguing that (a) the effect has been extensively replicated and (b) the amount of overtraining our pigeons received may not have been sufficient to produce it. We believe that our conclusion is warranted because (a) the original effect has not been replicated despite multiple attempts to do so and (b) the statement that more extended overtraining may be needed itself suggests that the original effect is not reliable. PMID:17575905

Dynamics of Leading-strand Lesion Skipping by the Replisome

PubMed Central

Yeeles, Joseph T.P.; Marians, Kenneth J.

2013-01-01

SUMMARY The E. coli replisome stalls transiently when it encounters a lesion in the leading-strand template, skipping over the damage by reinitiating replication at a new primer synthesized downstream by the primase. We report here that template unwinding and lagging-strand synthesis continue downstream of the lesion at a reduced rate after replisome stalling, that one replisome is capable of skipping multiple lesions, and that the rate limiting steps of replication restart involve the synthesis and activation of the new primer downstream. We also find little support for the concept that polymerase uncoupling, where extensive lagging-strand synthesis proceeds downstream in the absence of leading-strand synthesis, involves physical separation of the leading-strand polymerase from the replisome. Instead, our data indicate that extensive uncoupled replication likely results from a failure of the leading-strand polymerase still associated with the DNA helicase and the lagging-strand polymerase that are proceeding downstream to reinitiate synthesis. PMID:24268579

Regulation of DNA replication during development

PubMed Central

Nordman, Jared; Orr-Weaver, Terry L.

2012-01-01

As development unfolds, DNA replication is not only coordinated with cell proliferation, but is regulated uniquely in specific cell types and organs. This differential regulation of DNA synthesis requires crosstalk between DNA replication and differentiation. This dynamic aspect of DNA replication is highlighted by the finding that the distribution of replication origins varies between differentiated cell types and changes with differentiation. Moreover, differential DNA replication in some cell types can lead to increases or decreases in gene copy number along chromosomes. This review highlights the recent advances and technologies that have provided us with new insights into the developmental regulation of DNA replication. PMID:22223677

Draft Genome Sequences of Three European Laboratory Derivatives from Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933, Including Two Plasmids

PubMed Central

Fellner, Lea; Huptas, Christopher; Simon, Svenja; Mühlig, Anna; Neuhaus, Klaus

2016-01-01

Escherichia coli O157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagic E. coli (EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. PMID:27056239

Implications of down regulation of rcsA and rcsA-regulated colanic acid biosynthesis genes in increased acid sensitivity and enhanced curli and biofilm production in enterohemorrhagic Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

Enterohemorrhagic Escherichia coli (E. coli) O157:H7 strain 86-24, originally linked to a disease outbreak in the western USA in 1982, exhibits acid resistance as indicated by its ability to survive exposure to acidic conditions (pH2.5) for several hours. The strain 86-24 is a poor biofilm producer ...

Phosphorylated STAT5 directly facilitates parvovirus B19 DNA replication in human erythroid progenitors through interaction with the MCM complex

PubMed Central

Ganaie, Safder S.; Zou, Wei; Xu, Peng; Deng, Xuefeng; Kleiboeker, Steve

2017-01-01

Productive infection of human parvovirus B19 (B19V) exhibits high tropism for burst forming unit erythroid (BFU-E) and colony forming unit erythroid (CFU-E) progenitor cells in human bone marrow and fetal liver. This exclusive restriction of the virus replication to human erythroid progenitor cells is partly due to the intracellular factors that are essential for viral DNA replication, including erythropoietin signaling. Efficient B19V replication also requires hypoxic conditions, which upregulate the signal transducer and activator of transcription 5 (STAT5) pathway, and phosphorylated STAT5 is essential for virus replication. In this study, our results revealed direct involvement of STAT5 in B19V DNA replication. Consensus STAT5-binding elements were identified adjacent to the NS1-binding element within the minimal origins of viral DNA replication in the B19V genome. Phosphorylated STAT5 specifically interacted with viral DNA replication origins both in vivo and in vitro, and was actively recruited within the viral DNA replication centers. Notably, STAT5 interacted with minichromosome maintenance (MCM) complex, suggesting that STAT5 directly facilitates viral DNA replication by recruiting the helicase complex of the cellular DNA replication machinery to viral DNA replication centers. The FDA-approved drug pimozide dephosphorylates STAT5, and it inhibited B19V replication in ex vivo expanded human erythroid progenitors. Our results demonstrated that pimozide could be a promising antiviral drug for treatment of B19V-related diseases. PMID:28459842

Modeling the infection dynamics of bacteriophages in enteric Escherichia coli: estimating the contribution of transduction to antimicrobial gene spread.

PubMed

Volkova, Victoriya V; Lu, Zhao; Besser, Thomas; Gröhn, Yrjö T

2014-07-01

Animal-associated bacterial communities are infected by bacteriophages, although the dynamics of these infections are poorly understood. Transduction by bacteriophages may contribute to transfer of antimicrobial resistance genes, but the relative importance of transduction among other gene transfer mechanisms is unknown. We therefore developed a candidate deterministic mathematical model of the infection dynamics of enteric coliphages in commensal Escherichia coli in the large intestine of cattle. We assumed the phages were associated with the intestine and were predominantly temperate. Model simulations demonstrated how, given the bacterial ecology and infection dynamics, most (>90%) commensal enteric E. coli bacteria may become lysogens of enteric coliphages during intestinal transit. Using the model and the most liberal assumptions about transduction efficiency and resistance gene frequency, we approximated the upper numerical limits ("worst-case scenario") of gene transfer through specialized and generalized transduction in E. coli by enteric coliphages when the transduced genetic segment is picked at random. The estimates were consistent with a relatively small contribution of transduction to lateral gene spread; for example, generalized transduction delivered the chromosomal resistance gene to up to 8 E. coli bacteria/hour within the population of 1.47 × 10(8) E. coli bacteria/liter luminal contents. In comparison, the plasmidic blaCMY-2 gene carried by ~2% of enteric E. coli was transferred by conjugation at a rate at least 1.4 × 10(3) times greater than our generalized transduction estimate. The estimated numbers of transductants varied nonlinearly depending on the ecology of bacteria available for phages to infect, that is, on the assumed rates of turnover and replication of enteric E. coli. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Modeling the Infection Dynamics of Bacteriophages in Enteric Escherichia coli: Estimating the Contribution of Transduction to Antimicrobial Gene Spread

PubMed Central

Lu, Zhao; Besser, Thomas; Gröhn, Yrjö T.

2014-01-01

Animal-associated bacterial communities are infected by bacteriophages, although the dynamics of these infections are poorly understood. Transduction by bacteriophages may contribute to transfer of antimicrobial resistance genes, but the relative importance of transduction among other gene transfer mechanisms is unknown. We therefore developed a candidate deterministic mathematical model of the infection dynamics of enteric coliphages in commensal Escherichia coli in the large intestine of cattle. We assumed the phages were associated with the intestine and were predominantly temperate. Model simulations demonstrated how, given the bacterial ecology and infection dynamics, most (>90%) commensal enteric E. coli bacteria may become lysogens of enteric coliphages during intestinal transit. Using the model and the most liberal assumptions about transduction efficiency and resistance gene frequency, we approximated the upper numerical limits (“worst-case scenario”) of gene transfer through specialized and generalized transduction in E. coli by enteric coliphages when the transduced genetic segment is picked at random. The estimates were consistent with a relatively small contribution of transduction to lateral gene spread; for example, generalized transduction delivered the chromosomal resistance gene to up to 8 E. coli bacteria/hour within the population of 1.47 × 108 E. coli bacteria/liter luminal contents. In comparison, the plasmidic blaCMY-2 gene carried by ∼2% of enteric E. coli was transferred by conjugation at a rate at least 1.4 × 103 times greater than our generalized transduction estimate. The estimated numbers of transductants varied nonlinearly depending on the ecology of bacteria available for phages to infect, that is, on the assumed rates of turnover and replication of enteric E. coli. PMID:24814786

Replication of damaged DNA in vitro is blocked by p53

PubMed Central

Zhou, Jianmin; Prives, Carol

2003-01-01

The tumor suppressor protein p53 may have other roles and functions in addition to its well-documented ability to serve as a sequence-specific transcriptional activator in response to DNA damage. We showed previously that p53 can block the replication of polyomavirus origin-containing DNA (Py ori-DNA) in vitro when p53 binding sites are present on the late side of the Py ori. Here we have both further extended these observations and have also examined whether p53 might be able to bind directly to and inhibit the replication of damaged DNA. We found that p53 strongly inhibits replication of γ-irradiated Py ori-DNA and such inhibition requires both the central DNA binding domain and the extreme C-terminus of the p53 protein. An endogenous p53 binding site lies within the Py origin and is required for the ability of p53 to block initiation of replication from γ-irradiated Py ori-DNA, suggesting the possibility of DNA looping caused by p53 binding both non-specifically to sites of DNA damage and specifically to the endogenous site in the polyomavirus origin. Our results thus suggest the possibility that under some circumstances p53 might serve as a direct regulator of DNA replication and suggest as well an additional function for cooperation between its two autonomous DNA binding domains. PMID:12853603

The fork and the kinase: a DNA replication tale from a CHK1 perspective.

PubMed

González Besteiro, Marina A; Gottifredi, Vanesa

2015-01-01

Replication fork progression is being continuously hampered by exogenously introduced and naturally occurring DNA lesions and other physical obstacles. Checkpoint kinase 1 (Chk1) is activated at replication forks that encounter damaged DNA. Subsequently, Chk1 inhibits the initiation of new replication factories and stimulates the firing of dormant origins (those in the vicinity of stalled forks). Chk1 also avoids fork collapse into DSBs (double strand breaks) and promotes fork elongation. At the molecular level, the current model considers stalled forks as the site of Chk1 activation and the nucleoplasm as the location where Chk1 phosphorylates target proteins. This model certainly serves to explain how Chk1 modulates origin firing, but how Chk1 controls the fate of stalled forks is less clear. Interestingly, recent reports demonstrating that Chk1 phosphorylates chromatin-bound proteins and even holds kinase-independent functions might shed light on how Chk1 contributes to the elongation of damaged DNA. Indeed, such findings have unveiled a puzzling connection between Chk1 and DNA lesion bypass, which might be central to promoting fork elongation and checkpoint attenuation. In summary, Chk1 is a multifaceted and versatile signaling factor that acts at ongoing forks and replication origins to determine the extent and quality of the cellular response to replication stress. Copyright © 2014 Elsevier B.V. All rights reserved.

A structural role for the PHP domain in E. coli DNA polymerase III.

PubMed

Barros, Tiago; Guenther, Joel; Kelch, Brian; Anaya, Jordan; Prabhakar, Arjun; O'Donnell, Mike; Kuriyan, John; Lamers, Meindert H

2013-05-14

In addition to the core catalytic machinery, bacterial replicative DNA polymerases contain a Polymerase and Histidinol Phosphatase (PHP) domain whose function is not entirely understood. The PHP domains of some bacterial replicases are active metal-dependent nucleases that may play a role in proofreading. In E. coli DNA polymerase III, however, the PHP domain has lost several metal-coordinating residues and is likely to be catalytically inactive. Genomic searches show that the loss of metal-coordinating residues in polymerase PHP domains is likely to have coevolved with the presence of a separate proofreading exonuclease that works with the polymerase. Although the E. coli Pol III PHP domain has lost metal-coordinating residues, the structure of the domain has been conserved to a remarkable degree when compared to that of metal-binding PHP domains. This is demonstrated by our ability to restore metal binding with only three point mutations, as confirmed by the metal-bound crystal structure of this mutant determined at 2.9 Å resolution. We also show that Pol III, a large multi-domain protein, unfolds cooperatively and that mutations in the degenerate metal-binding site of the PHP domain decrease the overall stability of Pol III and reduce its activity. While the presence of a PHP domain in replicative bacterial polymerases is strictly conserved, its ability to coordinate metals and to perform proofreading exonuclease activity is not, suggesting additional non-enzymatic roles for the domain. Our results show that the PHP domain is a major structural element in Pol III and its integrity modulates both the stability and activity of the polymerase.

The Impact of Media, Phylogenetic Classification, and E. coli Pathotypes on Biofilm Formation in Extraintestinal and Commensal E. coli From Humans and Animals.

PubMed

Nielsen, Daniel W; Klimavicz, James S; Cavender, Tia; Wannemuehler, Yvonne; Barbieri, Nicolle L; Nolan, Lisa K; Logue, Catherine M

2018-01-01

Extraintestinal pathogenic Escherichia coli (ExPEC) include avian pathogenic E. coli (APEC), neonatal meningitis E. coli (NMEC), and uropathogenic E. coli (UPEC) and are responsible for significant animal and human morbidity and mortality. This study sought to investigate if biofilm formation by ExPEC likely contributes to these losses since biofilms are associated with recurrent urinary tract infections, antibiotic resistance, and bacterial exchange of genetic material. Therefore, the goal of this study was to examine differences in biofilm formation among a collection of ExPEC and to ascertain if there is a relationship between their ability to produce biofilms and their assignment to phylogenetic groups in three media types - M63, diluted TSB, and BHI. Our results suggest that ExPEC produce relatively different levels of biofilm formation in the media tested as APEC (70.4%, p = 0.0064) and NMEC (84.4%, p = 0.0093) isolates were poor biofilm formers in minimal medium M63 while UPEC isolates produced significantly higher ODs under nutrient-limited conditions with 25% of strains producing strong biofilms in diluted TSB ( p = 0.0204). Additionally, E. coli phylogenetic assignment using Clermont's original and revised typing scheme demonstrated significant differences among the phylogenetic groups in the different media. When the original phylogenetic group isolates previously typed as group D were phylogenetically typed under the revised scheme and examined, they showed substantial variation in their ability to form biofilms, which may explain the significant values of revised phylogenetic groups E and F in M63 ( p = 0.0291, p = 0.0024). Our data indicates that biofilm formation is correlated with phylogenetic classification and subpathotype or commensal grouping of E. coli strains.

Biochemistry and genetics of autotrophy in Methanococcus. Progress report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Whitman, W.B.

In the last two years of this research, the most exciting results have come from the work on the genetics of methanococci. First, the author demonstrated that the cryptic plasmid from Methanococcus maripaludis C5, pURB500, could be transformed into Methanococcus maripaludis JJ. Strain JJ is the type strain of M. maripaludis and has only about 65% DNA:DNA hybridization to strain C5. Because of the low relatedness of these strains, it was not obvious that pURB500 could be transferred between them. This goal was achieved by first transforming strain C5 with a series of suicide plasmids containing the pac cassette, whichmore » possessed the selectable puromycin resistance marker, and different cloned fragments of pURB500. From the puromycin-resistant transformants, a plasmid was isolated that transformed strain JJ. However, when this plasmid was electroporated into E. coli, only rearrangement products were obtained that contained small portions of the original pURB500. These plasmids no longer transformed Methanococcus. While these experiments did not yield a shuttle vector, they demonstrated that pURB500 could replicate in strain JJ.« less

A novel prokaryotic promoter identified in the genome of some monopartite begomoviruses.

PubMed

Wang, Wei-Chen; Hsu, Yau-Heiu; Lin, Na-Sheng; Wu, Chia-Ying; Lai, Yi-Chin; Hu, Chung-Chi

2013-01-01

Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses.

A Novel Prokaryotic Promoter Identified in the Genome of Some Monopartite Begomoviruses

PubMed Central

Wang, Wei-Chen; Hsu, Yau-Heiu; Lin, Na-Sheng; Wu, Chia-Ying; Lai, Yi-Chin; Hu, Chung-Chi

2013-01-01

Geminiviruses are known to exhibit both prokaryotic and eukaryotic features in their genomes, with the ability to express their genes and even replicate in bacterial cells. We have demonstrated previously the existence of unit-length single-stranded circular DNAs of Ageratum yellow vein virus (AYVV, a species in the genus Begomovirus, family Geminiviridae) in Escherichia coli cells, which prompted our search for unknown prokaryotic functions in the begomovirus genomes. By using a promoter trapping strategy, we identified a novel prokaryotic promoter, designated AV3 promoter, in nts 762-831 of the AYVV genome. Activity assays revealed that the AV3 promoter is strong, unidirectional, and constitutive, with an endogenous downstream ribosome binding site and a translatable short open reading frame of eight amino acids. Sequence analyses suggested that the AV3 promoter might be a remnant of prokaryotic ancestors that could be related to certain promoters of bacteria from marine or freshwater environments. The discovery of the prokaryotic AV3 promoter provided further evidence for the prokaryotic origin in the evolutionary history of geminiviruses. PMID:23936138

Bayesian evaluation of effect size after replicating an original study

PubMed Central

van Aert, Robbie C. M.; van Assen, Marcel A. L. M.

2017-01-01

The vast majority of published results in the literature is statistically significant, which raises concerns about their reliability. The Reproducibility Project Psychology (RPP) and Experimental Economics Replication Project (EE-RP) both replicated a large number of published studies in psychology and economics. The original study and replication were statistically significant in 36.1% in RPP and 68.8% in EE-RP suggesting many null effects among the replicated studies. However, evidence in favor of the null hypothesis cannot be examined with null hypothesis significance testing. We developed a Bayesian meta-analysis method called snapshot hybrid that is easy to use and understand and quantifies the amount of evidence in favor of a zero, small, medium and large effect. The method computes posterior model probabilities for a zero, small, medium, and large effect and adjusts for publication bias by taking into account that the original study is statistically significant. We first analytically approximate the methods performance, and demonstrate the necessity to control for the original study’s significance to enable the accumulation of evidence for a true zero effect. Then we applied the method to the data of RPP and EE-RP, showing that the underlying effect sizes of the included studies in EE-RP are generally larger than in RPP, but that the sample sizes of especially the included studies in RPP are often too small to draw definite conclusions about the true effect size. We also illustrate how snapshot hybrid can be used to determine the required sample size of the replication akin to power analysis in null hypothesis significance testing and present an easy to use web application (https://rvanaert.shinyapps.io/snapshot/) and R code for applying the method. PMID:28388646

UV Damage-Induced Phosphorylation of HBO1 Triggers CRL4DDB2-Mediated Degradation To Regulate Cell Proliferation

PubMed Central

Matsunuma, Ryoichi; Ohhata, Tatsuya; Kitagawa, Kyoko; Sakai, Satoshi; Uchida, Chiharu; Shiotani, Bunsyo; Matsumoto, Masaki; Nakayama, Keiichi I.; Ogura, Hiroyuki; Shiiya, Norihiko; Kitagawa, Masatoshi

2015-01-01

Histone acetyltransferase binding to ORC-1 (HBO1) is a critically important histone acetyltransferase for forming the prereplicative complex (pre-RC) at the replication origin. Pre-RC formation is completed by loading of the MCM2-7 heterohexameric complex, which functions as a helicase in DNA replication. HBO1 recruited to the replication origin by CDT1 acetylates histone H4 to relax the chromatin conformation and facilitates loading of the MCM complex onto replication origins. However, the acetylation status and mechanism of regulation of histone H3 at replication origins remain elusive. HBO1 positively regulates cell proliferation under normal cell growth conditions. Whether HBO1 regulates proliferation in response to DNA damage is poorly understood. In this study, we demonstrated that HBO1 was degraded after DNA damage to suppress cell proliferation. Ser50 and Ser53 of HBO1 were phosphorylated in an ATM/ATR DNA damage sensor-dependent manner after UV treatment. ATM/ATR-dependently phosphorylated HBO1 preferentially interacted with DDB2 and was ubiquitylated by CRL4DDB2. Replacement of endogenous HBO1 in Ser50/53Ala mutants maintained acetylation of histone H3K14 and impaired cell cycle regulation in response to UV irradiation. Our findings demonstrate that HBO1 is one of the targets in the DNA damage checkpoint. These results show that ubiquitin-dependent control of the HBO1 protein contributes to cell survival during UV irradiation. PMID:26572825

Human FEN1 Expression and Solubility Patterson in DNA Replication and Repair

DTIC Science & Technology

1999-11-03

following DNA replication from the simian virus 40 (SV40) origin of replication in vitro. Human FEN1, and FEN1 homologues from yeast to mammals, are...also implicated in different forms of DNA repair. In this thesis, I provide additional evidence supporting human FEN1’s role in nuclear DNA replication in...coincident with S phase DNA replication in both primary and transformed cells. Using novel antibodies that recognize human FEN1, I further show that very

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

PubMed

Naito, Y; Naito, T; Kobayashi, I

1998-01-01

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

High mutation rates limit evolutionary adaptation in Escherichia coli

PubMed Central

Wagner, Andreas

2018-01-01

Mutation is fundamental to evolution, because it generates the genetic variation on which selection can act. In nature, genetic changes often increase the mutation rate in systems that range from viruses and bacteria to human tumors. Such an increase promotes the accumulation of frequent deleterious or neutral alleles, but it can also increase the chances that a population acquires rare beneficial alleles. Here, we study how up to 100-fold increases in Escherichia coli’s genomic mutation rate affect adaptive evolution. To do so, we evolved multiple replicate populations of asexual E. coli strains engineered to have four different mutation rates for 3000 generations in the laboratory. We measured the ability of evolved populations to grow in their original environment and in more than 90 novel chemical environments. In addition, we subjected the populations to whole genome population sequencing. Although populations with higher mutation rates accumulated greater genetic diversity, this diversity conveyed benefits only for modestly increased mutation rates, where populations adapted faster and also thrived better than their ancestors in some novel environments. In contrast, some populations at the highest mutation rates showed reduced adaptation during evolution, and failed to thrive in all of the 90 alternative environments. In addition, they experienced a dramatic decrease in mutation rate. Our work demonstrates that the mutation rate changes the global balance between deleterious and beneficial mutational effects on fitness. In contrast to most theoretical models, our experiments suggest that this tipping point already occurs at the modest mutation rates that are found in the wild. PMID:29702649

RuvAB and RecG are not essential for the recovery of DNA synthesis following UV-induced DNA damage in Escherichia coli.

PubMed Central

Donaldson, Janet R; Courcelle, Charmain T; Courcelle, Justin

2004-01-01

Ultraviolet light induces DNA lesions that block the progression of the replication machinery. Several models speculate that the resumption of replication following disruption by UV-induced DNA damage requires regression of the nascent DNA or migration of the replication machinery away from the blocking lesion to allow repair or bypass of the lesion to occur. Both RuvAB and RecG catalyze branch migration of three- and four-stranded DNA junctions in vitro and are proposed to catalyze fork regression in vivo. To examine this possibility, we characterized the recovery of DNA synthesis in ruvAB and recG mutants. We found that in the absence of either RecG or RuvAB, arrested replication forks are maintained and DNA synthesis is resumed with kinetics that are similar to those in wild-type cells. The data presented here indicate that RecG- or RuvAB-catalyzed fork regression is not essential for DNA synthesis to resume following arrest by UV-induced DNA damage in vivo. PMID:15126385

Single molecular biology: coming of age in DNA replication.

PubMed

Liu, Xiao-Jing; Lou, Hui-Qiang

2017-09-20

DNA replication is an essential process of the living organisms. To achieve precise and reliable replication, DNA polymerases play a central role in DNA synthesis. Previous investigations have shown that the average rates of DNA synthesis on the leading and lagging strands in a replisome must be similar to avoid the formation of significant gaps in the nascent strands. The underlying mechanism has been assumed to be coordination between leading- and lagging-strand polymerases. However, Kowalczykowski's lab members recently performed single molecule techniques in E. coli and showed the real-time behavior of a replisome. The leading- and lagging-strand polymerases function stochastically and independently. Furthermore, when a DNA polymerase is paused, the helicase slows down in a self-regulating fail-safe mechanism, akin to a ''dead-man's switch''. Based on the real-time single-molecular observation, the authors propose that leading- and lagging-strand polymerases synthesize DNA stochastically within a Gaussian distribution. Along with the development and application of single-molecule techniques, we will witness a new age of DNA replication and other biological researches.

Draft Genome Sequences of Three European Laboratory Derivatives from Enterohemorrhagic Escherichia coli O157:H7 Strain EDL933, Including Two Plasmids.

PubMed

Fellner, Lea; Huptas, Christopher; Simon, Svenja; Mühlig, Anna; Scherer, Siegfried; Neuhaus, Klaus

2016-04-07

Escherichia coliO157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagicE. coli(EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. Copyright © 2016 Fellner et al.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

PubMed Central

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873