Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro.

PubMed Central

Bae, Y S; Kawasaki, I; Ikeda, H; Liu, L F

1988-01-01

We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system. The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro. Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination. The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions. Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase. The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits. Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes. Images PMID:2832845

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

PubMed

Zsurka, Gábor; Kraytsberg, Yevgenia; Kudina, Tatiana; Kornblum, Cornelia; Elger, Christian E; Khrapko, Konstantin; Kunz, Wolfram S

2005-08-01

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

Recombinant DNA for Teachers.

ERIC Educational Resources Information Center

Duvall, James G., III

1992-01-01

A science teacher describes his experience at a workshop to learn to teach the Cold Spring Harbor DNA Science Laboratory Protocols. These protocols lead students through processes for taking E. coli cells and transforming them into a new antibiotic resistant strain. The workshop featured discussions of the role of DNA recombinant technology in…

RPA homologs and ssDNA processing during meiotic recombination.

PubMed

Ribeiro, Jonathan; Abby, Emilie; Livera, Gabriel; Martini, Emmanuelle

2016-06-01

Meiotic homologous recombination is a specialized process that involves homologous chromosome pairing and strand exchange to guarantee proper chromosome segregation and genetic diversity. The formation and repair of DNA double-strand breaks (DSBs) during meiotic recombination differs from those during mitotic recombination in that the homologous chromosome rather than the sister chromatid is the preferred repair template. The processing of single-stranded DNA (ssDNA) formed on intermediate recombination structures is central to driving the specific outcomes of DSB repair during meiosis. Replication protein A (RPA) is the main ssDNA-binding protein complex involved in DNA metabolism. However, the existence of RPA orthologs in plants and the recent discovery of meiosis specific with OB domains (MEIOB), a widely conserved meiosis-specific RPA1 paralog, strongly suggest that multiple RPA complexes evolved and specialized to subdivide their roles during DNA metabolism. Here we review ssDNA formation and maturation during mitotic and meiotic recombination underlying the meiotic specific features. We describe and discuss the existence and properties of MEIOB and multiple RPA subunits in plants and highlight how they can provide meiosis-specific fates to ssDNA processing during homologous recombination. Understanding the functions of these RPA homologs and how they interact with the canonical RPA subunits is of major interest in the fields of meiosis and DNA repair.

Homeologous plastid DNA transformation in tobacco is mediated by multiple recombination events.

PubMed Central

Kavanagh, T A; Thanh, N D; Lao, N T; McGrath, N; Peter, S O; Horváth, E M; Dix, P J; Medgyesy, P

1999-01-01

Efficient plastid transformation has been achieved in Nicotiana tabacum using cloned plastid DNA of Solanum nigrum carrying mutations conferring spectinomycin and streptomycin resistance. The use of the incompletely homologous (homeologous) Solanum plastid DNA as donor resulted in a Nicotiana plastid transformation frequency comparable with that of other experiments where completely homologous plastid DNA was introduced. Physical mapping and nucleotide sequence analysis of the targeted plastid DNA region in the transformants demonstrated efficient site-specific integration of the 7.8-kb Solanum plastid DNA and the exclusion of the vector DNA. The integration of the cloned Solanum plastid DNA into the Nicotiana plastid genome involved multiple recombination events as revealed by the presence of discontinuous tracts of Solanum-specific sequences that were interspersed between Nicotiana-specific markers. Marked position effects resulted in very frequent cointegration of the nonselected peripheral donor markers located adjacent to the vector DNA. Data presented here on the efficiency and features of homeologous plastid DNA recombination are consistent with the existence of an active RecA-mediated, but a diminished mismatch, recombination/repair system in higher-plant plastids. PMID:10388829

Mitochondrial DNA recombination in a free-ranging Australian lizard.

PubMed

Ujvari, Beata; Dowton, Mark; Madsen, Thomas

2007-04-22

Mitochondrial DNA (mtDNA) is the traditional workhorse for reconstructing evolutionary events. The frequent use of mtDNA in such analyses derives from the apparent simplicity of its inheritance: maternal and lacking bi-parental recombination. However, in hybrid zones, the reproductive barriers are often not completely developed, resulting in the breakdown of male mitochondrial elimination mechanisms, leading to leakage of paternal mitochondria and transient heteroplasmy, resulting in an increased possibility of recombination. Despite the widespread occurrence of heteroplasmy and the presence of the molecular machinery necessary for recombination, we know of no documented example of recombination of mtDNA in any terrestrial wild vertebrate population. By sequencing the entire mitochondrial genome (16761bp), we present evidence for mitochondrial recombination in the hybrid zone of two mitochondrial haplotypes in the Australian frillneck lizard (Chlamydosaurus kingii).

Cloning of habutobin cDNA and antithrombotic activity of recombinant protein

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

Sunagawa, Masanori; Nakamura, Mariko; Kosugi, Tadayoshi

2007-11-03

The habutobin cDNA was cloned from total RNA extracted from venom glands of Trimeresurus flavoviridis (the habu snake). The conceptual translation of 1539 bp of habutobin cDNA consists of 236 amino acids and its molecular weight is 25.7 kDa. Histidine (His)-tagged recombinant habutobin fusion protein, pET-r-habutobin and AcNPV-r-habutobin, was purified by bacterial system and baculoviral system, respectively. After refolding pET-r-habutobin, there were two protein bands at about 32 kDa and 65 kDa, indicating that habutobin might be produced as a monomer protein and processed to form two concatenated protein. Purified AcNPV-r-habutobin dose-dependently increased fibrin forming activity and inhibited collagen-induced aggregationmore » of rabbit washed platelets. Thus, AcNPV-r-habutobin produced by baculoviral system is very useful for study on structure-function relationship, which is necessary for developing an antithrombotic drug from habutobin.« less

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

PubMed Central

Zahn, Astrid; Eranki, Anil K.; Patenaude, Anne-Marie; Methot, Stephen P.; Fifield, Heather; Cortizas, Elena M.; Foster, Paul; Imai, Kohsuke; Durandy, Anne; Larijani, Mani; Verdun, Ramiro E.; Di Noia, Javier M.

2014-01-01

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR. PMID:24591601

Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1.

PubMed

Pryce, David W; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J

2009-03-24

DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion.

Recombination at DNA replication fork barriers is not universal and is differentially regulated by Swi1

PubMed Central

Pryce, David W.; Ramayah, Soshila; Jaendling, Alessa; McFarlane, Ramsay J.

2009-01-01

DNA replication stress has been implicated in the etiology of genetic diseases, including cancers. It has been proposed that genomic sites that inhibit or slow DNA replication fork progression possess recombination hotspot activity and can form potential fragile sites. Here we used the fission yeast, Schizosaccharomyces pombe, to demonstrate that hotspot activity is not a universal feature of replication fork barriers (RFBs), and we propose that most sites within the genome that form RFBs do not have recombination hotspot activity under nonstressed conditions. We further demonstrate that Swi1, the TIMELESS homologue, differentially controls the recombination potential of RFBs, switching between being a suppressor and an activator of recombination in a site-specific fashion. PMID:19273851

Direct evidence for homologous recombination in mussel (Mytilus galloprovincialis) mitochondrial DNA.

PubMed

Ladoukakis, E D; Zouros, E

2001-07-01

The assumption that animal mitochondrial DNA (mtDNA) does not undergo homologous recombination is based on indirect evidence, yet it has had an important influence on our understanding of mtDNA repair and mutation accumulation (and thus mitochondrial disease and aging) and on biohistorical inferences made from population data. Recently, several studies have suggested recombination in primate mtDNA on the basis of patterns of frequency distribution and linkage associations of mtDNA mutations in human populations, but others have failed to produce similar evidence. Here, we provide direct evidence for homologous mtDNA recombination in mussels, where heteroplasmy is the rule in males. Our results indicate a high rate of mtDNA recombination. Coupled with the observation that mammalian mitochondria contain the enzymes needed for the catalysis of homologous recombination, these findings suggest that animal mtDNA molecules may recombine regularly and that the extent to which this generates new haplotypes may depend only on the frequency of biparental inheritance of the mitochondrial genome. This generalization must, however, await evidence from animal species with typical maternal mtDNA inheritance.

The RECG1 DNA Translocase Is a Key Factor in Recombination Surveillance, Repair, and Segregation of the Mitochondrial DNA in Arabidopsis[OPEN

PubMed Central

Le Ret, Monique; Bergdoll, Marc; Bichara, Marc; Dietrich, André

2015-01-01

The mitochondria of flowering plants have considerably larger and more complex genomes than the mitochondria of animals or fungi, mostly due to recombination activities that modulate their genomic structures. These activities most probably participate in the repair of mitochondrial DNA (mtDNA) lesions by recombination-dependent processes. Rare ectopic recombination across short repeats generates new genomic configurations that contribute to mtDNA heteroplasmy, which drives rapid evolution of the sequence organization of plant mtDNAs. We found that Arabidopsis thaliana RECG1, an ortholog of the bacterial RecG translocase, is an organellar protein with multiple roles in mtDNA maintenance. RECG1 targets to mitochondria and plastids and can complement a bacterial recG mutant that shows defects in repair and replication control. Characterization of Arabidopsis recG1 mutants showed that RECG1 is required for recombination-dependent repair and for suppression of ectopic recombination in mitochondria, most likely because of its role in recovery of stalled replication forks. The analysis of alternative mitotypes present in a recG1 line and of their segregation following backcross allowed us to build a model to explain how a new stable mtDNA configuration, compatible with normal plant development, can be generated by stoichiometric shift. PMID:26462909

High-Efficiency Ligation and Recombination of DNA Fragments by Vertebrate Cells

NASA Astrophysics Data System (ADS)

Miller, Cynthia K.; Temin, Howard M.

1983-05-01

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.

Immunoglobulin class switch DNA recombination: induction, targeting and beyond

PubMed Central

Xu, Zhenming; Zan, Hong; Pone, Egest J.; Mai, Thach; Casali, Paolo

2012-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus is central to the maturation of the antibody response and critically requires the AID cytidine deaminase. CSR entails changes of the chromatin state and transcriptional activation of the IgH locus upstream and downstream switch (S) regions that are to undergo S-S DNA recombination, induction of AID, and targeting of CSR factors to S regions by 14-3-3 adaptors and as enabled by the transcription machinery and histone modifications. In this Review, we focus on recent advances in CSR induction and targeting. We also outline an integrated model of the assembly of macromolecular complexes that transduce critical epigenetic information to enzymatic effectors of the CSR machinery. PMID:22728528

Specific Modifications of Histone Tails, but Not DNA Methylation, Mirror the Temporal Variation of Mammalian Recombination Hotspots

PubMed Central

Zeng, Jia; Yi, Soojin V.

2014-01-01

Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called “recombination hotspot paradox”) remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy “bivalent” chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. PMID:25326136

Three Decades of Recombinant DNA.

ERIC Educational Resources Information Center

Palmer, Jackie

1985-01-01

Discusses highlights in the development of genetic engineering, examining techniques with recombinant DNA, legal and ethical issues, GenBank (a national database of nucleic acid sequences), and other topics. (JN)

Recombination or mutational hot spots in human mtDNA?

PubMed

Innan, Hideki; Nordborg, Magnus

2002-07-01

Awadalla, Eyre-Walker, and Maynard Smith (1999) recently argued that there might be recombination in human mitochondrial DNA (mtDNA). Their claim was based on their observation of decaying linkage disequilibrium (LD) as a function of physical distance. Their study was much criticized, and follow-up studies have failed to find any evidence for recombination. We argue that the criticisms levied, even if correct, could not possibly explain the findings of Awadalla, Eyre-Walker, and Maynard Smith (1999). Nonetheless, the test proposed by Awadalla, Eyre-Walker, and Maynard Smith (1999 ) is not robust because recombination is not the only explanation for decay of LD. We show that such a pattern can be caused by mutational hot spots as well. However, a closer look at the data suggests that the pattern observed was not caused by mutational hot spots but rather by chance. Thus, there appears to be no evidence for recombination in the mtDNA polymorphism data. In conclusion, we discuss the possibility of detecting recombination in mtDNA and the implications of its existence.

Evidence for recombination in scorpion mitochondrial DNA (Scorpiones: Buthidae).

PubMed

Gantenbein, Benjamin; Fet, Victor; Gantenbein-Ritter, Iris A; Balloux, François

2005-04-07

There has been very little undisputed evidence for recombination in animal mitochondrial DNA (mtDNA) provided so far. Previous unpublished results suggestive of mtDNA recombination in the scorpion family Buthidae, together with cytological evidence for a unique mechanism of mitochondrial fusion in that family, prompted us to investigate this group in more details. First, we sequenced the complete mtDNA genome of Mesobuthus gibbosus, and chose two genes opposing each other (16S and coxI). We then sequenced 150 individuals from the natural populations of four species of Buthidae (Old World genera Buthus and Mesobuthus). We observed strong evidence for widespread recombination through highly significant negative correlations between linkage disequilibrium and physical distance in three out of four species. The evidence is further confirmed when using five other tests for recombination and by the presence of a high amount of homoplasy in phylogenetic trees.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

PubMed

Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

2014-01-01

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

PubMed Central

Fritsch, Emilie S.; Chabbert, Christophe D.; Klaus, Bernd; Steinmetz, Lars M.

2014-01-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

A genome-wide map of mitochondrial DNA recombination in yeast.

PubMed

Fritsch, Emilie S; Chabbert, Christophe D; Klaus, Bernd; Steinmetz, Lars M

2014-10-01

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome

Specific modifications of histone tails, but not DNA methylation, mirror the temporal variation of mammalian recombination hotspots.

PubMed

Zeng, Jia; Yi, Soojin V

2014-10-16

Recombination clusters nonuniformly across mammalian genomes at discrete genomic loci referred to as recombination hotspots. Despite their ubiquitous presence, individual hotspots rapidly lose their activities, and the molecular and evolutionary mechanisms underlying such frequent hotspot turnovers (the so-called "recombination hotspot paradox") remain unresolved. Even though some sequence motifs are significantly associated with hotspots, multiple lines of evidence indicate that factors other than underlying sequences, such as epigenetic modifications, may affect the evolution of recombination hotspots. Thus, identifying epigenetic factors that covary with recombination at fine-scale is a promising step for this important research area. It was previously reported that recombination rates correlate with indirect measures of DNA methylation in the human genome. Here, we analyze experimentally determined DNA methylation and histone modification of human sperms, and show that the correlation between DNA methylation and recombination in long-range windows does not hold with respect to the spatial and temporal variation of recombination at hotspots. On the other hand, two histone modifications (H3K4me3 and H3K27me3) overlap extensively with recombination hotspots. Similar trends were observed in mice. These results indicate that specific histone modifications rather than DNA methylation are associated with the rapid evolution of recombination hotspots. Furthermore, many human recombination hotspots occupy "bivalent" chromatin regions that harbor both active (H3K4me3) and repressive (H3K27me3) marks. This may explain why human recombination hotspots tend to occur in nongenic regions, in contrast to yeast and Arabidopsis hotspots that are characterized by generally active chromatins. Our results highlight the dynamic epigenetic underpinnings of recombination hotspot evolution. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for

Development of the National Institutes of Health Guidelines for Recombinant DNA Research.

PubMed Central

Talbot, B

1983-01-01

Recombinant DNA is a technique of major importance in basic biomedical research and, increasingly, in industrial applications. Although the risks of this research remain hypothetical, scientists working in the field have spearheaded discussions of safety. The original National Institutes of Health (NIH) Guidelines for Recombinant DNA Research were issued in June 1976. They assigned each type of recombinant DNA experiment a specific level of "physical containment" and of "biological containment." Responsibility for overseeing the application of the guidelines belongs to the NIH Recombinant DNA Advisory Committee (RAC)--composed of scientists and laymen, including non-voting representatives from many Federal agencies--and local institutional biosafety committees at each university where recombinant DNA research is conducted. The NIH guidelines were subsequently adopted by other Federal agencies, but congressional proposals aimed at extending the guidelines to private industry did not result in national legislation. Some States and localities regulate recombinant DNA research, however, and many private companies have voluntarily submitted information on their recombinant DNA work for RAC and NIH approval. The NIH guidelines underwent a major revision in December 1978 and have been revised approximately every 3 months since then. NIH supports experiments to assess recombinant DNA risks and publishes and updates a plan for a risk assessment program. PMID:6611823

Ribosomal DNA replication fork barrier and HOT1 recombination hot spot: shared sequences but independent activities.

PubMed

Ward, T R; Hoang, M L; Prusty, R; Lau, C K; Keil, R L; Fangman, W L; Brewer, B J

2000-07-01

In the ribosomal DNA of Saccharomyces cerevisiae, sequences in the nontranscribed spacer 3' of the 35S ribosomal RNA gene are important to the polar arrest of replication forks at a site called the replication fork barrier (RFB) and also to the cis-acting, mitotic hyperrecombination site called HOT1. We have found that the RFB and HOT1 activity share some but not all of their essential sequences. Many of the mutations that reduce HOT1 recombination also decrease or eliminate fork arrest at one of two closely spaced RFB sites, RFB1 and RFB2. A simple model for the juxtaposition of RFB and HOT1 sequences is that the breakage of strands in replication forks arrested at RFB stimulates recombination. Contrary to this model, we show here that HOT1-stimulated recombination does not require the arrest of forks at the RFB. Therefore, while HOT1 activity is independent of replication fork arrest, HOT1 and RFB require some common sequences, suggesting the existence of a common trans-acting factor(s).

DNA Sequence-Mediated, Evolutionarily Rapid Redistribution of Meiotic Recombination Hotspots

PubMed Central

Wahls, Wayne P.; Davidson, Mari K.

2011-01-01

Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor. PMID:22084420

Can indirect tests detect a known recombination event in human mtDNA?

PubMed

White, Daniel James; Gemmell, Neil John

2009-07-01

Whether human mitochondrial DNA (mtDNA) recombines sufficiently to influence its evolution, evolutionary analysis, and disease etiology, remains equivocal. Overall, evidence from indirect studies of population genetic data suggests that recombination is not occurring at detectable levels. This may be explained by no, or low, recombination or, alternatively, current indirect tests may be incapable of detecting recombination in human mtDNA. To investigate the latter, we have tested whether six well-established indirect tests of recombination could detect recombination in a human mtDNA data set, in which its occurrence had been empirically confirmed. Three showed statistical evidence for recombination (r(2) vs. distance, the Homoplasy test, Neighborhood Similarity Score), and three did not (D' vs. distance, Max Chi Squared, Pairwise Homoplasy Index). Possible reasons for detection failure are discussed. Further, evidence from earlier studies suggesting a lack of recombination in mtDNA in humans is reconsidered, taking into account the appropriateness of the tests used, based on our new findings.

Single Molecule Study of DNA Organization and Recombination

NASA Astrophysics Data System (ADS)

Xiao, Botao

We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

Marker-Dependent Recombination in T4 Bacteriophage. IV. Recombinational Effects of Antimutator T4 DNA Polymerase

PubMed Central

Shcherbakov, V. P.; Plugina, L. A.; Kudryashova, E. A.

1995-01-01

Recombinational effects of the antimutator allele tsL42 of gene 43 of phage T4, encoding DNA polymerase, were studied in crosses between rIIB mutants. Recombination under tsL42-restricted conditions differed from the normal one in several respects: (1) basic recombination was enhanced, especially within very short distances; (2) mismatch repair tracts were shortened, while the contribution of mismatch repair to recombination was not changed; (3) marker interference at very short distances was augmented. We infer that the T4 DNA polymerase is directly involved in mismatch repair, performing both excision of a nonmatched single strand (by its 3' -> 5' exonuclease) and filling the resulting gap. A pathway for the mismatch repair was substantiated; it includes sequential action of endo VII (gp49) -> 3'->5' exonuclease (gp43) -> DNA polymerase (gp43) -> DNA ligase (gp30). It is argued that the marker interference at very short distances may result from the same sequence of events during the final processing of recombinational intermediates. PMID:7635281

Recombination Promoted by DNA Viruses: Phage λ to Herpes Simplex Virus

PubMed Central

Weller, Sandra K.; Sawitzke, James A.

2015-01-01

The purpose of this review is to explore recombination strategies in DNA viruses. Homologous recombination is a universal genetic process that plays multiple roles in the biology of all organisms, including viruses. Recombination and DNA replication are interconnected, with recombination being essential for repairing DNA damage and supporting replication of the viral genome. Recombination also creates genetic diversity, and viral recombination mechanisms have important implications for understanding viral origins as well as the dynamic nature of viral-host interactions. Both bacteriophage λ and herpes simplex virus (HSV) display high rates of recombination, both utilizing their own proteins and commandeering cellular proteins to promote recombination reactions. We focus primarily on λ and HSV, as they have proven amenable to both genetic and biochemical analysis and have recently been shown to exhibit some surprising similarities that will guide future studies. PMID:25002096

How good are indirect tests at detecting recombination in human mtDNA?

PubMed

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-07-08

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D' and r(2), Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ(2)) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7-70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed.

How Good Are Indirect Tests at Detecting Recombination in Human mtDNA?

PubMed Central

White, Daniel James; Bryant, David; Gemmell, Neil John

2013-01-01

Empirical proof of human mitochondrial DNA (mtDNA) recombination in somatic tissues was obtained in 2004; however, a lack of irrefutable evidence exists for recombination in human mtDNA at the population level. Our inability to demonstrate convincingly a signal of recombination in population data sets of human mtDNA sequence may be due, in part, to the ineffectiveness of current indirect tests. Previously, we tested some well-established indirect tests of recombination (linkage disequilibrium vs. distance using D′ and r2, Homoplasy Test, Pairwise Homoplasy Index, Neighborhood Similarity Score, and Max χ2) on sequence data derived from the only empirically confirmed case of human mtDNA recombination thus far and demonstrated that some methods were unable to detect recombination. Here, we assess the performance of these six well-established tests and explore what characteristics specific to human mtDNA sequence may affect their efficacy by simulating sequence under various parameters with levels of recombination (ρ) that vary around an empirically derived estimate for human mtDNA (population parameter ρ = 5.492). No test performed infallibly under any of our scenarios, and error rates varied across tests, whereas detection rates increased substantially with ρ values > 5.492. Under a model of evolution that incorporates parameters specific to human mtDNA, including rate heterogeneity, population expansion, and ρ = 5.492, successful detection rates are limited to a range of 7−70% across tests with an acceptable level of false-positive results: the neighborhood similarity score incompatibility test performed best overall under these parameters. Population growth seems to have the greatest impact on recombination detection probabilities across all models tested, likely due to its impact on sequence diversity. The implications of our findings on our current understanding of mtDNA recombination in humans are discussed. PMID:23665874

Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

PubMed

Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

2012-03-01

Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

An Overview of the Molecular Mechanisms of Recombinational DNA Repair

PubMed Central

Kowalczykowski, Stephen C.

2015-01-01

Recombinational DNA repair is a universal aspect of DNA metabolism and is essential for genomic integrity. It is a template-directed process that uses a second chromosomal copy (sister, daughter, or homolog) to ensure proper repair of broken chromosomes. The key steps of recombination are conserved from phage through human, and an overview of those steps is provided in this review. The first step is resection by helicases and nucleases to produce single-stranded DNA (ssDNA) that defines the homologous locus. The ssDNA is a scaffold for assembly of the RecA/RAD51 filament, which promotes the homology search. On finding homology, the nucleoprotein filament catalyzes exchange of DNA strands to form a joint molecule. Recombination is controlled by regulating the fate of both RecA/RAD51 filaments and DNA pairing intermediates. Finally, intermediates that mature into Holliday structures are disjoined by either nucleolytic resolution or topological dissolution. PMID:26525148

The yeast MSH1 gene is not involved in DNA repair or recombination during meiosis.

PubMed

Sia, Elaine A; Kirkpatrick, David T

2005-02-03

Six strong homologs of the bacterial MutS DNA mismatch repair (MMR) gene have been identified in the yeast Saccharomyces cerevisiae. With the exception of the MSH1 gene, the involvement of each homolog in DNA repair and recombination during meiosis has been determined previously. Five of the homologs have been demonstrated to act in meiotic DNA repair (MSH2, MSH3, MSH6 and MSH4) and/or meiotic recombination (MSH4 and MSH5). Unfortunately the loss of mitochondrial function that results from deletion of MSH1 disrupts meiotic progression, precluding an analysis of MSH1 function in meiotic DNA repair and recombination. However, the recent identification of two separation-of-function alleles of MSH1 that interfere with protein function but still maintain functional mitochondria allow the meiotic activities of MSH1 to be determined. We show that the G776D and F105A alleles of MSH1 exhibit no defects in meiotic recombination, repair base-base mismatches and large loop mismatches efficiently during meiosis, and have high levels of spore viability. These data indicate that the MSH1 protein, unlike other MutS homologs in yeast, plays no role in DNA repair or recombination during meiosis.

Transcription and Recombination: When RNA Meets DNA

PubMed Central

Aguilera, Andrés; Gaillard, Hélène

2014-01-01

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription–replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. PMID:25085910

Role of Recombinant DNA Technology to Improve Life.

PubMed

Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad; Hou, Hongwei

2016-01-01

In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life.

Role of Recombinant DNA Technology to Improve Life

PubMed Central

Khan, Suliman; Ullah, Muhammad Wajid; Siddique, Rabeea; Nabi, Ghulam; Manan, Sehrish; Yousaf, Muhammad

2016-01-01

In the past century, the recombinant DNA technology was just an imagination that desirable characteristics can be improved in the living bodies by controlling the expressions of target genes. However, in recent era, this field has demonstrated unique impacts in bringing advancement in human life. By virtue of this technology, crucial proteins required for health problems and dietary purposes can be produced safely, affordably, and sufficiently. This technology has multidisciplinary applications and potential to deal with important aspects of life, for instance, improving health, enhancing food resources, and resistance to divergent adverse environmental effects. Particularly in agriculture, the genetically modified plants have augmented resistance to harmful agents, enhanced product yield, and shown increased adaptability for better survival. Moreover, recombinant pharmaceuticals are now being used confidently and rapidly attaining commercial approvals. Techniques of recombinant DNA technology, gene therapy, and genetic modifications are also widely used for the purpose of bioremediation and treating serious diseases. Due to tremendous advancement and broad range of application in the field of recombinant DNA technology, this review article mainly focuses on its importance and the possible applications in daily life. PMID:28053975

Self-regulation of recombinant DNA technology in Japan in the 1970s.

PubMed

Nagai, Hiroyuki; Nukaga, Yoshio; Saeki, Koji; Akabayashi, Akira

2009-07-01

Recombinant DNA technology was developed in the United States in the early 1970s. Leading scientists held an international Asilomar Conference in 1975 to examine the self regulation of recombinant DNA technology, followed by the U.S. National Institutes of Health drafting the Recombinant DNA Research Guidelines in 1976. The result of this conference significantly affected many nations, including Japan. However, there have been few historical studies on the self-regulation of recombinant technologies conducted by scientists and government officials in Japan. The purpose of this paper is to analyze how the Science Council of Japan, the Ministry of Education, Science adn Culture, and the Science and Technology Agency developed self-regulation policies for recombinant DNA technology in Japan in the 1970s. Groups of molecular biologist and geneticists played a key role in establishing guidelines in cooperation with government officials. Our findings suggest that self-regulation policies on recombinant DNA technology have influenced safety management for the life sciences and establishment of institutions for review in Japan.

Escherichia coli ArgR mutants defective in cer/Xer recombination, but not in DNA binding.

PubMed

Sénéchal, Hélène; Delesques, Jérémy; Szatmari, George

2010-04-01

The Escherichia coli arginine repressor (ArgR) is an L-arginine-dependent DNA-binding protein that controls the expression of the arginine biosynthetic genes and is required as an accessory factor for Xer site-specific recombination at cer and related recombination sites in plasmids. We used the technique of pentapeptide scanning mutagenesis to isolate a series of ArgR mutants that were considerably reduced in cer recombination, but were still able to repress an argA::lacZ fusion. DNA sequence analysis showed that all of the mutants mapped to the same nucleotide, resulting in a five amino acid insertion between residues 149 and 150 of ArgR, corresponding to the end of the alpha6 helix. A truncated ArgR containing a stop codon at residue 150 displayed the same phenotype as the protein with the five amino acid insertion, and both mutants displayed sequence-specific DNA-binding activity that was L-arginine dependent. These results show that the C-terminus of ArgR is more important in cer/Xer site-specific recombination than in DNA binding.

Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination.

PubMed

Mbantenkhu, MacMillan; Wang, Xiaowen; Nardozzi, Jonathan D; Wilkens, Stephan; Hoffman, Elizabeth; Patel, Anamika; Cosgrove, Michael S; Chen, Xin Jie

2011-12-09

Homologous recombination is a conserved molecular process that has primarily evolved for the repair of double-stranded DNA breaks and stalled replication forks. However, the recombination machinery in mitochondria is poorly understood. Here, we show that the yeast mitochondrial nucleoid protein, Mgm101, is related to the Rad52-type recombination proteins that are widespread in organisms from bacteriophage to humans. Mgm101 is required for repeat-mediated recombination and suppression of mtDNA fragmentation in vivo. It preferentially binds to single-stranded DNA and catalyzes the annealing of ssDNA precomplexed with the mitochondrial ssDNA-binding protein, Rim1. Transmission electron microscopy showed that Mgm101 forms large oligomeric rings of ∼14-fold symmetry and highly compressed helical filaments. Specific mutations affecting ring formation reduce protein stability in vitro. The data suggest that the ring structure may provide a scaffold for stabilization of Mgm101 by preventing the aggregation of the otherwise unstable monomeric conformation. Upon binding to ssDNA, Mgm101 is remobilized from the rings to form distinct nucleoprotein filaments. These studies reveal a recombination protein of likely bacteriophage origin in mitochondria and support the notion that recombination is indispensable for mtDNA integrity.

Mechanism of homologous recombination and implications for aging-related deletions in mitochondrial DNA.

PubMed

Chen, Xin Jie

2013-09-01

Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells.

Mechanism of Homologous Recombination and Implications for Aging-Related Deletions in Mitochondrial DNA

PubMed Central

2013-01-01

SUMMARY Homologous recombination is a universal process, conserved from bacteriophage to human, which is important for the repair of double-strand DNA breaks. Recombination in mitochondrial DNA (mtDNA) was documented more than 4 decades ago, but the underlying molecular mechanism has remained elusive. Recent studies have revealed the presence of a Rad52-type recombination system of bacteriophage origin in mitochondria, which operates by a single-strand annealing mechanism independent of the canonical RecA/Rad51-type recombinases. Increasing evidence supports the notion that, like in bacteriophages, mtDNA inheritance is a coordinated interplay between recombination, repair, and replication. These findings could have profound implications for understanding the mechanism of mtDNA inheritance and the generation of mtDNA deletions in aging cells. PMID:24006472

Prominent mitochondrial DNA recombination intermediates in human heart muscle.

PubMed

Kajander, O A; Karhunen, P J; Holt, I J; Jacobs, H T

2001-11-01

Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.

Transcription and recombination: when RNA meets DNA.

PubMed

Aguilera, Andrés; Gaillard, Hélène

2014-08-01

A particularly relevant phenomenon in cell physiology and proliferation is the fact that spontaneous mitotic recombination is strongly enhanced by transcription. The most accepted view is that transcription increases the occurrence of double-strand breaks and/or single-stranded DNA gaps that are repaired by recombination. Most breaks would arise as a consequence of the impact that transcription has on replication fork progression, provoking its stalling and/or breakage. Here, we discuss the mechanisms responsible for the cross talk between transcription and recombination, with emphasis on (1) the transcription-replication conflicts as the main source of recombinogenic DNA breaks, and (2) the formation of cotranscriptional R-loops as a major cause of such breaks. The new emerging questions and perspectives are discussed on the basis of the interference between transcription and replication, as well as the way RNA influences genome dynamics. Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.

Evidence for recombination of mitochondrial DNA in triploid crucian carp.

PubMed

Guo, Xinhong; Liu, Shaojun; Liu, Yun

2006-03-01

In this study, we report the complete mitochondrial DNA (mtDNA) sequences of the allotetraploid and triploid crucian carp and compare the complete mtDNA sequences between the triploid crucian carp and its female parent Japanese crucian carp and between the triploid crucian carp and its male parent allotetraploid. Our results indicate that the complete mtDNA nucleotide identity (98%) between the triploid crucian carp and its male parent allotetraploid was higher than that (93%) between the triploid crucian carp and its female parent Japanese crucian carp. Moreover, the presence of a pattern of identity and difference at synonymous sites of mitochondrial genomes between the triploid crucian carp and its parents provides direct evidence that triploid crucian carp possessed the recombination mtDNA fragment (12,759 bp) derived from the paternal fish. These results suggest that mtDNA recombination was derived from the fusion of the maternal and paternal mtDNAs. Compared with the haploid egg with one set of genome from the Japanese crucian carp, the diploid sperm with two sets of genomes from the allotetraploid could more easily make its mtDNA fuse with the mtDNA of the haploid egg. In addition, the triple hybrid nature of the triploid crucian carp probably allowed its better mtDNA recombination. In summary, our results provide the first evidence of mtDNA combination in polyploid fish.

In vitro excision of adeno-associated virus DNA from recombinant plasmids: Isolation of an enzyme fraction from HeLa cells that cleaves DNA at poly(G) sequences

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

Gottlieb, J.; Muzyczka, N.

1988-06-01

When circular recombinant plasmids containing adeno-associated virus (AAV) DNA sequences are transfected into human cells, the AAV provirus is rescued. Using these circular AAV plasmids as substrates, the authors isolated an enzyme fraction from HeLa cell nuclear extracts that excises intact AAV DNA in vitro from vector DNA and produces linear DNA products. The recognition signal for the enzyme is a polypurine-polypyrimidine sequence which is at least 9 residues long and rich in G . C base pairs. Such sequences are present in AAV recombinant plasmids as part of the first 15 base pairs of the AAV terminal repeat andmore » in some cases as the result of cloning the AAV genome by G . C tailing. The isolated enzyme fraction does not have significant endonucleolytic activity on single-stranded or double-stranded DNA. Plasmid DNA that is transfected into tissue culture cells is cleaved in vivo to produce a pattern of DNA fragments similar to that seen with purified enzyme in vitro. The activity has been called endo R for rescue, and its behavior suggests that it may have a role in recombination of cellular chromosomes.« less

DNA sequence alignment by microhomology sampling during homologous recombination

PubMed Central

Qi, Zhi; Redding, Sy; Lee, Ja Yil; Gibb, Bryan; Kwon, YoungHo; Niu, Hengyao; Gaines, William A.; Sung, Patrick

2015-01-01

Summary Homologous recombination (HR) mediates the exchange of genetic information between sister or homologous chromatids. During HR, members of the RecA/Rad51 family of recombinases must somehow search through vast quantities of DNA sequence to align and pair ssDNA with a homologous dsDNA template. Here we use single-molecule imaging to visualize Rad51 as it aligns and pairs homologous DNA sequences in real-time. We show that Rad51 uses a length-based recognition mechanism while interrogating dsDNA, enabling robust kinetic selection of 8-nucleotide (nt) tracts of microhomology, which kinetically confines the search to sites with a high probability of being a homologous target. Successful pairing with a 9th nucleotide coincides with an additional reduction in binding free energy and subsequent strand exchange occurs in precise 3-nt steps, reflecting the base triplet organization of the presynaptic complex. These findings provide crucial new insights into the physical and evolutionary underpinnings of DNA recombination. PMID:25684365

Kinetics of presynaptic filament assembly in the presence of single-stranded DNA binding protein and recombination mediator protein.

PubMed

Liu, Jie; Berger, Christopher L; Morrical, Scott W

2013-11-12

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and for the accurate repair of DNA double-strand breaks. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on single-stranded DNA (ssDNA), a process that is regulated by ssDNA binding protein (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate-limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. In this study, the well-defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator) is used to examine the kinetics of presynaptic filament assembly on native ssDNA in vitro. Results indicate that the ATP-dependent assembly of UvsX presynaptic filaments on Gp32-covered ssDNA is limited by a salt-sensitive nucleation step in the absence of mediator. Filament nucleation is selectively enhanced and rendered salt-resistant by mediator protein UvsY, which appears to stabilize a prenucleation complex. This mechanism potentially explains how UvsY promotes presynaptic filament assembly at physiologically relevant ionic strengths and Gp32 concentrations. Other data suggest that presynaptic filament assembly involves multiple nucleation events, resulting in many short UvsX-ssDNA filaments or clusters, which may be the relevant form for recombination in vivo. Together, these findings provide the first detailed kinetic model for presynaptic filament assembly involving all three major protein components (recombinase, mediator, and SSB) on native ssDNA.

The "Frankenplasmid" lab: an investigative exercise for teaching recombinant DNA methods.

PubMed

Dean, Derek M; Wilder, Jason A

2011-01-01

We describe an investigative laboratory module designed to give college undergraduates strong practical and theoretical experience with recombinant DNA methods within 3 weeks. After deducing restriction enzyme maps for two different plasmids, students ligate the plasmids together in the same reaction, transform E. coli with this mixture of ligated DNA, and plate the cells on media that specifically select for hybrid plasmids. The main goal of the assignment is for students to deduce the gene map of one hybrid "Frankenplasmid" using the LacZ phenotype of its transformants, PCR, and restriction mapping. Our protocol results in a number of possible outcomes, meaning that students are mapping truly unknown plasmids. The open-ended nature of this assignment results in an effective module that teaches recombinant DNA procedures while engaging students with its investigative approach, increasing complexity, and puzzle-like quality. Moreover, the modular design of the activity allows it to be adapted to a more limited schedule, introductory courses, or more advanced courses. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Science: The Recombinant DNA Advisory Committee.

ERIC Educational Resources Information Center

Wright, Susan

1979-01-01

Reports on the status of the Recombinant DNA Advisory Committee (RAC) and attempts to rationalize Suburban Highway Policy. Effective communication among members of the RAC is a current problem facing the committee. A federal transportation priority spending policy is suggested during these times of money and fuel shortages. (MA)

Breaks in the 45S rDNA Lead to Recombination-Mediated Loss of Repeats.

PubMed

Warmerdam, Daniël O; van den Berg, Jeroen; Medema, René H

2016-03-22

rDNA repeats constitute the most heavily transcribed region in the human genome. Tumors frequently display elevated levels of recombination in rDNA, indicating that the repeats are a liability to the genomic integrity of a cell. However, little is known about how cells deal with DNA double-stranded breaks in rDNA. Using selective endonucleases, we show that human cells are highly sensitive to breaks in 45S but not the 5S rDNA repeats. We find that homologous recombination inhibits repair of breaks in 45S rDNA, and this results in repeat loss. We identify the structural maintenance of chromosomes protein 5 (SMC5) as contributing to recombination-mediated repair of rDNA breaks. Together, our data demonstrate that SMC5-mediated recombination can lead to error-prone repair of 45S rDNA repeats, resulting in their loss and thereby reducing cellular viability. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

A role for recombination junctions in the segregation of mitochondrial DNA in yeast.

PubMed

Lockshon, D; Zweifel, S G; Freeman-Cook, L L; Lorimer, H E; Brewer, B J; Fangman, W L

1995-06-16

In S. cerevisiae, mitochondrial DNA (mtDNA) molecules, in spite of their high copy number, segregate as if there were a small number of heritable units. The rapid segregation of mitochondrial genomes can be analyzed using mtDNA deletion variants. These small, amplified genomes segregate preferentially from mixed zygotes relative to wild-type mtDNA. This segregation advantage is abolished by mutations in a gene, MGT1, that encodes a recombination junction-resolving enzyme. We show here that resolvase deficiency causes a larger proportion of molecules to be linked together by recombination junctions, resulting in the aggregation of mtDNA into a small number of cytological structures. This change in mtDNA structure can account for the increased mitotic loss of mtDNA and the altered pattern of mtDNA segregation from zygotes. We propose that the level of unresolved recombination junctions influences the number of heritable units of mtDNA.

An att site-based recombination reporter system for genome engineering and synthetic DNA assembly.

PubMed

Bland, Michael J; Ducos-Galand, Magaly; Val, Marie-Eve; Mazel, Didier

2017-07-14

Direct manipulation of the genome is a widespread technique for genetic studies and synthetic biology applications. The tyrosine and serine site-specific recombination systems of bacteriophages HK022 and ΦC31 are widely used for stable directional exchange and relocation of DNA sequences, making them valuable tools in these contexts. We have developed site-specific recombination tools that allow the direct selection of recombination events by embedding the attB site from each system within the β-lactamase resistance coding sequence (bla). The HK and ΦC31 tools were developed by placing the attB sites from each system into the signal peptide cleavage site coding sequence of bla. All possible open reading frames (ORFs) were inserted and tested for recombination efficiency and bla activity. Efficient recombination was observed for all tested ORFs (3 for HK, 6 for ΦC31) as shown through a cointegrate formation assay. The bla gene with the embedded attB site was functional for eight of the nine constructs tested. The HK/ΦC31 att-bla system offers a simple way to directly select recombination events, thus enhancing the use of site-specific recombination systems for carrying out precise, large-scale DNA manipulation, and adding useful tools to the genetics toolbox. We further show the power and flexibility of bla to be used as a reporter for recombination.

A reanalysis of the indirect evidence for recombination in human mitochondrial DNA.

PubMed

Piganeau, G; Eyre-Walker, A

2004-04-01

In an attempt to resolve the controversy about whether recombination occurs in human mtDNA, we have analysed three recently published data sets of complete mtDNA sequences along with 10 RFLP data sets. We have analysed the relationship between linkage disequilibrium (LD) and distance between sites under a variety of conditions using two measures of LD, r2 and /D'/. We find that there is a negative correlation between r2 and distance in the majority of data sets, but no overall trend for /D'/. Five out of six mtDNA sequence data sets show an excess of homoplasy, but this could be due to either recombination or hypervariable sites. Two additional recombination detection methods used, Geneconv and Maximum Chi-Square, showed nonsignificant results. The overall significance of these findings is hard to quantify because of nonindependence, but our results suggest a lack of evidence for recombination in human mtDNA.

Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

PubMed

Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

2015-01-01

Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

The FANC pathway is activated by adenovirus infection and promotes viral replication-dependent recombination

PubMed Central

Cherubini, Gioia; Naim, Valeria; Caruso, Paola; Burla, Romina; Bogliolo, Massimo; Cundari, Enrico; Benihoud, Karim; Saggio, Isabella; Rosselli, Filippo

2011-01-01

Deciphering the crosstalk between a host cell and a virus during infection is important not only to better define viral biology but also to improve our understanding of cellular processes. We identified the FANC pathway as a helper of viral replication and recombination by searching for cellular targets that are modified by adenovirus (Ad) infection and are involved in its outcome. This pathway, which is involved in the DNA damage response and checkpoint control, is altered in Fanconi anaemia, a rare cancer predisposition syndrome. We show here that Ad5 infection activates the FANC pathway independent of the classical DNA damage response. Infection with a non-replicating Ad shows that the presence of viral DNA is not sufficient to induce the monoubiquitination of FANCD2 but still activates the DNA damage response coordinated by phospho-NBS1 and phospho-CHK1. E1A expression alone fails to induce FANCD2 monoubiquitination, indicating that a productive viral infection and/or replication is required for FANC pathway activation. Our data indicate that Ad5 infection induces FANCD2 activation to promote its own replication. Specifically, we show that FANCD2 is involved in the recombination process that accompanies viral DNA replication. This study provides evidence of a DNA damage-independent function of the FANC pathway and identifies a cellular system involved in Ad5 recombination. PMID:21421559

21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

Code of Federal Regulations, 2011 CFR

2011-04-01

... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

Code of Federal Regulations, 2013 CFR

2013-04-01

... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

Code of Federal Regulations, 2014 CFR

2014-04-01

... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

Code of Federal Regulations, 2012 CFR

2012-04-01

... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

21 CFR 878.4494 - Absorbable poly(hydroxybutyrate) surgical suture produced by recombinant DNA technology.

Code of Federal Regulations, 2010 CFR

2010-04-01

... produced by recombinant DNA technology. 878.4494 Section 878.4494 Food and Drugs FOOD AND DRUG... recombinant DNA technology. (a) Identification. An absorbable poly(hydroxybutyrate) surgical suture is an... deoxyribonucleic acid (DNA) technology. The device is intended for use in general soft tissue approximation and...

The Mitochondrial DNA-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination.

PubMed

Blomme, Jonas; Van Aken, Olivier; Van Leene, Jelle; Jégu, Teddy; De Rycke, Riet; De Bruyne, Michiel; Vercruysse, Jasmien; Nolf, Jonah; Van Daele, Twiggy; De Milde, Liesbeth; Vermeersch, Mattias; des Francs-Small, Catherine Colas; De Jaeger, Geert; Benhamed, Moussa; Millar, A Harvey; Inzé, Dirk; Gonzalez, Nathalie

2017-05-01

In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development. © 2017 American Society of Plant Biologists. All rights reserved.

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

PubMed Central

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-01-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ− mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ− cells and increased deletion mutagenesis at the ori5 region in ρ+ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5. PMID:23598996

Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast.

PubMed

Ling, Feng; Hori, Akiko; Yoshitani, Ayako; Niu, Rong; Yoshida, Minoru; Shibata, Takehiko

2013-06-01

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small ρ(-) mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ(-) cells and increased deletion mutagenesis at the ori5 region in ρ(+) cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.

The recombinant expression and activity detection of MAF-1 fusion protein.

PubMed

Fu, Ping; Wu, Jianwei; Gao, Song; Guo, Guo; Zhang, Yong; Liu, Jian

2015-10-01

This study establishes the recombinant expression system of MAF-1 (Musca domestica antifungal peptide-1) and demonstrates the antifungal activity of the expression product and shows the relationship between biological activity and structure. The gene segments on mature peptide part of MAF-1 were cloned, based on the primers designed according to the cDNA sequence of MAF-1. We constructed the recombinant prokaryotic expression plasmid using prokaryotic expression vector (pET-28a(+)) and converted it to the competent cell of BL21(DE3) to gain recombinant MAF-1 fusion protein with His tag sequence through purifying affinity chromatographic column of Ni-NTA. To conduct the Western Blotting test, recombinant MAF-1 fusion protein was used to produce the polyclonal antibody of rat. The antifungal activity of the expression product was detected using Candida albicans (ATCC10231) as the indicator. The MAF-1 recombinant fusion protein was purified to exhibit obvious antifungal activity, which lays the foundation for the further study of MAF-1 biological activity, the relationship between structure and function, as well as control of gene expression.

Evidence for recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic.

PubMed

Burzyński, Artur; Zbawicka, Małgorzata; Skibinski, David O F; Wenne, Roman

2003-03-01

A number of studies have claimed that recombination occurs in animal mtDNA, although this evidence is controversial. Ladoukakis and Zouros (2001) provided strong evidence for mtDNA recombination in the COIII gene in gonadal tissue in the marine mussel Mytilus galloprovincialis from the Black Sea. The recombinant molecules they reported had not however become established in the population from which experimental animals were sampled. In the present study, we provide further evidence of the generality of mtDNA recombination in Mytilus by reporting recombinant mtDNA molecules in a related mussel species, Mytilus trossulus, from the Baltic. The mtDNA region studied begins in the 16S rRNA gene and terminates in the cytochrome b gene and includes a major noncoding region that may be analogous to the D-loop region observed in other animals. Many bivalve species, including some Mytilus species, are unusual in that they have two mtDNA genomes, one of which is inherited maternally (F genome) the other inherited paternally (M genome). Two recombinant variants reported in the present study have population frequencies of 5% and 36% and appear to be mosaic for F-like and M-like sequences. However, both variants have the noncoding region from the M genome, and both are transmitted to sperm like the M genome. We speculate that acquisition of the noncoding region by the recombinant molecules has conferred a paternal role on mtDNA genomes that otherwise resemble the F genome in sequence.

The Mitochondrial DNA-Associated Protein SWIB5 Influences mtDNA Architecture and Homologous Recombination[OPEN

PubMed Central

Vercruysse, Jasmien; Van Daele, Twiggy; De Milde, Liesbeth; Benhamed, Moussa; Inzé, Dirk

2017-01-01

In addition to the nucleus, mitochondria and chloroplasts in plant cells also contain genomes. Efficient DNA repair pathways are crucial in these organelles to fix damage resulting from endogenous and exogenous factors. Plant organellar genomes are complex compared with their animal counterparts, and although several plant-specific mediators of organelle DNA repair have been reported, many regulators remain to be identified. Here, we show that a mitochondrial SWI/SNF (nucleosome remodeling) complex B protein, SWIB5, is capable of associating with mitochondrial DNA (mtDNA) in Arabidopsis thaliana. Gain- and loss-of-function mutants provided evidence for a role of SWIB5 in influencing mtDNA architecture and homologous recombination at specific intermediate-sized repeats both under normal and genotoxic conditions. SWIB5 interacts with other mitochondrial SWIB proteins. Gene expression and mutant phenotypic analysis of SWIB5 and SWIB family members suggests a link between organellar genome maintenance and cell proliferation. Taken together, our work presents a protein family that influences mtDNA architecture and homologous recombination in plants and suggests a link between organelle functioning and plant development. PMID:28420746

Highly sensitive detection of mutations in CHO cell recombinant DNA using multi-parallel single molecule real-time DNA sequencing.

PubMed

Cartwright, Joseph F; Anderson, Karin; Longworth, Joseph; Lobb, Philip; James, David C

2018-06-01

High-fidelity replication of biologic-encoding recombinant DNA sequences by engineered mammalian cell cultures is an essential pre-requisite for the development of stable cell lines for the production of biotherapeutics. However, immortalized mammalian cells characteristically exhibit an increased point mutation frequency compared to mammalian cells in vivo, both across their genomes and at specific loci (hotspots). Thus unforeseen mutations in recombinant DNA sequences can arise and be maintained within producer cell populations. These may affect both the stability of recombinant gene expression and give rise to protein sequence variants with variable bioactivity and immunogenicity. Rigorous quantitative assessment of recombinant DNA integrity should therefore form part of the cell line development process and be an essential quality assurance metric for instances where synthetic/multi-component assemblies are utilized to engineer mammalian cells, such as the assessment of recombinant DNA fidelity or the mutability of single-site integration target loci. Based on Pacific Biosciences (Menlo Park, CA) single molecule real-time (SMRT™) circular consensus sequencing (CCS) technology we developed a rDNA sequence analysis tool to process the multi-parallel sequencing of ∼40,000 single recombinant DNA molecules. After statistical filtering of raw sequencing data, we show that this analytical method is capable of detecting single point mutations in rDNA to a minimum single mutation frequency of 0.0042% (<1/24,000 bases). Using a stable CHO transfectant pool harboring a randomly integrated 5 kB plasmid construct encoding GFP we found that 28% of recombinant plasmid copies contained at least one low frequency (<0.3%) point mutation. These mutations were predominantly found in GC base pairs (85%) and that there was no positional bias in mutation across the plasmid sequence. There was no discernable difference between the mutation frequencies of coding and non

No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline

PubMed Central

Hagström, Erik; Freyer, Christoph; Battersby, Brendan J.; Stewart, James B.; Larsson, Nils-Göran

2014-01-01

Variants of mitochondrial DNA (mtDNA) are commonly used as markers to track human evolution because of the high sequence divergence and exclusive maternal inheritance. It is assumed that the inheritance is clonal, i.e. that mtDNA is transmitted between generations without germline recombination. In contrast to this assumption, a number of studies have reported the presence of recombinant mtDNA molecules in cell lines and animal tissues, including humans. If germline recombination of mtDNA is frequent, it would strongly impact phylogenetic and population studies by altering estimates of coalescent time and branch lengths in phylogenetic trees. Unfortunately, this whole area is controversial and the experimental approaches have been widely criticized as they often depend on polymerase chain reaction (PCR) amplification of mtDNA and/or involve studies of transformed cell lines. In this study, we used an in vivo mouse model that has had germline heteroplasmy for a defined set of mtDNA mutations for more than 50 generations. To assess recombination, we adapted and validated a method based on cloning of single mtDNA molecules in the λ phage, without prior PCR amplification, followed by subsequent mutation analysis. We screened 2922 mtDNA molecules and found no germline recombination after transmission of mtDNA under genetically and evolutionary relevant conditions in mammals. PMID:24163253

Biochemistry of homologous recombination in Escherichia coli.

PubMed Central

Kowalczykowski, S C; Dixon, D A; Eggleston, A K; Lauder, S D; Rehrauer, W M

1994-01-01

Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination. Images PMID:7968921

Biparental inheritance of organelles in Pelargonium: evidence for intergenomic recombination of mitochondrial DNA.

PubMed

Apitz, Janina; Weihe, Andreas; Pohlheim, Frank; Börner, Thomas

2013-02-01

While uniparental transmission of mtDNA is widespread and dominating in eukaryotes leaving mutation as the major source of genotypic diversity, recently, biparental inheritance of mitochondrial genes has been demonstrated in reciprocal crosses of Pelargonium zonale and P. inquinans. The thereby arising heteroplasmy carries the potential for recombination between mtDNAs of different descent, i.e. between the parental mitochondrial genomes. We have analyzed these Pelargonium hybrids for mitochondrial intergenomic recombination events by examining differences in DNA blot hybridization patterns of the mitochondrial genes atp1 and cob. Further investigation of these genes and their flanking regions using nucleotide sequence polymorphisms and PCR revealed DNA segments in the progeny, which contained both P. zonale and P. inquinans sequences suggesting an intergenomic recombination in hybrids of Pelargonium. This turns Pelargonium into an interesting subject for studies of recombination and evolutionary dynamics of mitochondrial genomes.

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

Construction of C35 gene bait recombinants and T47D cell cDNA library.

PubMed

Yin, Kun; Xu, Chao; Zhao, Gui-Hua; Liu, Ye; Xiao, Ting; Zhu, Song; Yan, Ge

2017-11-20

C35 is a novel tumor biomarker associated with metastasis progression. To investigate the interaction factors of C35 in its high expressed breast cancer cell lines, we constructed bait recombinant plasmids of C35 gene and T47D cell cDNA library for yeast two-hybrid screening. Full length C35 sequences were subcloned using RT-PCR from cDNA template extracted from T47D cells. Based on functional domain analysis, the full-length C35 1-348bp was also truncated into two fragments C351-153bp and C35154-348bp to avoid auto-activation. The three kinds of C35 genes were successfully amplified and inserted into pGBKT7 to construct bait recombinant plasmids pGBKT7-C351-348bp, pGBKT7-C351-153bp and pGBKT7-C35154-348bp, then transformed into Y187 yeast cells by the lithium acetate method. Auto-activation and toxicity of C35 baits were detected using nutritional deficient medium and X-α-Gal assays. The T47D cell ds cDNA was generated by SMART TM technology and the library was constructed using in vivo recombination-mediated cloning in the AH109 yeast strain using a pGADT7-Rec plasmid. The transformed Y187/pGBKT7-C351-348bp line was intensively inhibited while the truncated Y187/pGBKT7-C35 lines had no auto-activation and toxicity in yeast cells. The titer of established cDNA library was 2 × 10 7 pfu/mL with high transformation efficiency of 1.4 × 10 6 , and the insert size of ds cDNA was distributed homogeneously between 0.5-2.0 kb. Our research generated a T47D cell cDNA library with high titer, and the constructed two C35 "baits" contained a respective functional immunoreceptor tyrosine based activation motif (ITAM) and the conserved last four amino acids Cys-Ile-Leu-Val (CILV) motif, and therefore laid a foundation for screening the C35 interaction factors in a BC cell line.

Exploring optimization parameters to increase ssDNA recombineering in Lactococcus lactis and Lactobacillus reuteri

PubMed Central

van Pijkeren, Jan-Peter; Neoh, Kar Mun; Sirias, Denise; Findley, Anthony S.; Britton, Robert A.

2012-01-01

Single-stranded DNA (ssDNA) recombineering is a technology which is used to make subtle changes in the chromosome of several bacterial genera. Cells which express a single-stranded DNA binding protein (RecT or Bet) are transformed with an oligonucleotide which is incorporated via an annealing and replication-dependent mechanism. By in silico analysis we identified ssDNA binding protein homologs in the genus Lactobacillus and Lactococcus lactis. To assess whether we could further improve the recombineering efficiency in Lactobacillus reuteri ATCC PTA 6475 we expressed several RecT homologs in this strain. RecT derived from Enterococcus faecalis CRMEN 19 yielded comparable efficiencies compared with a native RecT protein, but none of the other proteins further increased the recombineering efficiency. We successfully improved recombineering efficiency 10-fold in L. lactis by increasing oligonucleotide concentration combined with the use of oligonucleotides containing phosphorothioate-linkages (PTOs). Surprisingly, neither increased oligonucleotide concentration nor PTO linkages enhanced recombineering in L. reuteri 6475. To emphasize the utility of this technology in improving probiotic features we modified six bases in a transcriptional regulatory element region of the pdu-operon of L. reuteri 6475, yielding a 3-fold increase in the production of the antimicrobial compound reuterin. Directed genetic modification of lactic acid bacteria through ssDNA recombineering will simplify strain improvement in a way that, when mutating a single base, is genetically indistinguishable from strains obtained through directed evolution. PMID:22750793

Regulating infidelity: RNA-mediated recruitment of AID to DNA during class switch recombination.

PubMed

DiMenna, Lauren J; Chaudhuri, Jayanta

2016-03-01

The mechanism by which the DNA deaminase activation-induced cytidine deaminase (AID) is specifically recruited to repetitive switch region DNA during class switch recombination is still poorly understood. Work over the past decade has revealed a strong link between transcription and RNA polymerase-associated factors in AID recruitment, yet none of these processes satisfactorily explain how AID specificity is affected. Here, we review a recent finding wherein AID is guided to switch regions not by a protein factor but by an RNA moiety, and especially one associated with a noncoding RNA that has been long thought of as being inert. This work explains the long-standing requirement of splicing of noncoding transcripts during class switching, and has implications in both B cell-mediated immunity as well as the underlying pathological syndromes associated with the recombination reaction. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination.

PubMed

Bonnefoy, Nathalie; Fox, Thomas D

2007-01-01

Saccharomyces cerevisiae is currently the only species in which genetic transformation of mitochondria can be used to generate a wide variety of defined alterations in mitochondrial deoxyribonucleic acid (mtDNA). DNA sequences can be delivered into yeast mitochondria by microprojectile bombardment (biolistic transformation) and subsequently incorporated into mtDNA by the highly active homologous recombination machinery present in the organelle. Although transformation frequencies are relatively low, the availability of strong mitochondrial selectable markers for the yeast system, both natural and synthetic, makes the isolation of transformants routine. The strategies and procedures reviewed here allow the researcher to insert defined mutations into endogenous mitochondrial genes and to insert new genes into mtDNA. These methods provide powerful in vivo tools for the study of mitochondrial biology.

A non-canonical DNA structure enables homologous recombination in various genetic systems.

PubMed

Masuda, Tokiha; Ito, Yutaka; Terada, Tohru; Shibata, Takehiko; Mikawa, Tsutomu

2009-10-30

Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins.

Diversity of the Arabidopsis mitochondrial genome occurs via nuclear-controlled recombination activity.

PubMed

Arrieta-Montiel, Maria P; Shedge, Vikas; Davila, Jaime; Christensen, Alan C; Mackenzie, Sally A

2009-12-01

The plant mitochondrial genome is recombinogenic, with DNA exchange activity controlled to a large extent by nuclear gene products. One nuclear gene, MSH1, appears to participate in suppressing recombination in Arabidopsis at every repeated sequence ranging in size from 108 to 556 bp. Present in a wide range of plant species, these mitochondrial repeats display evidence of successful asymmetric DNA exchange in Arabidopsis when MSH1 is disrupted. Recombination frequency appears to be influenced by repeat sequence homology and size, with larger size repeats corresponding to increased DNA exchange activity. The extensive mitochondrial genomic reorganization of the msh1 mutant produced altered mitochondrial transcription patterns. Comparison of mitochondrial genomes from the Arabidopsis ecotypes C24, Col-0, and Ler suggests that MSH1 activity accounts for most or all of the polymorphisms distinguishing these genomes, producing ecotype-specific stoichiometric changes in each line. Our observations suggest that MSH1 participates in mitochondrial genome evolution by influencing the lineage-specific pattern of mitochondrial genetic variation in higher plants.

Rogue athletes and recombinant DNA technology: challenges for doping control.

PubMed

Azzazy, Hassan M E; Mansour, Mai M H

2007-10-01

The quest for athletic excellence holds no limit for some athletes, and the advances in recombinant DNA technology have handed these athletes the ultimate doping weapons: recombinant proteins and gene doping. Some detection methods are now available for several recombinant proteins that are commercially available as pharmaceuticals and being abused by dopers. However, researchers are struggling to come up with efficient detection methods in preparation for the imminent threat of gene doping, expected in the 2008 Olympics. This Forum article presents the main detection strategies for recombinant proteins and the forthcoming detection strategies for gene doping as well as the prime analytical challenges facing them.

Recombinant DNA. Rifkin's regulatory revivalism runs riot.

PubMed

David, P

Jeremy Rifkin, activist opponent of genetic engineering, has adopted tactics of litigation, persuasion, and confrontation in his campaign to halt genetic experimentation. The Recombinant DNA Advisory Committee of the National Institutes of Health has often been the target of his criticism, most recently for its failure to prepare an environmental risk assessment for some DNA tests it approved. Rifkin has won support for his position from religious organizations in the United States, and in June 1983 persuaded an ecumenical group of religious leaders to ask Congress to ban genetic experiments that would affect the human germ line.

The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo.

PubMed

MacAlpine, D M; Perlman, P S; Butow, R A

1998-06-09

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (rho+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in rho+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (rho-). mtDNA recombination junctions are not observed in rho+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Deltamgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in rho+ mtDNA of Deltamgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by >/= 10-fold in wild-type rho+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of rho+ mtDNA.

In utero and acute exposure to benzene: investigation of DNA double-strand breaks and DNA recombination in mice.

PubMed

Lau, Annette; Belanger, Christine Lea; Winn, Louise M

2009-05-31

Benzene, a ubiquitous pollutant, has been identified as a human leukemogen and early exposure to environmental carcinogens such as benzene has been linked to childhood leukemia. It is known that genotoxic agents can increase the frequency of DNA double-strand breaks (DSBs), which can initiate DNA recombinational repair mechanisms. In this study we investigated the induction of micronuclei, the formation of gamma-H2A.X as a marker of DNA DSBs, and the induction of somatic DNA recombination events in hematopoietic tissue from pKZ1 transgenic mice exposed acutely or in utero to benzene. Adult male C57Bl/6N mice were treated with a single i.p. injection of benzene, and timed-pregnant females pKZ1 were treated with daily i.p. injections of 200 mg/kg or 400 mg/kg benzene through gestational days 7-15. Acute exposure to 400 mg/kg benzene resulted in a statistically significant increase in the percentage of micronucleated cells in adult male bone marrow cells and in fetal liver and post-natal day 9 bone marrow cells of mice exposed in utero. Immunoblotting techniques did not detect benzene-induced increases in the formation of gamma-H2A.X in bone marrow cells of adult male mice and in maternal bone marrow, fetal liver, and post-natal bone marrow cells after specific time-point exposures. Finally, no recombination events were detected in adult pKZ1 mouse tissue; however, in post-natal day 9 pups in utero exposure to 400 mg/kg of benzene caused a trend towards increasing recombination frequency although this did not reach statistical significance. These results demonstrate that in utero exposure increases the frequency of micronuclei and DNA recombination events in hematopoietic tissue of fetal and post-natal mice and may be an initiating event in the etiology of childhood leukemias. Further investigations into different types of DNA damage and repair pathways are warranted to fully elucidate the role of genotoxic mechanisms in the etiology of benzene-induced childhood

mtDNA lineage analysis of mouse L-cell lines reveals the accumulation of multiple mtDNA mutants and intermolecular recombination

PubMed Central

Fan, Weiwei; Lin, Chun Shi; Potluri, Prasanth; Procaccio, Vincent; Wallace, Douglas C.

2012-01-01

The role of mitochondrial DNA (mtDNA) mutations and mtDNA recombination in cancer cell proliferation and developmental biology remains controversial. While analyzing the mtDNAs of several mouse L cell lines, we discovered that every cell line harbored multiple mtDNA mutants. These included four missense mutations, two frameshift mutations, and one tRNA homopolymer expansion. The LA9 cell lines lacked wild-type mtDNAs but harbored a heteroplasmic mixture of mtDNAs, each with a different combination of these variants. We isolated each of the mtDNAs in a separate cybrid cell line. This permitted determination of the linkage phase of each mtDNA and its physiological characteristics. All of the polypeptide mutations inhibited their oxidative phosphorylation (OXPHOS) complexes. However, they also increased mitochondrial reactive oxygen species (ROS) production, and the level of ROS production was proportional to the cellular proliferation rate. By comparing the mtDNA haplotypes of the different cell lines, we were able to reconstruct the mtDNA mutational history of the L–L929 cell line. This revealed that every heteroplasmic L-cell line harbored a mtDNA that had been generated by intracellular mtDNA homologous recombination. Therefore, deleterious mtDNA mutations that increase ROS production can provide a proliferative advantage to cancer or stem cells, and optimal combinations of mutant loci can be generated through recombination. PMID:22345519

A phosphorylation-and-ubiquitylation circuitry driving ATR activation and homologous recombination

PubMed Central

Dubois, Jean-Christophe; Yates, Maïlyn; Gaudreau-Lapierre, Antoine; Clément, Geneviève; Cappadocia, Laurent; Gaudreau, Luc

2017-01-01

Abstract RPA-coated single-stranded DNA (RPA–ssDNA), a nucleoprotein structure induced by DNA damage, promotes ATR activation and homologous recombination (HR). RPA is hyper-phosphorylated and ubiquitylated after DNA damage. The ubiquitylation of RPA by PRP19 and RFWD3 facilitates ATR activation and HR, but how it is stimulated by DNA damage is still unclear. Here, we show that RFWD3 binds RPA constitutively, whereas PRP19 recognizes RPA after DNA damage. The recruitment of PRP19 by RPA depends on PIKK-mediated RPA phosphorylation and a positively charged pocket in PRP19. An RPA32 mutant lacking phosphorylation sites fails to recruit PRP19 and support RPA ubiquitylation. PRP19 mutants unable to bind RPA or lacking ubiquitin ligase activity also fail to support RPA ubiquitylation and HR. These results suggest that RPA phosphorylation enhances the recruitment of PRP19 to RPA–ssDNA and stimulates RPA ubiquitylation through a process requiring both PRP19 and RFWD3, thereby triggering a phosphorylation-ubiquitylation circuitry that promotes ATR activation and HR. PMID:28666352

76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-01-19

... recombinant DNA technology, or breeding one or more transgenic rodents to create a new transgenic rodent (i.e., breeding of two different transgenic rodents or the breeding of a transgenic rodent and a non-transgenic... NIH Guidelines so as to exempt breeding of almost all transgenic rodents that can be housed at BL1...

Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications

PubMed Central

Cassier-Chauvat, Corinne; Veaudor, Théo; Chauvat, Franck

2016-01-01

Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess many E. coli-like DNA recombination and repair genes, and possibly other genes not yet identified. E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marine Prochlorococcus and Synechococcus strains harbor recBCD (DNA recombination), umuCD (mutational DNA replication), as well as the key SOS genes lexA (regulation of the SOS system) and sulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess an E. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studied Gloeobacter strains lack alkB, lexA, and sulA; and Synechococcus PCC7942 has neither lexA nor recCD. Furthermore, the Synechocystis PCC6803 lexA product does not regulate DNA repair genes. Collectively, these findings

Generating and repairing genetically programmed DNA breaks during immunoglobulin class switch recombination

PubMed Central

Nicolas, Laura; Cols, Montserrat; Choi, Jee Eun; Chaudhuri, Jayanta; Vuong, Bao

2018-01-01

Adaptive immune responses require the generation of a diverse repertoire of immunoglobulins (Igs) that can recognize and neutralize a seemingly infinite number of antigens. V(D)J recombination creates the primary Ig repertoire, which subsequently is modified by somatic hypermutation (SHM) and class switch recombination (CSR). SHM promotes Ig affinity maturation whereas CSR alters the effector function of the Ig. Both SHM and CSR require activation-induced cytidine deaminase (AID) to produce dU:dG mismatches in the Ig locus that are transformed into untemplated mutations in variable coding segments during SHM or DNA double-strand breaks (DSBs) in switch regions during CSR. Within the Ig locus, DNA repair pathways are diverted from their canonical role in maintaining genomic integrity to permit AID-directed mutation and deletion of gene coding segments. Recently identified proteins, genes, and regulatory networks have provided new insights into the temporally and spatially coordinated molecular interactions that control the formation and repair of DSBs within the Ig locus. Unravelling the genetic program that allows B cells to selectively alter the Ig coding regions while protecting non-Ig genes from DNA damage advances our understanding of the molecular processes that maintain genomic integrity as well as humoral immunity. PMID:29744038

[Effect of endonuclease G depletion on plasmid DNA uptake and levels of homologous recombination in hela cells].

PubMed

Misic, V; El-Mogy, M; Geng, S; Haj-Ahmad, Y

2016-01-01

Endonuclease G (EndoG) is a mitochondrial apoptosis regulator that also has roles outside of programmed cell death. It has been implicated as a defence DNase involved in the degradation of exogenous DNA after transfection of mammalian cells and in homologous recombination of viral and endogenous DNA. In this study, we looked at the effect of EndoG depletion on plasmid DNA uptake and the levels of homologous recombination in HeLa cells. We show that the proposed defence role of EndoG against uptake of non-viral DNA vectors does not extend to the cervical carcinoma HeLa cells, as targeting of EndoG expression by RNA interference failed to increase intracellular plasmid DNA levels. However, reducing EndoG levels in HeLa cells resulted in a statistically significant reduction of homologous recombination between two plasmid DNA substrates. These findings suggest that non-viral DNA vectors are also substrates for EndoG in its role in homologous recombination.

DNA polymorphism in recombining and non-recombing mating-type-specific loci of the smut fungus Microbotryum

PubMed Central

Votintseva, A A; Filatov, D A

2011-01-01

The population-genetic processes leading to the genetic degeneration of non-recombining regions have mainly been studied in animal and plant sex chromosomes. Here, we report population genetic analysis of the processes in the non-recombining mating-type-specific regions of the smut fungus Microbotryum violaceum. M. violaceum has A1 and A2 mating types, determined by mating-type-specific ‘sex chromosomes' that contain 1–2 Mb long non-recombining regions. If genetic degeneration were occurring, then one would expect reduced DNA polymorphism in the non-recombining regions of this fungus. The analysis of DNA diversity among 19 M. violaceum strains, collected across Europe from Silene latifolia flowers, revealed that (i) DNA polymorphism is relatively low in all 20 studied loci (π∼0.15%), (ii) it is not significantly different between the two mating-type-specific chromosomes nor between the non-recombining and recombining regions, (iii) there is substantial population structure in M. violaceum populations, which resembles that of its host species, S. latifolia, and (iv) there is significant linkage disequilibrium, suggesting that widespread selfing in this species results in a reduction of the effective recombination rate across the genome. We hypothesise that selfing-related reduction of recombination across the M. violaceum genome negates the difference in the level of DNA polymorphism between the recombining and non-recombining regions, and may possibly lead to similar levels of genetic degeneration in the mating-type-specific regions of the non-recombining ‘sex chromosomes' and elsewhere in the genome. PMID:21081967

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms.

PubMed

Yang, Peng; Wu, Min; Guo, Jing; Kwoh, Chee Keong; Przytycka, Teresa M; Zheng, Jie

2014-02-17

As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Recently, an algorithm called "LDsplit" has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that utilizes the genetic variation of

LDsplit: screening for cis-regulatory motifs stimulating meiotic recombination hotspots by analysis of DNA sequence polymorphisms

PubMed Central

2014-01-01

Background As a fundamental genomic element, meiotic recombination hotspot plays important roles in life sciences. Thus uncovering its regulatory mechanisms has broad impact on biomedical research. Despite the recent identification of the zinc finger protein PRDM9 and its 13-mer binding motif as major regulators for meiotic recombination hotspots, other regulators remain to be discovered. Existing methods for finding DNA sequence motifs of recombination hotspots often rely on the enrichment of co-localizations between hotspots and short DNA patterns, which ignore the cross-individual variation of recombination rates and sequence polymorphisms in the population. Our objective in this paper is to capture signals encoded in genetic variations for the discovery of recombination-associated DNA motifs. Results Recently, an algorithm called “LDsplit” has been designed to detect the association between single nucleotide polymorphisms (SNPs) and proximal meiotic recombination hotspots. The association is measured by the difference of population recombination rates at a hotspot between two alleles of a candidate SNP. Here we present an open source software tool of LDsplit, with integrative data visualization for recombination hotspots and their proximal SNPs. Applying LDsplit on SNPs inside an established 7-mer motif bound by PRDM9 we observed that SNP alleles preserving the original motif tend to have higher recombination rates than the opposite alleles that disrupt the motif. Running on SNP windows around hotspots each containing an occurrence of the 7-mer motif, LDsplit is able to guide the established motif finding algorithm of MEME to recover the 7-mer motif. In contrast, without LDsplit the 7-mer motif could not be identified. Conclusions LDsplit is a software tool for the discovery of cis-regulatory DNA sequence motifs stimulating meiotic recombination hotspots by screening and narrowing down to hotspot associated SNPs. It is the first computational method that

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination

PubMed Central

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A.; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S.; Lan, Li

2015-01-01

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome. PMID:26100862

DNA damage during the G0/G1 phase triggers RNA-templated, Cockayne syndrome B-dependent homologous recombination.

PubMed

Wei, Leizhen; Nakajima, Satoshi; Böhm, Stefanie; Bernstein, Kara A; Shen, Zhiyuan; Tsang, Michael; Levine, Arthur S; Lan, Li

2015-07-07

Damage repair mechanisms at transcriptionally active sites during the G0/G1 phase are largely unknown. To elucidate these mechanisms, we introduced genome site-specific oxidative DNA damage and determined the role of transcription in repair factor assembly. We find that KU and NBS1 are recruited to damage sites independent of transcription. However, assembly of RPA1, RAD51C, RAD51, and RAD52 at such sites is strictly governed by active transcription and requires both wild-type Cockayne syndrome protein B (CSB) function and the presence of RNA in the G0/G1 phase. We show that the ATPase activity of CSB is indispensable for loading and binding of the recombination factors. CSB counters radiation-induced DNA damage in both cells and zebrafish models. Taken together, our results have uncovered a novel, RNA-based recombination mechanism by which CSB protects genome stability from strand breaks at transcriptionally active sites and may provide insight into the clinical manifestations of Cockayne syndrome.

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination.

PubMed

Kobayashi, Maki; Aida, Masatoshi; Nagaoka, Hitoshi; Begum, Nasim A; Kitawaki, Yoko; Nakata, Mikiyo; Stanlie, Andre; Doi, Tomomitsu; Kato, Lucia; Okazaki, Il-mi; Shinkura, Reiko; Muramatsu, Masamichi; Kinoshita, Kazuo; Honjo, Tasuku

2009-12-29

To initiate class switch recombination (CSR) activation-induced cytidine deaminase (AID) induces staggered nick cleavage in the S region, which lies 5' to each Ig constant region gene and is rich in palindromic sequences. Topoisomerase 1 (Top1) controls the supercoiling of DNA by nicking, rotating, and religating one strand of DNA. Curiously, Top1 reduction or AID overexpression causes the genomic instability. Here, we report that the inactivation of Top1 by its specific inhibitor camptothecin drastically blocked both the S region cleavage and CSR, indicating that Top1 is responsible for the S region cleavage in CSR. Surprisingly, AID expression suppressed Top1 mRNA translation and reduced its protein level. In addition, the decrease in the Top1 protein by RNA-mediated knockdown augmented the AID-dependent S region cleavage, as well as CSR. Furthermore, Top1 reduction altered DNA structure of the Smu region. Taken together, AID-induced Top1 reduction alters S region DNA structure probably to non-B form, on which Top1 can introduce nicks but cannot religate, resulting in S region cleavage.

Evaluation of the effectiveness and safety of the thermo-treatment process to dispose of recombinant DNA waste from biological research laboratories.

PubMed

Li, Meng-Nan; Zheng, Guang-Hong; Wang, Lei; Xiao, Wei; Fu, Xiao-Hua; Le, Yi-Quan; Ren, Da-Ming

2009-01-01

The discharge of recombinant DNA waste from biological laboratories into the eco-system may be one of the pathways resulting in horizontal gene transfer or "gene pollution". Heating at 100 degrees C for 5-10 min is a common method for treating recombinant DNA waste in biological research laboratories in China. In this study, we evaluated the effectiveness and the safety of the thermo-treatment method in the disposal of recombinant DNA waste. Quantitative PCR, plasmid transformation and electrophoresis technology were used to evaluate the decay/denaturation efficiency during the thermo-treatment process of recombinant plasmid, pET-28b. Results showed that prolonging thermo-treatment time could improve decay efficiency of the plasmid, and its decay half-life was 2.7-4.0 min during the thermo-treatment at 100 degrees C. However, after 30 min of thermo-treatment some transforming activity remained. Higher ionic strength could protect recombinant plasmid from decay during the treatment process. These results indicate that thermo-treatment at 100 degrees C cannot decay and inactivate pET-28b completely. In addition, preliminary results showed that thermo-treated recombinant plasmids were not degraded completely in a short period when they were discharged into an aquatic environment. This implies that when thermo-treated recombinant DNAs are discharged into the eco-system, they may have enough time to re-nature and transform, thus resulting in gene diffusion.

A general method to eliminate laboratory induced recombinants during massive, parallel sequencing of cDNA library.

PubMed

Waugh, Caryll; Cromer, Deborah; Grimm, Andrew; Chopra, Abha; Mallal, Simon; Davenport, Miles; Mak, Johnson

2015-04-09

Massive, parallel sequencing is a potent tool for dissecting the regulation of biological processes by revealing the dynamics of the cellular RNA profile under different conditions. Similarly, massive, parallel sequencing can be used to reveal the complexity of viral quasispecies that are often found in the RNA virus infected host. However, the production of cDNA libraries for next-generation sequencing (NGS) necessitates the reverse transcription of RNA into cDNA and the amplification of the cDNA template using PCR, which may introduce artefact in the form of phantom nucleic acids species that can bias the composition and interpretation of original RNA profiles. Using HIV as a model we have characterised the major sources of error during the conversion of viral RNA to cDNA, namely excess RNA template and the RNaseH activity of the polymerase enzyme, reverse transcriptase. In addition we have analysed the effect of PCR cycle on detection of recombinants and assessed the contribution of transfection of highly similar plasmid DNA to the formation of recombinant species during the production of our control viruses. We have identified RNA template concentrations, RNaseH activity of reverse transcriptase, and PCR conditions as key parameters that must be carefully optimised to minimise chimeric artefacts. Using our optimised RT-PCR conditions, in combination with our modified PCR amplification procedure, we have developed a reliable technique for accurate determination of RNA species using NGS technology.

Use of virion DNA as a cloning vector for the construction of mutant and recombinant herpesviruses.

PubMed

Duboise, S M; Guo, J; Desrosiers, R C; Jung, J U

1996-10-15

We have developed improved procedures for the isolation of deletion mutant, point mutant, and recombinant herpesvirus saimiri. These procedures take advantage of the absence of NotI and AscI restriction enzyme sites within the viral genome and use reporter genes for the identification of recombinant viruses. Genes for secreted engineered alkaline phosphatase and green fluorescent protein were placed under simian virus 40 early promoter control and flanked by NotI and AscI restriction sites. When permissive cells were cotransfected with herpesvirus saimiri virion DNA and one of the engineered reporter genes cloned within herpesvirus saimiri sequences, recombinant viruses were readily identified and purified on the basis of expression of the reporter gene. Digestion of recombinant virion DNA with NotI or AscI was used to delete the reporter gene from the recombinant herpesvirus saimiri. Replacement of the reporter gene can be achieved by NotI or AscI digestion of virion DNA and ligation with a terminally matched fragment or, alternatively, by homologous recombination in cotransfected cells. Any gene can, in theory, be cloned directly into the virion DNA when flanked by the appropriate NotI or AscI sites. These procedures should be widely applicable in their general form to most or all herpesviruses that replicate permissively in cultured cells.

Would Dissociative Recombination of DNA+ be a Possible Pathway of DNA Damage?

NASA Astrophysics Data System (ADS)

Kwon, H. C.; Chen, Z. P.; Strom, R. A.; Andrianarijaona, V. M.

2015-05-01

It is known that dissociative recombination (DR) is one of the very efficient processes of destruction of molecular cations into neutral particles. During the past few years, the focus of DR has been expanded from small inorganic molecules to macromolecular cation. We are probing the possibility of the DR of DNA+ after ionization of DNA, for example due to ionizing radiation. Therefore we are investigating the existence of autoionization states within nucleotide bases (Guanine, Adenine, Cytosine, and Thymine). Our results from computational analysis using the modern electronic structure program ORCA will be presented. Authors wish to give special thanks to Pacific Union College Student Senate for their financial support.

Action of ethyl and methyl methane sulfonates on DNA injection and genetic recombination in T7 bacteriophage.

PubMed Central

Karska-Wysocki, B; Mamet-Bratley, M D; Verly, W G

1976-01-01

After treatment with methyl or ethyl methane sulfonate, T7 amber mutants display a reduced capacity for recombination. Moreover, alkylation reduces recombination frequency involving markers on the right-hand side of the genetic map more than it reduces recombination frequency involving markers on the left-hand side. We interpret this to mean that alkylation can stop DNA injection at any point along the DNA molecule, and that T7 phage injects its DNA in a unique fashion starting from the end carrying the genes for early proteins. PMID:183007

Correlated Template-Switching Events during Minus-Strand DNA Synthesis: a Mechanism for High Negative Interference during Retroviral Recombination

PubMed Central

Anderson, Jeffrey A.; Teufel, Ronald J.; Yin, Philip D.; Hu, Wei-Shau

1998-01-01

Two models for the mechanism of retroviral recombination have been proposed: forced copy choice (minus-strand recombination) and strand displacement-assimilation (plus-strand recombination). Each minus-strand recombination event results in one template switch, whereas each plus-strand recombination event results in two template switches. Recombinant proviruses with one and more than one template switches were previously observed. Recombinants with one template switch were generated by minus-strand recombination, while recombinants containing more than one template switch may have been generated by plus-strand recombination or by correlated minus-strand recombination. We recently observed that retroviral recombination exhibits high negative interference whereby the frequency of recombinants containing multiple template-switching events is higher than expected. To delineate the mechanism that generates recombinants with more than one template switch, we devised a system that permits only minus-strand recombination. Two highly homologous vectors, WH204 and WH221, containing eight different restriction site markers were used. The primer binding site (PBS) of WH221 was deleted; although reverse transcription cannot initiate from WH221 RNA, it can serve as a template for DNA synthesis in heterozygotic virions. After one round of retroviral replication, the structures of the recombinant proviruses were examined. Recombinants containing two, three, four, and five template switches were observed at 1.4-, 10-, 65-, and 50-fold-higher frequencies, respectively, than expected. This indicates that minus-strand recombination events are correlated and can generate proviruses with multiple template switches efficiently. The frequencies of recombinants containing multiple template switches were similar to those observed in the previous system, which allowed both minus- and plus-strand recombination. Thus, the previously reported high negative interference during retroviral

Successful development of recombinant DNA-derived pharmaceuticals.

PubMed

Werner, R G; Pommer, C H

1990-11-01

Successful development of recombinant DNA-derived pharmaceuticals, a new class of therapeutic agents, is determined by a variety of factors affecting the selection and positioning of the compound under development. For an efficient development it is of utmost importance that the mechanism of action of the compound selected be understood on a molecular level. The compound's potential therapeutical profile and a strong patent position are key positioning considerations, as well as vital elements in shortening the development phase and protecting innovation. Installation of an interdisciplinary project management team, along with a clear definition of team members' responsibilities, is required to avoid delays and improve communication during development. Selection of the organism to be used in production must take into consideration both the structure of the protein and the quality and safety of the final product. New technologies require a considerable investment in new manufacturing facilities and equipment. Often, the decision for such an investment must be made early and with a high degree of uncertainty. Desired product yield, expected dosage, and estimated market potential are the most important considerations in this decision. Following public disclosure of the plan to develop recombinant DNA-derived products, approval of the production plant and expansion or adaptation to the new process and technology may be delayed. For this reason, they should be considered as a critical step in the overall development phase. Recruitment of qualified staff is a time-consuming and critical element of the production process. Its impact on the product timeline should not be underestimated, especially if such technologies are new to the company. The entire production process must be validated in respect to identity, purity, and safety of the product to guarantee constant product quality, as well as for safety aspects in the environment. Adequate in-process and final product

Collaborative Learning in Biology: Debating the Ethics of Recombinant DNA Technology.

ERIC Educational Resources Information Center

Anderson, Rodney P.

1998-01-01

Discusses applications of recombinant DNA technology and the controversies surrounding that technique. Provides a cooperative learning project idea that involves teams of students investigating and debating these issues. (DDR)

RPA accumulation during class switch recombination represents 5'-3' DNA-end resection during the S-G2/M phase of the cell cycle.

PubMed

Yamane, Arito; Robbiani, Davide F; Resch, Wolfgang; Bothmer, Anne; Nakahashi, Hirotaka; Oliveira, Thiago; Rommel, Philipp C; Brown, Eric J; Nussenzweig, Andre; Nussenzweig, Michel C; Casellas, Rafael

2013-01-31

Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

A Collaborative, Investigative Recombinant DNA Technology Course with Laboratory

ERIC Educational Resources Information Center

Pezzementi, Leo; Johnson, Joy F.

2002-01-01

A recombinant DNA technology course was designed to promote contextual, collaborative, inquiry-based learning of science where students learn from one another and have a sense of ownership of their education. The class stressed group presentations and critical reading and discussion of scientific articles. The laboratory consisted of two research…

Strand invasion structures in the inverted repeat of Candida albicans mitochondrial DNA reveal a role for homologous recombination in replication.

PubMed

Gerhold, Joachim M; Aun, Anu; Sedman, Tiina; Jõers, Priit; Sedman, Juhan

2010-09-24

Molecular recombination and transcription are proposed mechanisms to initiate mitochondrial DNA (mtDNA) replication in yeast. We conducted a comprehensive analysis of mtDNA from the yeast Candida albicans. Two-dimensional agarose gel electrophoresis of mtDNA intermediates reveals no bubble structures diagnostic of specific replication origins, but rather supports recombination-driven replication initiation of mtDNA in yeast. Specific species of Y structures together with DNA copy number analyses of a C. albicans mutant strain provide evidence that a region in a mainly noncoding inverted repeat is predominantly involved in replication initiation via homologous recombination. Our further findings show that the C. albicans mtDNA forms a complex branched network that does not contain detectable amounts of circular molecules. We provide topological evidence for recombination-driven mtDNA replication initiation and introduce C. albicans as a suitable model organism to study wild-type mtDNA maintenance in yeast. Copyright © 2010 Elsevier Inc. All rights reserved.

Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

The C terminus of Ku80 activates the DNA-dependent protein kinase catalytic subunit.

PubMed

Singleton, B K; Torres-Arzayus, M I; Rottinghaus, S T; Taccioli, G E; Jeggo, P A

1999-05-01

Ku is a heterodimeric protein with double-stranded DNA end-binding activity that operates in the process of nonhomologous end joining. Ku is thought to target the DNA-dependent protein kinase (DNA-PK) complex to the DNA and, when DNA bound, can interact and activate the DNA-PK catalytic subunit (DNA-PKcs). We have carried out a 3' deletion analysis of Ku80, the larger subunit of Ku, and shown that the C-terminal 178 amino acid residues are dispensable for DNA end-binding activity but are required for efficient interaction of Ku with DNA-PKcs. Cells expressing Ku80 proteins that lack the terminal 178 residues have low DNA-PK activity, are radiation sensitive, and can recombine the signal junctions but not the coding junctions during V(D)J recombination. These cells have therefore acquired the phenotype of mouse SCID cells despite expressing DNA-PKcs protein, suggesting that an interaction between DNA-PKcs and Ku, involving the C-terminal region of Ku80, is required for DNA double-strand break rejoining and coding but not signal joint formation. To gain further insight into important domains in Ku80, we report a point mutational change in Ku80 in the defective xrs-2 cell line. This residue is conserved among species and lies outside of the previously reported Ku70-Ku80 interaction domain. The mutational change nonetheless abrogates the Ku70-Ku80 interaction and DNA end-binding activity.

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

PubMed Central

Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

2014-01-01

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

PubMed

Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

2013-04-01

In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

Phosphorylated (pT371)TRF1 is recruited to sites of DNA damage to facilitate homologous recombination and checkpoint activation

PubMed Central

McKerlie, Megan; Walker, John R.; Mitchell, Taylor R. H.; Wilson, Florence R.; Zhu, Xu-Dong

2013-01-01

TRF1, a duplex telomeric DNA-binding protein, plays an important role in telomere metabolism. We have previously reported that a fraction of endogenous TRF1 can stably exist free of telomere chromatin when it is phosphorylated at T371 by Cdk1; however, the role of this telomere-free (pT371)TRF1 has yet to be fully characterized. Here we show that phosphorylated (pT371)TRF1 is recruited to sites of DNA damage, forming damage-induced foci in response to ionizing radiation (IR), etoposide and camptothecin. We find that IR-induced (pT371)TRF1 foci formation is dependent on the ATM- and Mre11/Rad50/Nbs1-mediated DNA damage response. While loss of functional BRCA1 impairs the formation of IR-induced (pT371)TRF1 foci, depletion of either 53BP1 or Rif1 stimulates IR-induced (pT371)TRF1 foci formation. In addition, we show that TRF1 depletion or the lack of its phosphorylation at T371 impairs DNA end resection and repair of nontelomeric DNA double-strand breaks by homologous recombination. The lack of TRF1 phosphorylation at T371 also hampers the activation of the G2/M checkpoint and sensitizes cells to PARP inhibition, IR and camptothecin. Collectively, these results reveal a novel but important function of phosphorylated (pT371)TRF1 in facilitating DNA double-strand break repair and the maintenance of genome integrity. PMID:23997120

Mdt1 Facilitates Efficient Repair of Blocked DNA Double-Strand Breaks and Recombinational Maintenance of Telomeres▿

PubMed Central

Pike, Brietta L.; Heierhorst, Jörg

2007-01-01

DNA recombination plays critical roles in DNA repair and alternative telomere maintenance. Here we show that absence of the SQ/TQ cluster domain-containing protein Mdt1 (Ybl051c) renders Saccharomyces cerevisiae particularly hypersensitive to bleomycin, a drug that causes 3′-phospho-glycolate-blocked DNA double-strand breaks (DSBs). mdt1Δ also hypersensitizes partially recombination-defective cells to camptothecin-induced 3′-phospho-tyrosyl protein-blocked DSBs. Remarkably, whereas mdt1Δ cells are unable to restore broken chromosomes after bleomycin treatment, they efficiently repair “clean” endonuclease-generated DSBs. Epistasis analyses indicate that MDT1 acts in the repair of bleomycin-induced DSBs by regulating the efficiency of the homologous recombination pathway as well as telomere-related functions of the KU complex. Moreover, mdt1Δ leads to severe synthetic growth defects with a deletion of the recombination facilitator and telomere-positioning factor gene CTF18 already in the absence of exogenous DNA damage. Importantly, mdt1Δ causes a dramatic shift from the usually prevalent type II to the less-efficient type I pathway of recombinational telomere maintenance in the absence of telomerase in liquid senescence assays. As telomeres resemble protein-blocked DSBs, the results indicate that Mdt1 acts in a novel blocked-end-specific recombination pathway that is required for the efficiency of both drug-induced DSB repair and telomerase-independent telomere maintenance. PMID:17636027

Bacteriophage T5 encodes a homolog of the eukaryotic transcription coactivator PC4 implicated in recombination-dependent DNA replication.

PubMed

Steigemann, Birthe; Schulz, Annina; Werten, Sebastiaan

2013-11-15

The RNA polymerase II cofactor PC4 globally regulates transcription of protein-encoding genes through interactions with unwinding DNA, the basal transcription machinery and transcription activators. Here, we report the surprising identification of PC4 homologs in all sequenced representatives of the T5 family of bacteriophages, as well as in an archaeon and seven phyla of eubacteria. We have solved the crystal structure of the full-length T5 protein at 1.9Å, revealing a striking resemblance to the characteristic single-stranded DNA (ssDNA)-binding core domain of PC4. Intriguing novel structural features include a potential regulatory region at the N-terminus and a C-terminal extension of the homodimerisation interface. The genome organisation of T5-related bacteriophages points at involvement of the PC4 homolog in recombination-dependent DNA replication, strongly suggesting that the protein corresponds to the hitherto elusive replicative ssDNA-binding protein of the T5 family. Our findings imply that PC4-like factors intervene in multiple unwinding-related processes by acting as versatile modifiers of nucleic acid conformation and raise the possibility that the eukaryotic transcription coactivator derives from ancestral DNA replication, recombination and repair factors. © 2013.

Recombination of mitochondrial DNA without selection pressure among compatible strains of the Aspergillus niger species aggregate.

PubMed

Tóth, B; Hamari, Z; Ferenczy, L; Varga, J; Kevei, F

1998-03-01

Previous mitochondrial transmission experiments between oligomycin-resistant and oligomycin-sensitive incompatible strains of the A. niger aggregate bearing various mtDNA RFLP profiles resulted in a great variety of mitochondrial recombinants under selection pressure. Apart from the recombinant mtDNAs, resistant clones harbouring unchanged RFLP profiles of resistant donor mtDNAs with the recipient nuclear backgrounds were rarely isolated. These strains were anastomosed with nuclearly isogenic oligomycin-sensitive recipient partners and the mitochondria of the resulting progeny were examined under non-selective conditions. These experiments provide insights into events which are possibly similar to those occurring in nature. The heterokaryons obtained formed both oligomycin-resistant and -sensitive sectors, most of which were found to be homoplasmons. Progenies harbouring oligomycin-resistant and -sensitive mtDNAs may originate either from individual recombination events or be due to parental segregation. MtDNA recombination might take place in the heterokaryons without selection by oligomycin. The most frequent recombinant types of mtDNA RFLP profiles were indistinguishable from those recombinant mtDNAs which were frequently obtained under selection pressure from directed transfer experiments between incompatible strains. We present evidence that mixed mitochondrial populations may influence the compatibility reactions in the presence of an isogenic nuclear background, that recombination may take place without selection pressure, and that the process does not require specific nuclear sequences of both parental strains.

Exploration of the Dissociative Recombination following DNA ionization to DNA+ due to ionizing radiation

NASA Astrophysics Data System (ADS)

Strom, Richard A.; Zimmerly, Andrew T.; Andrianarijaona, Vola M.

2014-05-01

It is known that ionizing radiation generates low-energy secondary electrons, which may interact with the surrounding area, including biomolecules, such as triggering DNA single strand and double strand breaks as demonstrated by Sanche and coworkers (Radiat. Res. 157, 227(2002)). The bio-effects of low-energy electrons are currently a topic of high interest. Most of the studies are dedicated to dissociative electron attachments; however, the area is still mostly unexplored and still not well understood. We are computationally investigating the effect of ionizing radiation on DNA, such as its ionization to DNA+. More specifically, we are exploring the possibility of the dissociative recombination of the temporary DNA+ with one of the low-energy secondary electrons, produced by the ionizing radiation, to be another process of DNA strand breaks. Our preliminary results, which are performed with the binaries of ORCA, will be presented. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

Loop L1 governs the DNA-binding specificity and order for RecA-catalyzed reactions in homologous recombination and DNA repair

PubMed Central

Shinohara, Takeshi; Ikawa, Shukuko; Iwasaki, Wakana; Hiraki, Toshiki; Hikima, Takaaki; Mikawa, Tsutomu; Arai, Naoto; Kamiya, Nobuo; Shibata, Takehiko

2015-01-01

In all organisms, RecA-family recombinases catalyze homologous joint formation in homologous genetic recombination, which is essential for genome stability and diversification. In homologous joint formation, ATP-bound RecA/Rad51-recombinases first bind single-stranded DNA at its primary site and then interact with double-stranded DNA at another site. The underlying reason and the regulatory mechanism for this conserved binding order remain unknown. A comparison of the loop L1 structures in a DNA-free RecA crystal that we originally determined and in the reported DNA-bound active RecA crystals suggested that the aspartate at position 161 in loop L1 in DNA-free RecA prevented double-stranded, but not single-stranded, DNA-binding to the primary site. This was confirmed by the effects of the Ala-replacement of Asp-161 (D161A), analyzed directly by gel-mobility shift assays and indirectly by DNA-dependent ATPase activity and SOS repressor cleavage. When RecA/Rad51-recombinases interact with double-stranded DNA before single-stranded DNA, homologous joint-formation is suppressed, likely by forming a dead-end product. We found that the D161A-replacement reduced this suppression, probably by allowing double-stranded DNA to bind preferentially and reversibly to the primary site. Thus, Asp-161 in the flexible loop L1 of wild-type RecA determines the preference for single-stranded DNA-binding to the primary site and regulates the DNA-binding order in RecA-catalyzed recombinase reactions. PMID:25561575

Msh1p counteracts oxidative lesion-induced instability of mtDNA and stimulates mitochondrial recombination in Saccharomyces cerevisiae.

PubMed

Kaniak, Aneta; Dzierzbicki, Piotr; Rogowska, Agata T; Malc, Ewa; Fikus, Marta; Ciesla, Zygmunt

2009-03-01

The proximity of the mitochondrial genome to the respiratory chain, a major source of ROS (radical oxygen species), makes mtDNA more vulnerable to oxidative damage than nuclear DNA. Mitochondrial BER (base excision repair) is generally considered to be the main pathway involved in the prevention of oxidative lesion-induced mutations in mtDNA. However, we previously demonstrated that the increased frequency of mitochondrial Oli(r) mutants in an ogg1Delta strain, lacking the activity of a crucial mtBER glycosylase, is reduced in the presence of plasmids encoding Msh1p, the mitochondrial homologue of the bacterial mismatch protein MutS. This finding suggested that Msh1p might be involved in the prevention of mitochondrial mutagenesis induced by oxidative stress. Here we show that a double mutant carrying the msh1-R813W allele, encoding a variant of the protein defective in the ATP hydrolysis activity, combined with deletion of SOD2, encoding the mitochondrial superoxide dismutase, displays a synergistic effect on the frequency of Oli(r) mutants, indicating that Msh1p prevents generation of oxidative lesion-induced mitochondrial mutations. We also show that double mutants carrying the msh1-R813W allele, combined with deletion of either OGG1 or APN1, the latter resulting in deficiency of the Apn1 endonuclease, exhibit a synergistic effect on the frequency of respiration-defective mutants having gross rearrangements of the mitochondrial genome. This suggests that Msh1p, Ogg1p and Apn1p play overlapping functions in maintaining the stability of mtDNA. In addition, we demonstrate, using a novel ARG8(m) recombination assay, that a surplus of Msh1p results in enhanced mitochondrial recombination. Interestingly, the mutant forms of the protein, msh1p-R813W and msh1p-G776D, fail to stimulate recombination. We postulate that the Msh1p-enhanced homologous recombination may play an important role in the prevention of oxidative lesion-induced rearrangements of the mitochondrial

Activated recombinant adenovirus proteinases

DOEpatents

Anderson, C.W.; Mangel, W.F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

Activated recombinant adenovirus proteinases

DOEpatents

Anderson, Carl W.; Mangel, Walter F.

1999-08-10

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

The Saccharomyces cerevisiae DNA recombination and repair functions of the RAD52 epistasis group inhibit Ty1 transposition.

PubMed Central

Rattray, A J; Shafer, B K; Garfinkel, D J

2000-01-01

RNA transcribed from the Saccharomyces cerevisiae retrotransposon Ty1 accumulates to a high level in mitotically growing haploid cells, yet transposition occurs at very low frequencies. The product of reverse transcription is a linear double-stranded DNA molecule that reenters the genome by either Ty1-integrase-mediated insertion or homologous recombination with one of the preexisting genomic Ty1 (or delta) elements. Here we examine the role of the cellular homologous recombination functions on Ty1 transposition. We find that transposition is elevated in cells mutated for genes in the RAD52 recombinational repair pathway, such as RAD50, RAD51, RAD52, RAD54, or RAD57, or in the DNA ligase I gene CDC9, but is not elevated in cells mutated in the DNA repair functions encoded by the RAD1, RAD2, or MSH2 genes. The increase in Ty1 transposition observed when genes in the RAD52 recombinational pathway are mutated is not associated with a significant increase in Ty1 RNA or proteins. However, unincorporated Ty1 cDNA levels are markedly elevated. These results suggest that members of the RAD52 recombinational repair pathway inhibit Ty1 post-translationally by influencing the fate of Ty1 cDNA. PMID:10655210

Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

PubMed

Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

2015-02-01

There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection.

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation.

PubMed

De Tullio, Luisina; Kaniecki, Kyle; Kwon, Youngho; Crickard, J Brooks; Sung, Patrick; Greene, Eric C

2017-10-17

Srs2 is a super-family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single-molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (∼170 nt per second) in the 3'→5' direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA and, in doing so, promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Formation of (DNA)2-LNA triplet with recombinant base recognition: A quantum mechanical study

NASA Astrophysics Data System (ADS)

Mall, Vijaya Shri; Tiwari, Rakesh Kumar

2018-05-01

The formation of DNA triple helix offers the verity of new possibilities in molecular biology. However its applications are limited to purine and pyrimidine rich sequences recognized by forming Hoogsteen/Reverse Hoogsteen triplets in major groove sites of DNA duplex. To overcome this drawback modification in bases backbone and glucose of nucleotide unit of DNA have been proposed so that the third strand base recognized by both the bases of DNA duplex by forming Recombinant type(R-type) of bonding in mixed sequences. Here we performed Quanrum Mechanical (Hartree-Fock and DFT) methodology on natural DNA and Locked Nucleic Acids(LNA) triplets using 6-31G and some other new advance basis sets. Study suggests energetically stable conformation has been observed for recombinant triplets in order of G-C*G > A-T*A > G-C*C > T-A*T for both type of triplets. Interestingly LNA leads to more stable conformation in all set of triplets, clearly suggests an important biological tool to overcome above mentioned drawbacks.

DNA methylation epigenetically silences crossover hot spots and controls chromosomal domains of meiotic recombination in Arabidopsis.

PubMed

Yelina, Nataliya E; Lambing, Christophe; Hardcastle, Thomas J; Zhao, Xiaohui; Santos, Bruno; Henderson, Ian R

2015-10-15

During meiosis, homologous chromosomes undergo crossover recombination, which is typically concentrated in narrow hot spots that are controlled by genetic and epigenetic information. Arabidopsis chromosomes are highly DNA methylated in the repetitive centromeres, which are also crossover-suppressed. Here we demonstrate that RNA-directed DNA methylation is sufficient to locally silence Arabidopsis euchromatic crossover hot spots and is associated with increased nucleosome density and H3K9me2. However, loss of CG DNA methylation maintenance in met1 triggers epigenetic crossover remodeling at the chromosome scale, with pericentromeric decreases and euchromatic increases in recombination. We used recombination mutants that alter interfering and noninterfering crossover repair pathways (fancm and zip4) to demonstrate that remodeling primarily involves redistribution of interfering crossovers. Using whole-genome bisulfite sequencing, we show that crossover remodeling is driven by loss of CG methylation within the centromeric regions. Using cytogenetics, we profiled meiotic DNA double-strand break (DSB) foci in met1 and found them unchanged relative to wild type. We propose that met1 chromosome structure is altered, causing centromere-proximal DSBs to be inhibited from maturation into interfering crossovers. These data demonstrate that DNA methylation is sufficient to silence crossover hot spots and plays a key role in establishing domains of meiotic recombination along chromosomes. © 2015 Yelina et al.; Published by Cold Spring Harbor Laboratory Press.

Cancer, viruses, and mass migration: Paul Berg's venture into eukaryotic biology and the advent of recombinant DNA research and technology, 1967-1980.

PubMed

Yi, Doogab

2008-01-01

The existing literature on the development of recombinant DNA technology and genetic engineering tends to focus on Stanley Cohen and Herbert Boyer's recombinant DNA cloning technology and its commercialization starting in the mid-1970s. Historians of science, however, have pointedly noted that experimental procedures for making recombinant DNA molecules were initially developed by Stanford biochemist Paul Berg and his colleagues, Peter Lobban and A. Dale Kaiser in the early 1970s. This paper, recognizing the uneasy disjuncture between scientific authorship and legal invention in the history of recombinant DNA technology, investigates the development of recombinant DNA technology in its full scientific context. I do so by focusing on Stanford biochemist Berg's research on the genetic regulation of higher organisms. As I hope to demonstrate, Berg's new venture reflected a mass migration of biomedical researchers as they shifted from studying prokaryotic organisms like bacteria to studying eukaryotic organisms like mammalian and human cells. It was out of this boundary crossing from prokaryotic to eukaryotic systems through virus model systems that recombinant DNA technology and other significant new research techniques and agendas emerged. Indeed, in their attempt to reconstitute 'life' as a research technology, Stanford biochemists' recombinant DNA research recast genes as a sequence that could be rewritten thorough biochemical operations. The last part of this paper shifts focus from recombinant DNA technology's academic origins to its transformation into a genetic engineering technology by examining the wide range of experimental hybridizations which occurred as techniques and knowledge circulated between Stanford biochemists and the Bay Area's experimentalists. Situating their interchange in a dense research network based at Stanford's biochemistry department, this paper helps to revise the canonized history of genetic engineering's origins that emerged during

Refined protocols of tamoxifen injection for inducible DNA recombination in mouse astroglia.

PubMed

Jahn, Hannah M; Kasakow, Carmen V; Helfer, Andreas; Michely, Julian; Verkhratsky, Alexei; Maurer, Hans H; Scheller, Anja; Kirchhoff, Frank

2018-04-12

Inducible DNA recombination of floxed alleles in vivo by liver metabolites of tamoxifen (TAM) is an important tool to study gene functions. Here, we describe protocols for optimal DNA recombination in astrocytes, based on the GLAST-Cre ERT2 /loxP system. In addition, we demonstrate that quantification of genomic recombination allows to determine the proportion of cell types in various brain regions. We analyzed the presence and clearance of TAM and its metabolites (N-desmethyl-tamoxifen, 4-hydroxytamoxifen and endoxifen) in brain and serum of mice by liquid chromatographic-high resolution-tandem mass spectrometry (LC-HR-MS/MS) and assessed optimal injection protocols by quantitative RT-PCR of several floxed target genes (p2ry1, gria1, gabbr1 and Rosa26-tdTomato locus). Maximal recombination could be achieved in cortex and cerebellum by single daily injections for five and three consecutive days, respectively. Furthermore, quantifying the loss of floxed alleles predicted the percentage of GLAST-positive cells (astroglia) per brain region. We found that astrocytes contributed 20 to 30% of the total cell number in cortex, hippocampus, brainstem and optic nerve, while in the cerebellum Bergmann glia, velate astrocytes and white matter astrocytes accounted only for 8% of all cells.

Inhibitors of the proteasome suppress homologous DNA recombination in mammalian cells.

PubMed

Murakawa, Yasuhiro; Sonoda, Eiichiro; Barber, Louise J; Zeng, Weihua; Yokomori, Kyoko; Kimura, Hiroshi; Niimi, Atsuko; Lehmann, Alan; Zhao, Guang Yu; Hochegger, Helfrid; Boulton, Simon J; Takeda, Shunichi

2007-09-15

Proteasome inhibitors are novel antitumor agents against multiple myeloma and other malignancies. Despite the increasing clinical application, the molecular basis of their antitumor effect has been poorly understood due to the involvement of the ubiquitin-proteasome pathway in multiple cellular metabolisms. Here, we show that treatment of cells with proteasome inhibitors has no significant effect on nonhomologous end joining but suppresses homologous recombination (HR), which plays a key role in DNA double-strand break (DSB) repair. In this study, we treat human cells with proteasome inhibitors and show that the inhibition of the proteasome reduces the efficiency of HR-dependent repair of an artificial HR substrate. We further show that inhibition of the proteasome interferes with the activation of Rad51, a key factor for HR, although it does not affect the activation of ATM, gammaH2AX, or Mre11. These data show that the proteasome-mediated destruction is required for the promotion of HR at an early step. We suggest that the defect in HR-mediated DNA repair caused by proteasome inhibitors contributes to antitumor effect, as HR plays an essential role in cellular proliferation. Moreover, because HR plays key roles in the repair of DSBs caused by chemotherapeutic agents such as cisplatin and by radiotherapy, proteasome inhibitors may enhance the efficacy of these treatments through the suppression of HR-mediated DNA repair pathways.

Ectopic recombination between Ty elements in Saccharomyces cerevisiae is not induced by DNA damage.

PubMed

Parket, A; Kupiec, M

1992-10-01

Mitotic recombination is increased when cells are treated with a variety of physical and chemical agents that cause damage to their DNA. We show here, using Saccharomyces cerevisiae strains that carry marked Ty elements, that recombination between members of this family of retrotransposons is not increased by UV irradiation or by treatment with the radiomimetic drug methyl methanesulfonate. Both ectopic recombination and mutation events were elevated by these agents for non-Ty sequences in the same strain. We discuss possible mechanisms that can prevent the induction of recombination between Ty elements.

Process of labeling specific chromosomes using recombinant repetitive DNA

DOEpatents

Moyzis, R.K.; Meyne, J.

1988-02-12

Chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

Government Regulation of the Pursuit of Knowledge: The Recombinant DNA Controversy.

ERIC Educational Resources Information Center

Berger, Richard G.

1978-01-01

Government regulation of recombinant DNA research is addressed. Issues discussed include the potential of such research; National Institutes of Health guidelines; federal, state, and local regulation; the controversy over self-regulation; first amendment protection for scientific research; and problems in drafting legislation. (JMD)

Replication Intermediates of the Linear Mitochondrial DNA of Candida parapsilosis Suggest a Common Recombination Based Mechanism for Yeast Mitochondria*

PubMed Central

Gerhold, Joachim M.; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-01-01

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. PMID:24951592

Coexistence of minicircular and a highly rearranged mtDNA molecule suggests that recombination shapes mitochondrial genome organization.

PubMed

Mao, Meng; Austin, Andrew D; Johnson, Norman F; Dowton, Mark

2014-03-01

Recombination has been proposed as a possible mechanism to explain mitochondrial (mt) gene rearrangements, although the issue of whether mtDNA recombination occurs in animals has been controversial. In this study, we sequenced the entire mt genome of the megaspilid wasp Conostigmus sp., which possessed a highly rearranged mt genome. The sequence of the A+T-rich region contained a number of different types of repeats, similar to those reported previously in the nematode Meloidogyne javanica, in which recombination was discovered. In Conostigmus, we detected the end products of recombination: a range of minicircles. However, using isolated (cloned) fragments of the A+T-rich region, we established that some of these minicircles were found to be polymerase chain reaction (PCR) artifacts. It appears that regions with repeats are prone to PCR template switching or PCR jumping. Nevertheless, there is strong evidence that one minicircle is real, as amplification primers that straddle the putative breakpoint junction produce a single strong amplicon from genomic DNA but not from the cloned A+T-rich region. The results provide support for the direct link between recombination and mt gene rearrangement. Furthermore, we developed a model of recombination which is important for our understanding of mtDNA evolution.

Distinct DNA-binding surfaces in the ATPase and linker domains of MutLγ determine its substrate specificities and exert separable functions in meiotic recombination and mismatch repair

PubMed Central

2017-01-01

Mlh1-Mlh3 (MutLγ) is a mismatch repair factor with a central role in formation of meiotic crossovers, presumably through resolution of double Holliday junctions. MutLγ has DNA-binding, nuclease, and ATPase activities, but how these relate to one another and to in vivo functions are unclear. Here, we combine biochemical and genetic analyses to characterize Saccharomyces cerevisiae MutLγ. Limited proteolysis and atomic force microscopy showed that purified recombinant MutLγ undergoes ATP-driven conformational changes. In vitro, MutLγ displayed separable DNA-binding activities toward Holliday junctions (HJ) and, surprisingly, single-stranded DNA (ssDNA), which was not predicted from current models. MutLγ bound DNA cooperatively, could bind multiple substrates simultaneously, and formed higher-order complexes. FeBABE hydroxyl radical footprinting indicated that the DNA-binding interfaces of MutLγ for ssDNA and HJ substrates only partially overlap. Most contacts with HJ substrates were located in the linker regions of MutLγ, whereas ssDNA contacts mapped within linker regions as well as the N-terminal ATPase domains. Using yeast genetic assays for mismatch repair and meiotic recombination, we found that mutations within different DNA-binding surfaces exert separable effects in vivo. For example, mutations within the Mlh1 linker conferred little or no meiotic phenotype but led to mismatch repair deficiency. Interestingly, mutations in the N-terminal domain of Mlh1 caused a stronger meiotic defect than mlh1Δ, suggesting that the mutant proteins retain an activity that interferes with alternative recombination pathways. Furthermore, mlh3Δ caused more chromosome missegregation than mlh1Δ, whereas mlh1Δ but not mlh3Δ partially alleviated meiotic defects of msh5Δ mutants. These findings illustrate functional differences between Mlh1 and Mlh3 during meiosis and suggest that their absence impinges on chromosome segregation not only via reduced formation of

Effect of DNA Binding on Geminate CO Recombination Kinetics in CO-sensing Transcription Factor CooA*

PubMed Central

Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L.; Champion, Paul M.

2012-01-01

Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role. PMID:22544803

Effect of DNA binding on geminate CO recombination kinetics in CO-sensing transcription factor CooA.

PubMed

Benabbas, Abdelkrim; Karunakaran, Venugopal; Youn, Hwan; Poulos, Thomas L; Champion, Paul M

2012-06-22

Carbon monoxide oxidation activator (CooA) proteins are heme-based CO-sensing transcription factors. Here we study the ultrafast dynamics of geminate CO rebinding in two CooA homologues, Rhodospirillum rubrum (RrCooA) and Carboxydothermus hydrogenoformans (ChCooA). The effects of DNA binding and the truncation of the DNA-binding domain on the CO geminate recombination kinetics were specifically investigated. The CO rebinding kinetics in these CooA complexes take place on ultrafast time scales but remain non-exponential over many decades in time. We show that this non-exponential kinetic response is due to a quenched enthalpic barrier distribution resulting from a distribution of heme geometries that is frozen or slowly evolving on the time scale of CO rebinding. We also show that, upon CO binding, the distal pocket of the heme in the CooA proteins relaxes to form a very efficient hydrophobic trap for CO. DNA binding further tightens the narrow distal pocket and slightly weakens the iron-proximal histidine bond. Comparison of the CO rebinding kinetics of RrCooA, truncated RrCooA, and DNA-bound RrCooA proteins reveals that the uncomplexed and inherently flexible DNA-binding domain adds additional structural heterogeneity to the heme doming coordinate. When CooA forms a complex with DNA, the flexibility of the DNA-binding domain decreases, and the distribution of the conformations available in the heme domain becomes restricted. The kinetic studies also offer insights into how the architecture of the heme environment can tune entropic barriers in order to control the geminate recombination of CO in heme proteins, whereas spin selection rules play a minor or non-existent role.

Mixed infection of Sida jamaicensis in Jamaica reveals the presence of three recombinant begomovirus DNA A components.

PubMed

Stewart, Cheryl; Kon, Tatsuya; Rojas, Maria; Graham, André; Martin, Darren; Gilbertson, Robert; Roye, Marcia

2014-09-01

Begomoviruses impose serious constraints on agriculture throughout the temperate, tropical and subtropical regions. Previously, we characterised a sida golden yellow vein virus isolate, SiGYVV-[JM:Lig2:08] (HQ009519-20) from a symptomatic Sida jamaicensis plant. With the aim of establishing whether it was hosting a mixed infection that could facilitate recombination, PCR-RFLP was done on DNA extracted from this plant, and the results suggested the presence of two additional genetically distinct DNA-A molecules. Sequence analysis of these two DNA-A molecules (relying on BLAST searches and the CLUSTAL V algorithm within the DNASTAR MegAlign module) revealed that they belonged to novel species, and we have tentatively named these viruses sida golden mosaic Braco virus-[Jamaica:Liguanea:2008] and sida golden mosaic Liguanea virus-[Jamaica:1:2008]. Using RDP4 (recombination detection program), we determined that all three viruses were recombinant, with bases ~10 to ~440 of both SiGMLigV-[JM:Lig:08] and SiGYVV-[JM:Lig2:08] having been derived from a relative of SiGMBV-[JM:Lig:08] (P<2.070×10(-7) for all seven of the recombination detection methods). SiGMBV-[JM:Lig:08] was itself a product of recombination, deriving bases ~490-1195 from a virus that was ~92% similar to malvastrum yellow mosaic Helshire virus. Phylogenetically, these DNA-A components are most closely related to those of malvaceous weed-infecting begomoviruses from Jamaica, Cuba, Florida and Mexico. The SiGMBV DNA-A was able to elicit symptomatic infection in N. benthamiana.

Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

PubMed Central

Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

2011-01-01

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

Tankyrases Promote Homologous Recombination and Check Point Activation in Response to DSBs

PubMed Central

Furst, Audrey; Koch, Marc; Fischer, Benoit; Soutoglou, Evi

2016-01-01

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation. PMID:26845027

Identification and analysis of recombineering functions from Gram-negative and Gram-positive bacteria and their phages

PubMed Central

Datta, Simanti; Costantino, Nina; Zhou, Xiaomei; Court, Donald L.

2008-01-01

We report the identification and functional analysis of nine genes from Gram-positive and Gram-negative bacteria and their phages that are similar to lambda (λ) bet or Escherichia coli recT. Beta and RecT are single-strand DNA annealing proteins, referred to here as recombinases. Each of the nine other genes when expressed in E. coli carries out oligonucleotide-mediated recombination. To our knowledge, this is the first study showing single-strand recombinase activity from diverse bacteria. Similar to bet and recT, most of these other recombinases were found to be associated with putative exonuclease genes. Beta and RecT in conjunction with their cognate exonucleases carry out recombination of linear double-strand DNA. Among four of these foreign recombinase/exonuclease pairs tested for recombination with double-strand DNA, three had activity, albeit barely detectable. Thus, although these recombinases can function in E. coli to catalyze oligonucleotide recombination, the double-strand DNA recombination activities with their exonuclease partners were inefficient. This study also demonstrated that Gam, by inhibiting host RecBCD nuclease activity, helps to improve the efficiency of λ Red-mediated recombination with linear double-strand DNA, but Gam is not absolutely essential. Thus, in other bacterial species where Gam analogs have not been identified, double-strand DNA recombination may still work in the absence of a Gam-like function. We anticipate that at least some of the recombineering systems studied here will potentiate oligonucleotide and double-strand DNA-mediated recombineering in their native or related bacteria. PMID:18230724

Are High School Students Ready for Recombinant DNA?: The UOP Experience.

ERIC Educational Resources Information Center

Minch, Michael J.

1989-01-01

Discusses a three-week summer college honors course for talented high school juniors with three exams, lab six days a week, a research paper, field trips, and student panel discussions. Presents an overview of the course. Describes the lab which uses "E. coli" for DNA recombination. (MVL)

Replication intermediates of the linear mitochondrial DNA of Candida parapsilosis suggest a common recombination based mechanism for yeast mitochondria.

PubMed

Gerhold, Joachim M; Sedman, Tiina; Visacka, Katarina; Slezakova, Judita; Tomaska, Lubomir; Nosek, Jozef; Sedman, Juhan

2014-08-15

Variation in the topology of mitochondrial DNA (mtDNA) in eukaryotes evokes the question if differently structured DNAs are replicated by a common mechanism. RNA-primed DNA synthesis has been established as a mechanism for replicating the circular animal/mammalian mtDNA. In yeasts, circular mtDNA molecules were assumed to be templates for rolling circle DNA-replication. We recently showed that in Candida albicans, which has circular mapping mtDNA, recombination driven replication is a major mechanism for replicating a complex branched mtDNA network. Careful analyses of C. albicans-mtDNA did not reveal detectable amounts of circular DNA molecules. In the present study we addressed the question of how the unit sized linear mtDNA of Candida parapsilosis terminating at both ends with arrays of tandem repeats (mitochondrial telomeres) is replicated. Originally, we expected to find replication intermediates diagnostic of canonical bi-directional replication initiation at the centrally located bi-directional promoter region. However, we found that the linear mtDNA of Candida parapsilosis also employs recombination for replication initiation. The most striking findings were that the mitochondrial telomeres appear to be hot spots for recombination driven replication, and that stable RNA:DNA hybrids, with a potential role in mtDNA replication, are also present in the mtDNA preparations. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The "Frankenplasmid" Lab: An Investigative Exercise for Teaching Recombinant DNA Methods

ERIC Educational Resources Information Center

Dean, Derek M.; Wilder, Jason A.

2011-01-01

We describe an investigative laboratory module designed to give college undergraduates strong practical and theoretical experience with recombinant DNA methods within 3 weeks. After deducing restriction enzyme maps for two different plasmids, students ligate the plasmids together in the same reaction, transform "E. coli" with this mixture of…

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

PubMed Central

Bolderson, Emma; Tomimatsu, Nozomi; Richard, Derek J.; Boucher, Didier; Kumar, Rakesh; Pandita, Tej K.; Burma, Sandeep; Khanna, Kum Kum

2010-01-01

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability. PMID:20019063

Epstein-Barr virus recombinants from overlapping cosmid fragments.

PubMed

Tomkinson, B; Robertson, E; Yalamanchili, R; Longnecker, R; Kieff, E

1993-12-01

Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the

Heat-mediated activation of affinity-immobilized Taq DNA polymerase.

PubMed

Nilsson, J; Bosnes, M; Larsen, F; Nygren, P A; Uhlén, M; Lundeberg, J

1997-04-01

A novel strategy for heat-mediated activation of recombinant Taq DNA polymerase is described. A serum albumin binding protein tag is used to affinity-immobilize an E. coli-expressed Taq DNA polymerase fusion protein onto a solid support coated with human serum albumin (HSA). Analysis of heat-mediated elution showed that elevated temperatures (> 70 degrees C) were required to significantly release the fusion protein from the solid support. A primer-extension assay showed that immobilization of the fusion protein resulted in little or no extension product. In contrast, fusion protein released from the HSA ligand by heat showed high polymerase activity. Thus, a heat-mediated release and reactivation of the Taq DNA polymerase fusion protein from the solid support can be obtained to allow for hot-start PCR with improved amplification performance.

Induction of homologous recombination following in utero exposure to DNA-damaging agents.

PubMed

Karia, Bijal; Martinez, Jo Ann; Bishop, Alexander J R

2013-11-01

Much of our understanding of homologous recombination, as well as the development of the working models for these processes, has been derived from extensive work in model organisms, such as yeast and fruit flies, and mammalian systems by studying the repair of induced double strand breaks or repair following exposure to genotoxic agents in vitro. We therefore set out to expand this in vitro work to ask whether DNA-damaging agents with varying modes of action could induce somatic change in an in vivo mouse model of homologous recombination. We exposed pregnant dams to DNA-damaging agents, conferring a variety of lesions at a specific time in embryo development. To monitor homologous recombination frequency, we used the well-established retinal pigment epithelium pink-eyed unstable assay. Homologous recombination resulting in the deletion of a duplicated 70 kb fragment in the coding region of the Oca2 gene renders this gene functional and can be visualized as a pigmented eyespot in the retinal pigment epithelium. We observed an increased frequency of pigmented eyespots in resultant litters following exposure to cisplatin, methyl methanesulfonate, ethyl methanesulfonate, 3-aminobenzamide, bleomycin, and etoposide with a contrasting decrease in the frequency of detectable reversion events following camptothecin and hydroxyurea exposure. The somatic genomic rearrangements that result from such a wide variety of differently acting damaging agents implies long-term potential effects from even short-term in utero exposures. Copyright © 2013 Elsevier B.V. All rights reserved.

Parallel In Vivo DNA Assembly by Recombination: Experimental Demonstration and Theoretical Approaches

PubMed Central

Shi, Zhenyu; Wedd, Anthony G.; Gras, Sally L.

2013-01-01

The development of synthetic biology requires rapid batch construction of large gene networks from combinations of smaller units. Despite the availability of computational predictions for well-characterized enzymes, the optimization of most synthetic biology projects requires combinational constructions and tests. A new building-brick-style parallel DNA assembly framework for simple and flexible batch construction is presented here. It is based on robust recombination steps and allows a variety of DNA assembly techniques to be organized for complex constructions (with or without scars). The assembly of five DNA fragments into a host genome was performed as an experimental demonstration. PMID:23468883

Recombinant Salmonella Bacteria Vectoring HIV/AIDS Vaccines

PubMed Central

Chin’ombe, Nyasha; Ruhanya, Vurayai

2013-01-01

HIV/AIDS is an important public health problem globally. An affordable, easy-to-deliver and protective HIV vaccine is therefore required to curb the pandemic from spreading further. Recombinant Salmonella bacteria can be harnessed to vector HIV antigens or DNA vaccines to the immune system for induction of specific protective immunity. These are capable of activating the innate, humoral and cellular immune responses at both mucosal and systemic compartments. Several studies have already demonstrated the utility of live recombinant Salmonella in delivering expressed foreign antigens as well as DNA vaccines to the host immune system. This review gives an overview of the studies in which recombinant Salmonella bacteria were used to vector HIV/AIDS antigens and DNA vaccines. Most of the recombinant Salmonella-based HIV/AIDS vaccines developed so far have only been tested in animals (mainly mice) and are yet to reach human trials. PMID:24478808

How-to-Do-It: Teaching Recombinant DNA Technology in High School Biology Courses.

ERIC Educational Resources Information Center

Dixon, Linda

1988-01-01

Reports on the teaching of recombinant DNA technology in high school biology courses. Explains reactions of the public, students, and colleagues to the molecular genetics unit. Indicates equipment, curricular materials, training, workshops, and availability. (RT)

Ensuring an exit strategy: RTEL1 restricts rogue recombination.

PubMed

Villeneuve, Anne M

2008-10-17

Success of homologous recombination-based DNA repair depends not only on recombinases, which promote invasion of the homologous DNA duplex that serves as a template for repair, but also on antirecombinases, which dismantle recombination intermediates to allow completion of repair. In this issue, Barber et al. (2008) identify a previously elusive antirecombinase activity important for maintaining genome stability in animals.

Promotion of BRCA2-Dependent Homologous Recombination by DSS1 via RPA Targeting and DNA Mimicry.

PubMed

Zhao, Weixing; Vaithiyalingam, Sivaraja; San Filippo, Joseph; Maranon, David G; Jimenez-Sainz, Judit; Fontenay, Gerald V; Kwon, Youngho; Leung, Stanley G; Lu, Lucy; Jensen, Ryan B; Chazin, Walter J; Wiese, Claudia; Sung, Patrick

2015-07-16

The tumor suppressor BRCA2 is thought to facilitate the handoff of ssDNA from replication protein A (RPA) to the RAD51 recombinase during DNA break and replication fork repair by homologous recombination. However, we find that RPA-RAD51 exchange requires the BRCA2 partner DSS1. Biochemical, structural, and in vivo analyses reveal that DSS1 allows the BRCA2-DSS1 complex to physically and functionally interact with RPA. Mechanistically, DSS1 acts as a DNA mimic to attenuate the affinity of RPA for ssDNA. A mutation in the solvent-exposed acidic domain of DSS1 compromises the efficacy of RPA-RAD51 exchange. Thus, by targeting RPA and mimicking DNA, DSS1 functions with BRCA2 in a two-component homologous recombination mediator complex in genome maintenance and tumor suppression. Our findings may provide a paradigm for understanding the roles of DSS1 in other biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

A BAC-bacterial recombination method to generate physically linked multiple gene reporter DNA constructs.

PubMed

Maye, Peter; Stover, Mary Louise; Liu, Yaling; Rowe, David W; Gong, Shiaochin; Lichtler, Alexander C

2009-03-13

Reporter gene mice are valuable animal models for biological research providing a gene expression readout that can contribute to cellular characterization within the context of a developmental process. With the advancement of bacterial recombination techniques to engineer reporter gene constructs from BAC genomic clones and the generation of optically distinguishable fluorescent protein reporter genes, there is an unprecedented capability to engineer more informative transgenic reporter mouse models relative to what has been traditionally available. We demonstrate here our first effort on the development of a three stage bacterial recombination strategy to physically link multiple genes together with their respective fluorescent protein (FP) reporters in one DNA fragment. This strategy uses bacterial recombination techniques to: (1) subclone genes of interest into BAC linking vectors, (2) insert desired reporter genes into respective genes and (3) link different gene-reporters together. As proof of concept, we have generated a single DNA fragment containing the genes Trap, Dmp1, and Ibsp driving the expression of ECFP, mCherry, and Topaz FP reporter genes, respectively. Using this DNA construct, we have successfully generated transgenic reporter mice that retain two to three gene readouts. The three stage methodology to link multiple genes with their respective fluorescent protein reporter works with reasonable efficiency. Moreover, gene linkage allows for their common chromosomal integration into a single locus. However, the testing of this multi-reporter DNA construct by transgenesis does suggest that the linkage of two different genes together, despite their large size, can still create a positional effect. We believe that gene choice, genomic DNA fragment size and the presence of endogenous insulator elements are critical variables.

DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair.

PubMed

Serrano, M A; Li, Z; Dangeti, M; Musich, P R; Patrick, S; Roginskaya, M; Cartwright, B; Zou, Y

2013-05-09

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are two distinct DNA double-stranded break (DSB) repair pathways. Here, we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR.

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA.

PubMed

Ling, F; Morioka, H; Ohtsuka, E; Shibata, T

2000-12-15

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.

Evidence for mitochondrial DNA recombination in a human population of island Melanesia.

PubMed Central

Hagelberg, E; Goldman, N; Lió, P; Whelan, S; Schiefenhövel, W; Clegg, J B; Bowden, D K

1999-01-01

Mitochondrial DNA (mtDNA) analysis has proved useful in studies of recent human evolution and the genetic affinities of human groups of different geographical regions. As part of an extensive survey of mtDNA diversity in present-day Pacific populations, we obtained sequence information of the hypervariable mtDNA control region of 452 individuals from various localities in the western Pacific. The mtDNA types fell into three major groups which reflect the settlement history of the area. Interestingly, we detected an extremely rare point mutation at high frequency in the small island of Nguna in the Melanesian archipelago of Vanuatu. Phylogenetic analysis of the mtDNA data indicated that the mutation was present in individuals of separate mtDNA lineages. We propose that the multiple occurrence of a rare mutation event in one isolated locality is highly improbable, and that recombination between different mtDNA types is a more likely explanation for our observation. If correct, this conclusion has important implications for the use of mtDNA in phylogenetic and evolutionary studies. PMID:10189712

Evidence for mitochondrial DNA recombination in a human population of island Melanesia.

PubMed

Hagelberg, E; Goldman, N; Lió, P; Whelan, S; Schiefenhövel, W; Clegg, J B; Bowden, D K

1999-03-07

Mitochondrial DNA (mtDNA) analysis has proved useful in studies of recent human evolution and the genetic affinities of human groups of different geographical regions. As part of an extensive survey of mtDNA diversity in present-day Pacific populations, we obtained sequence information of the hypervariable mtDNA control region of 452 individuals from various localities in the western Pacific. The mtDNA types fell into three major groups which reflect the settlement history of the area. Interestingly, we detected an extremely rare point mutation at high frequency in the small island of Nguna in the Melanesian archipelago of Vanuatu. Phylogenetic analysis of the mtDNA data indicated that the mutation was present in individuals of separate mtDNA lineages. We propose that the multiple occurrence of a rare mutation event in one isolated locality is highly improbable, and that recombination between different mtDNA types is a more likely explanation for our observation. If correct, this conclusion has important implications for the use of mtDNA in phylogenetic and evolutionary studies.

Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

PubMed Central

Siaud, Nicolas; Lam, Isabel; Christ, Nicole; Schlacher, Katharina; Xia, Bing; Jasin, Maria

2011-01-01

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations. PMID:22194698

Evidence of reduced recombination rate in human regulatory domains.

PubMed

Liu, Yaping; Sarkar, Abhishek; Kheradpour, Pouya; Ernst, Jason; Kellis, Manolis

2017-10-20

Recombination rate is non-uniformly distributed across the human genome. The variation of recombination rate at both fine and large scales cannot be fully explained by DNA sequences alone. Epigenetic factors, particularly DNA methylation, have recently been proposed to influence the variation in recombination rate. We study the relationship between recombination rate and gene regulatory domains, defined by a gene and its linked control elements. We define these links using expression quantitative trait loci (eQTLs), methylation quantitative trait loci (meQTLs), chromatin conformation from publicly available datasets (Hi-C and ChIA-PET), and correlated activity links that we infer across cell types. Each link type shows a "recombination rate valley" of significantly reduced recombination rate compared to matched control regions. This recombination rate valley is most pronounced for gene regulatory domains of early embryonic development genes, housekeeping genes, and constitutive regulatory elements, which are known to show increased evolutionary constraint across species. Recombination rate valleys show increased DNA methylation, reduced doublestranded break initiation, and increased repair efficiency, specifically in the lineage leading to the germ line. Moreover, by using only the overlap of functional links and DNA methylation in germ cells, we are able to predict the recombination rate with high accuracy. Our results suggest the existence of a recombination rate valley at regulatory domains and provide a potential molecular mechanism to interpret the interplay between genetic and epigenetic variations.

Segregation and recombination of a multipartite mitochondrial DNA in populations of the potato cyst nematode Globodera pallida.

PubMed

Armstrong, Miles R; Husmeier, Dirk; Phillips, Mark S; Blok, Vivian C

2007-06-01

The discovery that the potato cyst nematode Globodera pallida has a multipartite mitochondrial DNA (mtDNA) composed, at least in part, of six small circular mtDNAs (scmtDNAs) raised a number of questions concerning the population-level processes that might act on such a complex genome. Here we report our observations on the distribution of some scmtDNAs among a sample of European and South American G. pallida populations. The occurrence of sequence variants of scmtDNA IV in population P4A from South America, and that particular sequence variants are common to the individuals within a single cyst, is described. Evidence for recombination of sequence variants of scmtDNA IV in P4A is also reported. The mosaic structure of P4A scmtDNA IV sequences was revealed using several detection methods and recombination breakpoints were independently detected by maximum likelihood and Bayesian MCMC methods.

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination.

PubMed

Marin-Vicente, Consuelo; Domingo-Prim, Judit; Eberle, Andrea B; Visa, Neus

2015-03-15

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs. © 2015. Published by The Company of Biologists Ltd.

DNA recombination-initiation plays a role in the extremely biased inheritance of yeast [rho-] mitochondrial DNA that contains the replication origin ori5.

PubMed

Ling, Feng; Hori, Akiko; Shibata, Takehiko

2007-02-01

Hypersuppressiveness, as observed in Saccharomyces cerevisiae, is an extremely biased inheritance of a small mitochondrial DNA (mtDNA) fragment that contains a replication origin (HS [rho(-)] mtDNA). Our previous studies showed that concatemers (linear head-to-tail multimers) are obligatory intermediates for mtDNA partitioning and are primarily formed by rolling-circle replication mediated by Mhr1, a protein required for homologous mtDNA recombination. In this study, we found that Mhr1 is required for the hypersuppressiveness of HS [ori5] [rho(-)] mtDNA harboring ori5, one of the replication origins of normal ([rho(+)]) mtDNA. In addition, we detected an Ntg1-stimulated double-strand break at the ori5 locus. Purified Ntg1, a base excision repair enzyme, introduced a double-stranded break by itself into HS [ori5] [rho(-)] mtDNA at ori5 isolated from yeast cells. Both hypersuppressiveness and concatemer formation of HS [ori5] [rho(-)] mtDNA are simultaneously suppressed by the ntg1 null mutation. These results support a model in which, like homologous recombination, rolling-circle HS [ori5] [rho(-)] mtDNA replication is initiated by double-stranded breakage in ori5, followed by Mhr1-mediated homologous pairing of the processed nascent DNA ends with circular mtDNA. The hypersuppressiveness of HS [ori5] [rho(-)] mtDNA depends on a replication advantage furnished by the higher density of ori5 sequences and on a segregation advantage furnished by the higher genome copy number on transmitted concatemers.

Recombineering: A Homologous Recombination-Based Method of Genetic Engineering

PubMed Central

Sharan, Shyam K.; Thomason, Lynn C.; Kuznetsov, Sergey G.; Court, Donald L.

2009-01-01

Recombineering is an efficient method of in vivo genetic engineering applicable to chromosomal as well as episomal replicons in E. coli. This method circumvents the need for most standard in vitro cloning techniques. Recombineering allows construction of DNA molecules with precise junctions without constraints being imposed by restriction enzyme site location. Bacteriophage homologous recombination proteins catalyze these recombineering reactions using double- and single-strand linear DNA substrates, so-called targeting constructs, introduced by electroporation. Gene knockouts, deletions and point mutations are readily made, gene tags can be inserted, and regions of bacterial artificial chromosomes (BACs) or the E. coli genome can be subcloned by gene retrieval using recombineering. Most of these constructs can be made within about a week's time. PMID:19180090

The cell pole: the site of cross talk between the DNA uptake and genetic recombination machinery.

PubMed

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann B; Graumann, Peter L; Alonso, Juan C

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as "guardians", protects ssDNA from degradation and limit the RecA recombinase loading. Then, the "mediators" overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by "modulators", catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or "resolver" cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the "rescuers" will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective.

The cell pole: The site of cross talk between the DNA uptake and genetic recombination machinery

PubMed Central

Kidane, Dawit; Ayora, Silvia; Sweasy, Joann; Graumann, Peter L.; Alonso, Juan C.

2012-01-01

Natural transformation is a programmed mechanism characterized by binding of free double-stranded (ds) DNA from the environment to the cell pole in rod-shaped bacteria. In Bacillus subtilis some competence proteins, which process the dsDNA and translocate single-stranded (ss) DNA into the cytosol, recruit a set of recombination proteins mainly to one of the cell poles. A subset of single-stranded binding proteins, working as “guardians”, protect ssDNA from degradation and limit the RecA recombinase loading. Then, the “mediators” overcome the inhibitory role of guardians, and recruit RecA onto ssDNA. A RecA·ssDNA filament searches for homology on the chromosome and, in a process that is controlled by “modulators”, catalyzes strand invasion with the generation of a displacement loop (D-loop). A D-loop resolvase or “resolver” cleaves this intermediate, limited DNA replication restores missing information and a DNA ligase seals the DNA ends. However, if any step fails, the “rescuers” will repair the broken end to rescue chromosomal transformation. If the ssDNA does not share homology with resident DNA, but it contains information for autonomous replication, guardian and mediator proteins catalyze plasmid establishment after inhibition of RecA. DNA replication and ligation reconstitute the molecule (plasmid transformation). In this review, the interacting network that leads to a cross talk between proteins of the uptake and genetic recombination machinery will be placed into prospective. PMID:23046409

Identification and Characterization of the V(D)J Recombination Activating Gene 1 in Long-Term Memory of Context Fear Conditioning

PubMed Central

Castro-Pérez, Edgardo; Soto-Soto, Emilio; Pérez-Carambot, Marizabeth; Dionisio-Santos, Dawling; Saied-Santiago, Kristian; Ortiz-Zuazaga, Humberto G.; Peña de Ortiz, Sandra

2016-01-01

An increasing body of evidence suggests that mechanisms related to the introduction and repair of DNA double strand breaks (DSBs) may be associated with long-term memory (LTM) processes. Previous studies from our group suggested that factors known to function in DNA recombination/repair machineries, such as DNA ligases, polymerases, and DNA endonucleases, play a role in LTM. Here we report data using C57BL/6 mice showing that the V(D)J recombination-activating gene 1 (RAG1), which encodes a factor that introduces DSBs in immunoglobulin and T-cell receptor genes, is induced in the amygdala, but not in the hippocampus, after context fear conditioning. Amygdalar induction of RAG1 mRNA, measured by real-time PCR, was not observed in context-only or shock-only controls, suggesting that the context fear conditioning response is related to associative learning processes. Furthermore, double immunofluorescence studies demonstrated the neuronal localization of RAG1 protein in amygdalar sections prepared after perfusion and fixation. In functional studies, intra-amygdalar injections of RAG1 gapmer antisense oligonucleotides, given 1 h prior to conditioning, resulted in amygdalar knockdown of RAG1 mRNA and a significant impairment in LTM, tested 24 h after training. Overall, these findings suggest that the V(D)J recombination-activating gene 1, RAG1, may play a role in LTM consolidation. PMID:26843989

SV40 host-substituted variants: a new look at the monkey DNA inserts and recombinant junctions.

PubMed

Singer, Maxine; Winocour, Ernest

2011-04-10

The available monkey genomic data banks were examined in order to determine the chromosomal locations of the host DNA inserts in 8 host-substituted SV40 variant DNAs. Five of the 8 variants contained more than one linked monkey DNA insert per tandem repeat unit and in all cases but one, the 19 monkey DNA inserts in the 8 variants mapped to different locations in the monkey genome. The 50 parental DNAs (32 monkey and 18 SV40 DNA segments) which spanned the crossover and flanking regions that participated in monkey/monkey and monkey/SV40 recombinations were characterized by substantial levels of microhomology of up to 8 nucleotides in length; the parental DNAs also exhibited direct and inverted repeats at or adjacent to the crossover sequences. We discuss how the host-substituted SV40 variants arose and the nature of the recombination mechanisms involved. Copyright © 2011 Elsevier Inc. All rights reserved.

Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner.

PubMed

Loutre, Romuald; Heckel, Anne-Marie; Jeandard, Damien; Tarassov, Ivan; Entelis, Nina

2018-01-01

Mutations in mitochondrial DNA are an important source of severe and incurable human diseases. The vast majority of these mutations are heteroplasmic, meaning that mutant and wild-type genomes are present simultaneously in the same cell. Only a very high proportion of mutant mitochondrial DNA (heteroplasmy level) leads to pathological consequences. We previously demonstrated that mitochondrial targeting of small RNAs designed to anneal with mutant mtDNA can decrease the heteroplasmy level by specific inhibition of mutant mtDNA replication, thus representing a potential therapy. We have also shown that 5S ribosomal RNA, partially imported into human mitochondria, can be used as a vector to deliver anti-replicative oligoribonucleotides into human mitochondria. So far, the efficiency of cellular expression of recombinant 5S rRNA molecules bearing therapeutic insertions remained very low. In the present study, we designed new versions of anti-replicative recombinant 5S rRNA targeting a large deletion in mitochondrial DNA which causes the KSS syndrome, analyzed their specific annealing to KSS mitochondrial DNA and demonstrated their import into mitochondria of cultured human cells. To obtain an increased level of the recombinant 5S rRNA stable expression, we created transmitochondrial cybrid cell line bearing a site for Flp-recombinase and used this system for the recombinase-mediated integration of genes coding for the anti-replicative recombinant 5S rRNAs into nuclear genome. We demonstrated that stable expression of anti-replicative 5S rRNA versions in human transmitochondrial cybrid cells can induce a shift in heteroplasmy level of KSS mutation in mtDNA. This shift was directly dependent on the level of the recombinant 5S rRNA expression and the sequence of the anti-replicative insertion. Quantification of mtDNA copy number in transfected cells revealed the absence of a non-specific effect on wild type mtDNA replication, indicating that the decreased proportion

DNA-PK, ATM and ATR collaboratively regulate p53-RPA interaction to facilitate homologous recombination DNA repair

PubMed Central

Serrano, Moises A.; Li, Zhengke; Dangeti, Mohan; Musich, Phillip R.; Patrick, Steve; Roginskaya, Marina; Cartwright, Brian; Zou, Yue

2012-01-01

Homologous recombination (HR) and nonhomologous end-joining (NHEJ) are two distinct DNA double-strand break (DSB) repair pathways. Here we report that DNA-dependent protein kinase (DNA-PK), the core component of NHEJ, partnering with DNA-damage checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR), regulates HR repair of DSBs. The regulation was accomplished through modulation of the p53 and replication protein A (RPA) interaction. We show that upon DNA damage, p53 and RPA were freed from a p53-RPA complex by simultaneous phosphorylations of RPA at the N-terminus of RPA32 subunit by DNA-PK and of p53 at Ser37 and Ser46 in a Chk1/Chk2-independent manner by ATR and ATM, respectively. Neither the phosphorylation of RPA nor of p53 alone could dissociate p53 and RPA. Furthermore, disruption of the release significantly compromised HR repair of DSBs. Our results reveal a mechanism for the crosstalk between HR repair and NHEJ through the co-regulation of p53-RPA interaction by DNA-PK, ATM and ATR. PMID:22797063

Rad52/Rad59-dependent Recombination as a Means to Rectify Faulty Okazaki Fragment Processing*

PubMed Central

Lee, Miju; Lee, Chul-Hwan; Demin, Annie Albert; Munashingha, Palinda Ruvan; Amangyeld, Tamir; Kwon, Buki; Formosa, Tim; Seo, Yeon-Soo

2014-01-01

The correct removal of 5′-flap structures by Rad27 and Dna2 during Okazaki fragment maturation is crucial for the stable maintenance of genetic materials and cell viability. In this study, we identified RAD52, a key recombination protein, as a multicopy suppressor of dna2-K1080E, a lethal helicase-negative mutant allele of DNA2 in yeasts. In contrast, the overexpression of Rad51, which works conjointly with Rad52 in canonical homologous recombination, failed to suppress the growth defect of the dna2-K1080E mutation, indicating that Rad52 plays a unique and distinct role in Okazaki fragment metabolism. We found that the recombination-defective Rad52-QDDD/AAAA mutant did not rescue dna2-K1080E, suggesting that Rad52-mediated recombination is important for suppression. The Rad52-mediated enzymatic stimulation of Dna2 or Rad27 is not a direct cause of suppression observed in vivo, as both Rad52 and Rad52-QDDD/AAAA proteins stimulated the endonuclease activities of both Dna2 and Rad27 to a similar extent. The recombination mediator activity of Rad52 was dispensable for the suppression, whereas both the DNA annealing activity and its ability to interact with Rad59 were essential. In addition, we found that several cohesion establishment factors, including Rsc2 and Elg1, were required for the Rad52-dependent suppression of dna2-K1080E. Our findings suggest a novel Rad52/Rad59-dependent, but Rad51-independent recombination pathway that could ultimately lead to the removal of faulty flaps in conjunction with cohesion establishment factors. PMID:24711454

Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination.

PubMed

Sammler, Svenja; Bleidorn, Christoph; Tiedemann, Ralph

2011-01-14

Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken mitochondrial genome. As this RFB

Full mitochondrial genome sequences of two endemic Philippine hornbill species (Aves: Bucerotidae) provide evidence for pervasive mitochondrial DNA recombination

PubMed Central

2011-01-01

Background Although nowaday it is broadly accepted that mitochondrial DNA (mtDNA) may undergo recombination, the frequency of such recombination remains controversial. Its estimation is not straightforward, as recombination under homoplasmy (i.e., among identical mt genomes) is likely to be overlooked. In species with tandem duplications of large mtDNA fragments the detection of recombination can be facilitated, as it can lead to gene conversion among duplicates. Although the mechanisms for concerted evolution in mtDNA are not fully understood yet, recombination rates have been estimated from "one per speciation event" down to 850 years or even "during every replication cycle". Results Here we present the first complete mt genome of the avian family Bucerotidae, i.e., that of two Philippine hornbills, Aceros waldeni and Penelopides panini. The mt genomes are characterized by a tandemly duplicated region encompassing part of cytochrome b, 3 tRNAs, NADH6, and the control region. The duplicated fragments are identical to each other except for a short section in domain I and for the length of repeat motifs in domain III of the control region. Due to the heteroplasmy with regard to the number of these repeat motifs, there is some size variation in both genomes; with around 21,657 bp (A. waldeni) and 22,737 bp (P. panini), they significantly exceed the hitherto longest known avian mt genomes, that of the albatrosses. We discovered concerted evolution between the duplicated fragments within individuals. The existence of differences between individuals in coding genes as well as in the control region, which are maintained between duplicates, indicates that recombination apparently occurs frequently, i.e., in every generation. Conclusions The homogenised duplicates are interspersed by a short fragment which shows no sign of recombination. We hypothesize that this region corresponds to the so-called Replication Fork Barrier (RFB), which has been described from the chicken

Production of biologically active recombinant human factor H in Physcomitrella.

PubMed

Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

2011-04-01

The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Co-factor activated recombinant adenovirus proteinases

DOEpatents

Anderson, Carl W.; Mangel, Walter F.

1996-08-06

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

Redundant function of DNA ligase 1 and 3 in alternative end-joining during immunoglobulin class switch recombination.

PubMed

Masani, Shahnaz; Han, Li; Meek, Katheryn; Yu, Kefei

2016-02-02

Nonhomologous end-joining (NHEJ) is the major DNA double-strand break (DSB) repair pathway in mammals and resolves the DSBs generated during both V(D)J recombination in developing lymphocytes and class switch recombination (CSR) in antigen-stimulated B cells. In contrast to the absolute requirement for NHEJ to resolve DSBs associated with V(D)J recombination, DSBs associated with CSR can be resolved in NHEJ-deficient cells (albeit at a reduced level) by a poorly defined alternative end-joining (A-EJ) pathway. Deletion of DNA ligase IV (Lig4), a core component of the NHEJ pathway, reduces CSR efficiency in a mouse B-cell line capable of robust cytokine-stimulated CSR in cell culture. Here, we report that CSR levels are not further reduced by deletion of either of the two remaining DNA ligases (Lig1 and nuclear Lig3) in Lig4(-/-) cells. We conclude that in the absence of Lig4, Lig1, and Lig3 function in a redundant manner in resolving switch region DSBs during CSR.

Meiotic recombination hotspots - a comparative view.

PubMed

Choi, Kyuha; Henderson, Ian R

2015-07-01

During meiosis homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover. Meiotic recombination has a profound effect on patterns of genetic variation and is an important tool during crop breeding. Crossovers initiate from programmed DNA double-stranded breaks that are processed to form single-stranded DNA, which can invade a homologous chromosome. Strand invasion events mature into double Holliday junctions that can be resolved as crossovers. Extensive variation in the frequency of meiotic recombination occurs along chromosomes and is typically focused in narrow hotspots, observed both at the level of DNA breaks and final crossovers. We review methodologies to profile hotspots at different steps of the meiotic recombination pathway that have been used in different eukaryote species. We then discuss what these studies have revealed concerning specification of hotspot locations and activity and the contributions of both genetic and epigenetic factors. Understanding hotspots is important for interpreting patterns of genetic variation in populations and how eukaryotic genomes evolve. In addition, manipulation of hotspots will allow us to accelerate crop breeding, where meiotic recombination distributions can be limiting. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

PubMed

Mukherjee, Anirban; Vasquez, Karen M

2011-08-01

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

PubMed

Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

2012-04-01

cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

Co-factor activated recombinant adenovirus proteinases

DOEpatents

Anderson, C.W.; Mangel, W.F.

1996-08-06

This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

The xeroderma pigmentosum group B protein ERCC3 produced in the baculovirus system exhibits DNA helicase activity.

PubMed Central

Ma, L; Siemssen, E D; Noteborn, H M; van der Eb, A J

1994-01-01

The XPB/ERCC3 gene corrects the nucleotide excision-repair defect in the human hereditary disease xeroderma pigmentosum group B and encodes the largest subunit of the basal transcription factor BTF2/TFIIH. The primary sequence of the XPB/ERCC3 protein features the hallmarks of seven helicase motifs found in many known and putative helicases or helicase-related proteins. Recently, the multiprotein BTF2/TFIIH complex has been found to be associated with DNA helicase activity. To explore the properties and functions of XPB/ERCC3, we have used the baculovirus/insect-cell expression system to produce recombinant protein. We report here the construction and analysis of recombinant baculovirus expressing XPB/ERCC3. The XPB/ERCC3 protein is synthesized at a relatively high level in baculovirus-infected insect cells. While the majority of XPB/ERCC3 end up in the insoluble fraction of insect cell lysates, a minor fraction of recombinant protein is present in soluble form which can be purified under native conditions. We have found that a DNA helicase activity is associated with the purified XPB/ERCC3 protein, suggesting that XPB/ERCC3 may function as a DNA helicase in local unwinding of DNA template both in the context of transcription and nucleotide excision repair. Images PMID:7937133

Regulation of Immunoglobulin Class-Switch Recombination: Choreography of Noncoding Transcription, Targeted DNA Deamination, and Long-Range DNA Repair

PubMed Central

Matthews, Allysia J.; Zheng, Simin; DiMenna, Lauren J.; Chaudhuri, Jayanta

2014-01-01

Upon encountering antigens, mature IgM-positive B lymphocytes undergo class-switch recombination (CSR) wherein exons encoding the default Cμ constant coding gene segment of the immunoglobulin (Ig) heavy-chain (Igh) locus are excised and replaced with a new constant gene segment (referred to as “Ch genes”, e.g., Cγ, Cε, or Cα). The B cell thereby changes from expressing IgM to one producing IgG, IgE, or IgA, with each antibody isotype having a different effector function during an immune reaction. CSR is a DNA deletional-recombination reaction that proceeds through the generation of DNA double-strand breaks (DSBs) in repetitive switch (S) sequences preceding each Ch gene and is completed by end-joining between donor Sμ and acceptor S regions. CSR is a multistep reaction requiring transcription through S regions, the DNA cytidine deaminase AID, and the participation of several general DNA repair pathways including base excision repair, mismatch repair, and classical nonhomologous end-joining. In this review, we discuss our current understanding of how transcription through S regions generates substrates for AID-mediated deamination and how AID participates not only in the initiation of CSR but also in the conversion of deaminated residues into DSBs. Additionally, we review the multiple processes that regulate AID expression and facilitate its recruitment specifically to the Ig loci, and how deregulation of AID specificity leads to oncogenic translocations. Finally, we summarize recent data on the potential role of AID in the maintenance of the pluripotent stem cell state during epigenetic reprogramming. PMID:24507154

DNA-independent PARP-1 activation by phosphorylated ERK2 increases Elk1 activity: a link to histone acetylation.

PubMed

Cohen-Armon, Malka; Visochek, Leonid; Rozensal, Dana; Kalal, Adi; Geistrikh, Ilona; Klein, Rodika; Bendetz-Nezer, Sarit; Yao, Zhong; Seger, Rony

2007-01-26

PolyADP-ribose polymerases (PARPs) catalyze a posttranslational modification of nuclear proteins by polyADP-ribosylation. The catalytic activity of the abundant nuclear protein PARP-1 is stimulated by DNA strand breaks, and PARP-1 activation is required for initiation of DNA repair. Here we show that PARP-1 also acts within extracellular signal-regulated kinase (ERK) signaling cascade that mediates growth and differentiation. The findings reveal an alternative mode of PARP-1 activation, which does not involve binding to DNA or DNA damage. In a cell-free system, recombinant PARP-1 was intensively activated and thereby polyADP-ribosylated by a direct interaction with phosphorylated ERK2, and the activated PARP-1 dramatically increased ERK2-catalyzed phosphorylation of the transcription factor Elk1. In cortical neurons treated with nerve growth factors and in stimulated cardiomyocytes, PARP-1 activation enhanced ERK-induced Elk1-phosphorylation, core histone acetylation, and transcription of the Elk1-target gene c-fos. These findings constitute evidence for PARP-1 activity within the ERK signal-transduction pathway.

Genetic recombination induced by DNA double-strand break in bacteriophage T4: nature of the left/right bias.

PubMed

Shcherbakov, Victor P; Shcherbakova, Tamara; Plugina, Lidiya; Sizova, Svetlana; Kudryashova, Elena; Granovsky, Igor

2008-06-01

The experimental system combining double-strand breaks (DSBs), produced site-specifically by SegC endonuclease, with the famous advantages of the bacteriophage T4 rII mutant recombination analysis was used here to elucidate the origin of the recombination bias on two sides of the DSB, especially pronounced in gene 39 (topoisomerase II) and gene 59 (41-helicase loader) mutants. Three sources were found to contribute to the bias: (1) the SegC endonuclease may remain bound to the end of the broken DNA and thus protect it from exonuclease degradation; (2) in heteroduplex heterozygotes (HHs), arising as the recombinant products in the left-hand crosses, the transcribed strands are of rII mutant phenotype, so they, in contrast to the right-hand HHs, do not produce plaques on the lawn of the lambda-lysogenic host; and (3) the intrinsic polarity of T4 chromosome, reflected in transcription, may be a cause for discrimination of promoter-proximal and promoter-distal DNA sequences. It is shown that the apparent recombination bias does not imply one-sidedness of the DSB repair but just reflects a different depth of the end processing. It is inferred that the cause, underlying the "intrinsic" bias, might be interference between strand exchange and transcription. Topoisomerase and helicase functions are necessary to turn the process in favor of strand exchange. The idea is substantiated that the double-stranded to single-stranded DNA transition edge (not ss-DNA tip) serves as an actual recombinogenic element.

Impaired mitochondrial Fe-S cluster biogenesis activates the DNA damage response through different signaling mediators.

PubMed

Pijuan, Jordi; María, Carlos; Herrero, Enrique; Bellí, Gemma

2015-12-15

Fe-S cluster biogenesis machinery is required for multiple DNA metabolism processes. In this work, we show that, in Saccharomyces cerevisiae, defects at different stages of the mitochondrial Fe-S cluster assembly machinery (ISC) result in increased spontaneous mutation rate and hyper-recombination, accompanied by an increment in Rad52-associated DNA repair foci and a higher phosphorylated state of γH2A histone, altogether supporting the presence of constitutive DNA lesions. Furthermore, ISC assembly machinery deficiency elicits a DNA damage response that upregulates ribonucleotide reductase activity by promoting the reduction of Sml1 levels and the cytosolic redistribution of Rnr2 and Rnr4 enzyme subunits. Depending on the impaired stage of the ISC machinery, different signaling pathway mediators contribute to such a response, converging on Dun1. Thus, cells lacking the glutaredoxin Grx5, which are compromised at the core ISC system, show Mec1- and Rad53-independent Dun1 activation, whereas both Mec1 and Chk1 are required when the non-core ISC member Iba57 is absent. Grx5-null cells exhibit a strong dependence on the error-free post-replication repair and the homologous recombination pathways, demonstrating that a DNA damage response needs to be activated upon ISC impairment to preserve cell viability. © 2015. Published by The Company of Biologists Ltd.

[Improvement of thermal adaptability and fermentation of industrial ethanologenic yeast by genomic DNA mutagenesis-based genetic recombination].

PubMed

Liu, Xiuying; He, Xiuping; Lu, Ying; Zhang, Borun

2011-07-01

Ethanol is an attractive alternative to fossil fuels. Saccharomyces cerevisiae is the most important ethanol producer. However, in the process of industrial production of ethanol, both cell growth and fermentation of ethanologenic S. cerevisiae are dramatically affected by environmental stresses, such as thermal stress. In this study, we improved both the thermotolerance and fermentation performance of industrial ethanologenic S. cerevisiae by combined usage of chemical mutagenesis and genomic DNA mutagenesis-based genetic recombination method. The recombinant S. cerevisiae strain T44-2 could grow at 44 degrees C, 3 degrees C higher than that of the original strain CE6. The survival rate of T44-2 was 1.84 and 1.87-fold of that of CE6 when heat shock at 48 degrees C and 52 degrees C for 1 h respectively. At temperature higher than 37 degrees C, recombinant strain T44-2 always gave higher cell growth and ethanol production than those of strain CE6. Meanwhile, from 30 degrees C to 40 degrees C, recombinant strain T44-2 produces 91.2-83.8 g/L of ethanol from 200 g/L of glucose, which indicated that the recombinant strain T44-2 had both thermotolerance and broad thermal adaptability. The work offers a novel method, called genomic DNA mutagenesis-based genetic recombination, to improve the physiological functions of S. cerevisiae.

Recombinant Saccharomyces cerevisiae serves as novel carrier for oral DNA vaccines in Carassius auratus.

PubMed

Yan, Nana; Xu, Kun; Li, Xinyi; Liu, Yuwan; Bai, Yichun; Zhang, Xiaohan; Han, Baoquan; Chen, Zhilong; Zhang, Zhiying

2015-12-01

Oral delivery of DNA vaccines represents a promising vaccinating method for fish. Recombinant yeast has been proved to be a safe carrier for delivering antigen proteins and DNAs to some species in vivo. However, whether recombinant yeast can be used to deliver functional DNAs for vaccination to fish is still unknown. In this study, red crucian carp (Carassius auratus) was orally administrated with recombinant Saccharomyces cerevisiae harboring CMV-EGFP expression cassette. On day 5 post the first vaccination, EGFP expression in the hindgut was detected under fluorescence microscope. To further study whether the delivered gene could induce specific immune responses, the model antigen ovalbumin (OVA) was used as immunogen, and oral administrations were conducted with recombinant S. cerevisiae harboring pCMV-OVA mammalian gene expression cassette as gene delivery or pADH1-OVA yeast gene expression cassette as protein delivery. Each administration was performed with three different doses, and the OVA-specific serum antibody was detected in all the experimental groups by western blotting and enzyme-linked immunosorbent assay (ELISA). ELISA assay also revealed that pCMV-OVA group with lower dose (pCMV-OVA-L) and pADH1-OVA group with moderate dose (pADH1-OVA-M) triggered relatively stronger antibody response than the other two doses. Moreover, the antibody level induced by pCMV-OVA-L group was significantly higher than pADH1-OVA-M group at the same serum dilutions. All the results suggested that recombinant yeast can be used as a potential carrier for oral DNA vaccines and would help to develop more practical strategies to control infectious diseases in aquaculture. Copyright © 2015 Elsevier Ltd. All rights reserved.

Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe.

PubMed

Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K; Levin, Henry L

2016-01-01

Transposition and homologous recombination assays are valuable genetic tools to measure the production and integration of cDNA from the long terminal repeat (LTR) retrotransposon Tf1 in the fission yeast (Schizosaccharomyces pombe). Here we describe two genetic assays, one that measures the transposition activity of Tf1 by monitoring the mobility of a drug resistance marked Tf1 element expressed from a multi-copy plasmid and another assay that measures homologous recombination between Tf1 cDNA and the expression plasmid. While the transposition assay measures insertion of full-length Tf1 cDNA mediated by the transposon integrase, the homologous recombination assay measures levels of cDNA present in the nucleus and is independent of integrase activity. Combined, these assays can be used to systematically screen large collections of strains to identify mutations that specifically inhibit the integration step in the retroelement life cycle. Such mutations can be identified because they reduce transposition activity but nevertheless have wild-type frequencies of homologous recombination. Qualitative assays of yeast patches on agar plates detect large defects in integration and recombination, while the quantitative approach provides a precise method of determining integration and recombination frequencies.

Recombinant activated factor VII in cardiac surgery: single-center experience.

PubMed

Singh, Sarvesh Pal; Chauhan, Sandeep; Choudhury, Minati; Malik, Vishwas; Choudhary, Shiv Kumar

2014-02-01

The widespread off-label use of recombinant activated factor VII for the control of refractory postoperative hemorrhage continues despite a warning from the Food and Drug Administration. Although effective in reducing the need for transfusion of blood and blood products, safety concerns still prevail. To compare the dosing and efficacy of recombinant activated factor VII between pediatric and adult patients, and in the operating room and intensive care unit. The records of 69 patients (33 children and 36 adults) who underwent cardiovascular surgery and received recombinant activated factor VII were reviewed retrospectively. The dose of recombinant activated factor VII, mediastinal drainage, use of blood and blood products, incidence of thrombosis, and 28-day mortality were studied. the efficacy of recombinant activated factor VII was comparable in adults and children, despite the lower dose in adults. Prophylactic use of recombinant activated factor VII decreased the incidence of mediastinal exploration and the duration of intensive care unit stay. A 4.3% incidence of thrombotic complications was observed in this study. The efficacious dose of recombinant activated factor VII is much less in adults compared to children. Prophylactic use of recombinant activated factor VII decreases the dose required, the incidence of mediastinal exploration, and intensive care unit stay, with no survival benefit.

Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination

PubMed Central

Rowley, Paul A.; Kachroo, Aashiq H.; Ma, Chien-Hui; Maciaszek, Anna D.; Guga, Piotr; Jayaram, Makkuni

2015-01-01

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions. PMID:25999343

Heat exposure enhances radiosensitivity by depressing DNA-PK kinase activity during double strand break repair.

PubMed

Ihara, Makoto; Takeshita, Satoshi; Okaichi, Kumio; Okumura, Yutaka; Ohnishi, Takeo

2014-03-01

From the role of double strand DNA dependent protein kinase (DNA-PKcs) activity of non-homologous end joining (NHEJ) repair for DNA double strand breaks (DSBs), we aim to define possible associations between thermo-sensitisation and the enzyme activities in X-ray irradiated cells. DNA-PKcs deficient mouse, Chinese hamster and human cultured cells were compared to the parental wild-type cells. The radiosensitivities, the number of DSBs and DNA-PKcs activities after heat-treatment were measured. Both DNA-PKcs deficient cells and the wild-type cells showed increased radiosensitivities after heat-treatment. The wild-type cells have two repair processes; fast repair and slow repair. In contrast, DNA-PKcs deficient cells have only the slow repair process. The fast repair component apparently disappeared by heat-treatment in the wild-type cells. In both cell types, additional heat exposure enhanced radiosensitivities. Although DNA-PKcs activity was depressed by heat, the inactivated DNA-PKcs activity recovered during an incubation at 37 °C. DSB repair efficiency was dependent on the reactivation of DNA-PKcs activity. It was suggested that NHEJ is the major process used to repair X-ray-induced DSBs and utilises DNA-PKcs activity, but homologous recombination repair provides additional secondary levels of DSB repair. The thermo-sensitisation in X-ray-irradiated cells depends on the inhibition of NHEJ repair through the depression of DNA-PKcs activities.

Reconstitution of the yeast RNA polymerase III transcription system with all recombinant factors.

PubMed

Ducrot, Cécile; Lefebvre, Olivier; Landrieux, Emilie; Guirouilh-Barbat, Josée; Sentenac, André; Acker, Joel

2006-04-28

Transcription factor TFIIIC is a multisubunit complex required for promoter recognition and transcriptional activation of class III genes. We describe here the reconstitution of complete recombinant yeast TFIIIC and the molecular characterization of its two DNA-binding domains, tauA and tauB, using the baculovirus expression system. The B block-binding module, rtauB, was reconstituted with rtau138, rtau91, and rtau60 subunits. rtau131, rtau95, and rtau55 formed also a stable complex, rtauA, that displayed nonspecific DNA binding activity. Recombinant rTFIIIC was functionally equivalent to purified yeast TFIIIC, suggesting that the six recombinant subunits are necessary and sufficient to reconstitute a transcriptionally active TFIIIC complex. The formation and the properties of rTFIIIC-DNA complexes were affected by dephosphorylation treatments. The combination of complete recombinant rTFIIIC and rTFIIIB directed a low level of basal transcription, much weaker than with the crude B'' fraction, suggesting the existence of auxiliary factors that could modulate the yeast RNA polymerase III transcription system.

Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing.

PubMed

Valdés-Alemán, Javier; Téllez-Sosa, Juan; Ovilla-Muñoz, Marbella; Godoy-Lozano, Elizabeth; Velázquez-Ramírez, Daniel; Valdovinos-Torres, Humberto; Gómez-Barreto, Rosa E; Martinez-Barnetche, Jesús

2014-01-01

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.

Summary and critique of the new NIH guidelines for recombinant DNA research.

PubMed

Szybalski, W

1979-03-01

New NIH Guidelines for research involving recombinant DNA (R-DNA) molecules were issued on December 15, 1978. These are composed of four main parts, the first defining R-DNA and specifying prohibitions and exemptions, the second describing physical and biological containment, the third assigning the containment levels for many R-DNA experiments, and the fourth detailing the roles and responsibilities of the investigator, research institutions and NIH. Although the new Guidelines reduce restrictions, principally on those R-DNA experiments that use Escherichia coli K-12 host-vector systems, and exempt from the Guidelines several classes of experiments on prokaryotes that naturally exchange their DNA, most of their provisions are unjustified by the present assessment of the absence of any practical risks; many totally innocuous experiments are unnecessarily restricted and even virtually prohibited mainly because no host-vector systems were officially certified. The term Guidelines is a misnomer since they are mandatory regulations, even without any statutory basis. They impose large but unnecessary bureaucratic burdens on scientists, research institutions, research committees and NIH, and represent unwarranted censorship of basic research, which is antithetical to the creativity of human thought, thus posing serious dangers to the traditional freedom of inquiry.

Loss of Ubp3 increases silencing, decreases unequal recombination in rDNA, and shortens the replicative life span in Saccharomyces cerevisiae.

PubMed

Oling, David; Masoom, Rehan; Kvint, Kristian

2014-06-15

Ubp3 is a conserved ubiquitin protease that acts as an antisilencing factor in MAT and telomeric regions. Here we show that ubp3∆ mutants also display increased silencing in ribosomal DNA (rDNA). Consistent with this, RNA polymerase II occupancy is lower in cells lacking Ubp3 than in wild-type cells in all heterochromatic regions. Moreover, in a ubp3∆ mutant, unequal recombination in rDNA is highly suppressed. We present genetic evidence that this effect on rDNA recombination, but not silencing, is entirely dependent on the silencing factor Sir2. Further, ubp3∆ sir2∆ mutants age prematurely at the same rate as sir2∆ mutants. Thus our data suggest that recombination negatively influences replicative life span more so than silencing. However, in ubp3∆ mutants, recombination is not a prerequisite for aging, since cells lacking Ubp3 have a shorter life span than isogenic wild-type cells. We discuss the data in view of different models on how silencing and unequal recombination affect replicative life span and the role of Ubp3 in these processes. © 2014 Öling et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

The N-terminal DNA-binding domain of Rad52 promotes RAD51-independent recombination in Saccharomyces cerevisiae.

PubMed Central

Tsukamoto, Mariko; Yamashita, Kentaro; Miyazaki, Toshiko; Shinohara, Miki; Shinohara, Akira

2003-01-01

In Saccharomyces cerevisiae, the Rad52 protein plays a role in both RAD51-dependent and RAD51-independent recombination pathways. We characterized a rad52 mutant, rad52-329, which lacks the C-terminal Rad51-interacting domain, and studied its role in RAD51-independent recombination. The rad52-329 mutant is completely defective in mating-type switching, but partially proficient in recombination between inverted repeats. We also analyzed the effect of the rad52-329 mutant on telomere recombination. Yeast cells lacking telomerase maintain telomere length by recombination. The rad52-329 mutant is deficient in RAD51-dependent telomere recombination, but is proficient in RAD51-independent telomere recombination. In addition, we examined the roles of other recombination genes in the telomere recombination. The RAD51-independent recombination in the rad52-329 mutant is promoted by a paralogue of Rad52, Rad59. All components of the Rad50-Mre11-Xrs2 complex are also important, but not essential, for RAD51-independent telomere recombination. Interestingly, RAD51 inhibits the RAD51-independent, RAD52-dependent telomere recombination. These findings indicate that Rad52 itself, and more precisely its N-terminal DNA-binding domain, promote an essential reaction in recombination in the absence of RAD51. PMID:14704160

Dual inhibition of ATR and ATM potentiates the activity of trabectedin and lurbinectedin by perturbing the DNA damage response and homologous recombination repair.

PubMed

Lima, Michelle; Bouzid, Hana; Soares, Daniele G; Selle, Frédéric; Morel, Claire; Galmarini, Carlos M; Henriques, João A P; Larsen, Annette K; Escargueil, Alexandre E

2016-05-03

Trabectedin (Yondelis®, ecteinascidin-743, ET-743) is a marine-derived natural product approved for treatment of advanced soft tissue sarcoma and relapsed platinum-sensitive ovarian cancer. Lurbinectedin is a novel anticancer agent structurally related to trabectedin. Both ecteinascidins generate DNA double-strand breaks that are processed through homologous recombination repair (HRR), thereby rendering HRR-deficient cells particularly sensitive. We here characterize the DNA damage response (DDR) to trabectedin and lurbinectedin in HeLa cells. Our results show that both compounds activate the ATM/Chk2 (ataxia-telangiectasia mutated/checkpoint kinase 2) and ATR/Chk1 (ATM and RAD3-related/checkpoint kinase 1) pathways. Interestingly, pharmacological inhibition of Chk1/2, ATR or ATM is not accompanied by any significant improvement of the cytotoxic activity of the ecteinascidins while dual inhibition of ATM and ATR strongly potentiates it. Accordingly, concomitant inhibition of both ATR and ATM is an absolute requirement to efficiently block the formation of γ-H2AX, MDC1, BRCA1 and Rad51 foci following exposure to the ecteinascidins. These results are not restricted to HeLa cells, but are shared by cisplatin-sensitive and -resistant ovarian carcinoma cells. Together, our data identify ATR and ATM as central coordinators of the DDR to ecteinascidins and provide a mechanistic rationale for combining these compounds with ATR and ATM inhibitors.

Complex formation by the human Rad51B and Rad51C DNA repair proteins and their activities in vitro

NASA Technical Reports Server (NTRS)

Lio, Yi-Ching; Mazin, Alexander V.; Kowalczykowski, Stephen C.; Chen, David J.

2003-01-01

The human Rad51 protein is essential for DNA repair by homologous recombination. In addition to Rad51 protein, five paralogs have been identified: Rad51B/Rad51L1, Rad51C/Rad51L2, Rad51D/Rad51L3, XRCC2, and XRCC3. To further characterize a subset of these proteins, recombinant Rad51, Rad51B-(His)(6), and Rad51C proteins were individually expressed employing the baculovirus system, and each was purified from Sf9 insect cells. Evidence from nickel-nitrilotriacetic acid pull-down experiments demonstrates a highly stable Rad51B.Rad51C heterodimer, which interacts weakly with Rad51. Rad51B and Rad51C proteins were found to bind single- and double-stranded DNA and to preferentially bind 3'-end-tailed double-stranded DNA. The ability to bind DNA was elevated with mixed Rad51 and Rad51C, as well as with mixed Rad51B and Rad51C, compared with that of the individual protein. In addition, both Rad51B and Rad51C exhibit DNA-stimulated ATPase activity. Rad51C displays an ATP-independent apparent DNA strand exchange activity, whereas Rad51B shows no such activity; this apparent strand exchange ability results actually from a duplex DNA destabilization capability of Rad51C. By analogy to the yeast Rad55 and Rad57, our results suggest that Rad51B and Rad51C function through interactions with the human Rad51 recombinase and play a crucial role in the homologous recombinational repair pathway.

Cloning, sequence, and expression of a blood group B active recombinant alpha-D-galactosidase from pinto bean (Phaseolus vulgaris).

PubMed

Davis, M O; Hata, D J; Johnson, S A; Jones, D E; Harmata, M A; Evans, M L; Walker, J C; Smith, D S

1997-07-01

A cDNA encoding pinto bean alpha-D-galactosidase [E.C. 3.2.1.22] was obtained by amplification of cDNA using highly conserved sequences found in eucaryotic alpha-D-galactosidases. Subsequently a full length Phaseolus cDNA clone was obtained that is 1537 nt long and contains untranslated 5' and 3' sequences. The nucleotide sequence of the cDNA has a high degree of homology with other eucaryotic alpha-D-galactosidase genes. The recombinant alpha-D-galactosidase (rGal) was expressed in Escherichia coli and purified by ion exchange and affinity chromatography. Purified rGal was homogeneous by SDS-PAGE and had relative masses of 40.1 and 45.4 kDa under nonreducing and reducing conditions, respectively. The N-terminal sequence of the expressed protein contained the sequence GNGLGQTPPMG corresponding to that deduced from the cDNA sequence. The native molecular weight for rGal was determined to be 32.18 kDa by Sephacryl S-200 chromatography. The specific activity of the rGal was 349 mu moles of PNP-alpha-D-galactopyranoside hydrolyzed per mg of pure rGal per min. rGal was highly specific for alpha-D-galactosyl residues and degraded B oligosaccharide. No detectable hemagglutinin or protease activity was present in the preparations. Furthermore, rGal was active against the blood group B antigen on native human erythrocytes in cell suspension assays. The only detectable RBC phenotypic change was loss of the B and P1 epitopes. Recombinant Phaseolus vulgaris alpha-D-galactosidase may have useful biotechnical applications in the potential mass production of enzymatically converted, universally transfusable type O RBCs. alpha-D-galactosidase [E.C. 3.2.1.22] has been purified from a variety of procaryotic and eucaryotic species. Most alpha-D-galactosidases have similar low molecular weight substrate specificities, but activity against high molecular weight substrates is variable. Terminal alpha-D-galactoside residues are present in glycoproteins and glycolipids. Some alpha

Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination

PubMed Central

Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R.; Guerrero, Justin A.; Shen, Tian; Casali, Paolo

2017-01-01

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52−/− B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S–S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S–S recombination, as emphasized by the significantly greater CSR reduction in Rad52−/− versus Rad52+/+ B cells on Ku86 knockdown. PMID:28176781

Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends to modulate antibody class-switch DNA recombination.

PubMed

Zan, Hong; Tat, Connie; Qiu, Zhifang; Taylor, Julia R; Guerrero, Justin A; Shen, Tian; Casali, Paolo

2017-02-08

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52 +/+ counterparts, which display normal CSR, Rad52 -/- B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52 -/- versus Rad52 +/+ B cells on Ku86 knockdown.

Quantitative real-time PCR technique for the identification of E. coli residual DNA in streptokinase recombinant product.

PubMed

Fazelahi, Mansoureh; Kia, Vahid; Kaghazian, Hooman; Paryan, Mahdi

2017-11-26

Recombinant streptokinase is a biopharmaceutical which is usually produced in E. coli. Residual DNA as a contamination and risk factor may remain in the product. It is necessary to control the production procedure to exclude any possible contamination. The aim of the present study was to develop a highly specific and sensitive quantitative real-time PCR-based method to determine the amount of E. coli DNA in recombinant streptokinase. A specific primers and a probe was designed to detect all strains of E. coli. To determine the specificity, in addition to using NCBI BLASTn, 28 samples including human, bacterial, and viral genomes were used. The results confirmed that the assay detects no genomic DNA but E. coli's and the specificity was determined to be 100%. To determine the sensitivity and limit of detection of the assay, a 10-fold serial dilution (10 1 to 10 7 copies/µL) was tested in triplicate. The sensitivity of the test was determined to be 101 copies/µL or 35 fg/µL. Inter-assay and intra-assay were determined to be 0.86 and 1.69%, respectively. Based on the results, this assay can be used as an accurate method to evaluate the contamination of recombinant streptokinase in E. coli.

Scaffold Functions of 14-3-3 Adaptors in B Cell Immunoglobulin Class Switch DNA Recombination

PubMed Central

White, Clayton A.; Li, Guideng; Pone, Egest J.; Xu, Zhenming; Casali, Paolo

2013-01-01

Class switch DNA recombination (CSR) of the immunoglobulin heavy chain (IgH) locus crucially diversifies antibody biological effector functions. CSR involves the induction of activation-induced cytidine deaminase (AID) expression and AID targeting to switch (S) regions by 14-3-3 adaptors. 14-3-3 adaptors specifically bind to 5′-AGCT-3′ repeats, which make up for the core of all IgH locus S regions. They selectively target the upstream and downstream S regions that are set to undergo S–S DNA recombination. We hypothesized that 14-3-3 adaptors function as scaffolds to stabilize CSR enzymatic elements on S regions. Here we demonstrate that all seven 14-3-3β, 14-3-3ε, 14-3-3γ, 14-3-3η, 14-3-3σ, 14-3-3τ and 14-3-3ζ adaptors directly interacted with AID, PKA-Cα (catalytic subunit) and PKA-RIα (regulatory inhibitory subunit) and uracil DNA glycosylase (Ung). 14-3-3 adaptors, however, did not interact with AID C-terminal truncation mutant AIDΔ(180–198) or AIDF193A and AIDL196A point-mutants (which have been shown not to bind to S region DNA and fail to mediate CSR). 14-3-3 adaptors colocalized with AID and replication protein A (RPA) in B cells undergoing CSR. 14-3-3 and AID binding to S region DNA was disrupted by viral protein R (Vpr), an accessory protein of human immunodeficiency virus type-1 (HIV-1), which inhibited CSR without altering AID expression or germline IH-CH transcription. Accordingly, we demonstrated that 14-3-3 directly interact with Vpr, which in turn, also interact with AID, PKA-Cα and Ung. Altogether, our findings suggest that 14-3-3 adaptors play important scaffold functions and nucleate the assembly of multiple CSR factors on S regions. They also show that such assembly can be disrupted by a viral protein, thereby allowing us to hypothesize that small molecule compounds that specifically block 14-3-3 interactions with AID, PKA and/or Ung can be used to inhibit unwanted CSR. PMID:24282540

Oligonucleotide recombination enabled site-specific mutagenesis in bacteria

USDA-ARS?s Scientific Manuscript database

Recombineering refers to a strategy for engineering DNA sequences using a specialized mode of homologous recombination. This technology can be used for rapidly constructing precise changes in bacterial genome sequences in vivo. Oligo recombination is one type of recombineering that uses ssDNA olig...

Monitoring Recombination During Meiosis in Budding Yeast.

PubMed

Owens, Shannon; Tang, Shangming; Hunter, Neil

2018-01-01

Homologous recombination is fundamental to sexual reproduction, facilitating accurate segregation of homologous chromosomes at the first division of meiosis, and creating novel allele combinations that fuel evolution. Following initiation of meiotic recombination by programmed DNA double-strand breaks (DSBs), homologous pairing and DNA strand exchange form joint molecule (JM) intermediates that are ultimately resolved into crossover and noncrossover repair products. Physical monitoring of the DNA steps of meiotic recombination in Saccharomyces cerevisiae (budding yeast) cultures undergoing synchronous meiosis has provided seminal insights into the molecular basis of meiotic recombination and affords a powerful tool for dissecting the molecular roles of recombination factors. This chapter describes a suit of electrophoretic and Southern hybridization techniques used to detect and quantify the DNA intermediates of meiotic recombination at recombination hotspots in budding yeast. DSBs and recombination products (crossovers and noncrossovers) are resolved using one-dimensional electrophoresis and distinguished by restriction site polymorphisms between the parental chromosomes. Psoralen cross-linking is used to stabilize branched JMs, which are resolved from linear species by native/native two-dimensional electrophoresis. Native/denaturing two-dimensional electrophoresis is employed to determine the component DNA strands of JMs and to measure the processing of DSBs. These techniques are generally applicable to any locus where the frequency of recombination is high enough to detect intermediates by Southern hybridization. © 2018 Elsevier Inc. All rights reserved.

Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

DOEpatents

Lewis, Norman G.; Davin, Laurence B.; Dinkova-Kostova, Albena T.; Fujita, Masayuki; Gang, David R.; Sarkanen, Simo; Ford, Joshua D.

2001-04-03

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

Ca2+ improves organization of single-stranded DNA bases in human Rad51 filament, explaining stimulatory effect on gene recombination.

PubMed

Fornander, Louise H; Frykholm, Karolin; Reymer, Anna; Renodon-Cornière, Axelle; Takahashi, Masayuki; Nordén, Bengt

2012-06-01

Human RAD51 protein (HsRad51) catalyses the DNA strand exchange reaction for homologous recombination. To clarify the molecular mechanism of the reaction in vitro being more effective in the presence of Ca(2+) than of Mg(2+), we have investigated the effect of these ions on the structure of HsRad51 filament complexes with single- and double-stranded DNA, the reaction intermediates. Flow linear dichroism spectroscopy shows that the two ionic conditions induce significantly different structures in the HsRad51/single-stranded DNA complex, while the HsRad51/double-stranded DNA complex does not demonstrate this ionic dependence. In the HsRad51/single-stranded DNA filament, the primary intermediate of the strand exchange reaction, ATP/Ca(2+) induces an ordered conformation of DNA, with preferentially perpendicular orientation of nucleobases relative to the filament axis, while the presence of ATP/Mg(2+), ADP/Mg(2+) or ADP/Ca(2+) does not. A high strand exchange activity is observed for the filament formed with ATP/Ca(2+), whereas the other filaments exhibit lower activity. Molecular modelling suggests that the structural variation is caused by the divalent cation interfering with the L2 loop close to the DNA-binding site. It is proposed that the larger Ca(2+) stabilizes the loop conformation and thereby the protein-DNA interaction. A tight binding of DNA, with bases perpendicularly oriented, could facilitate strand exchange.

Roles of Bacillus subtilis DprA and SsbA in RecA-mediated genetic recombination.

PubMed

Yadav, Tribhuwan; Carrasco, Begoña; Serrano, Ester; Alonso, Juan C

2014-10-03

Bacillus subtilis competence-induced RecA, SsbA, SsbB, and DprA are required to internalize and to recombine single-stranded (ss) DNA with homologous resident duplex. RecA, in the ATP · Mg(2+)-bound form (RecA · ATP), can nucleate and form filament onto ssDNA but is inactive to catalyze DNA recombination. We report that SsbA or SsbB bound to ssDNA blocks the RecA filament formation and fails to activate recombination. DprA facilitates RecA filamentation; however, the filaments cannot engage in DNA recombination. When ssDNA was preincubated with SsbA, but not SsbB, DprA was able to activate DNA strand exchange dependent on RecA · ATP. This work demonstrates that RecA · ATP, in concert with SsbA and DprA, catalyzes DNA strand exchange, and SsbB is an accessory factor in the reaction. In contrast, RecA · dATP efficiently catalyzes strand exchange even in the absence of single-stranded binding proteins or DprA, and addition of the accessory factors marginally improved it. We proposed that the RecA-bound nucleotide (ATP and to a lesser extent dATP) might dictate the requirement for accessory factors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Analysis of branched DNA replication and recombination intermediates from prokaryotic cells by two-dimensional (2D) native-native agarose gel electrophoresis.

PubMed

Robinson, Nicholas P

2013-01-01

Branched DNA molecules are generated by the essential processes of replication and recombination. Owing to their distinctive extended shapes, these intermediates migrate differently from linear double-stranded DNA under certain electrophoretic conditions. However, these branched species exist in the cell at much low abundance than the bulk linear DNA. Consequently, branched molecules cannot be visualized by conventional electrophoresis and ethidium bromide staining. Two-dimensional native-native agarose electrophoresis has therefore been developed as a method to facilitate the separation and visualization of branched replication and recombination intermediates. A wide variety of studies have employed this technique to examine branched molecules in eukaryotic, archaeal, and bacterial cells, providing valuable insights into how DNA is duplicated and repaired in all three domains of life.

IDN2 Interacts with RPA and Facilitates DNA Double-Strand Break Repair by Homologous Recombination in Arabidopsis.

PubMed

Liu, Mingming; Ba, Zhaoqing; Costa-Nunes, Pedro; Wei, Wei; Li, Lanxia; Kong, Fansi; Li, Yan; Chai, Jijie; Pontes, Olga; Qi, Yijun

2017-03-01

Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. We previously showed that DSB-induced small RNAs (diRNAs) facilitate homologous recombination-mediated DSB repair in Arabidopsis thaliana Here, we show that INVOLVED IN DE NOVO2 (IDN2), a double-stranded RNA binding protein involved in small RNA-directed DNA methylation, is required for DSB repair in Arabidopsis. We find that IDN2 interacts with the heterotrimeric replication protein A (RPA) complex. Depletion of IDN2 or the diRNA binding ARGONAUTE2 leads to increased accumulation of RPA at DSB sites and mislocalization of the recombination factor RAD51. These findings support a model in which IDN2 interacts with RPA and facilitates the release of RPA from single-stranded DNA tails and subsequent recruitment of RAD51 at DSB sites to promote DSB repair. © 2017 American Society of Plant Biologists. All rights reserved.

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates.

PubMed

Jung, Hana; Lee, Jin A; Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

2014-01-01

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

The numbers of individual mitochondrial DNA molecules and mitochondrial DNA nucleoids in yeast are co-regulated by the general amino acid control pathway.

PubMed

MacAlpine, D M; Perlman, P S; Butow, R A

2000-02-15

Mitochondrial DNA (mtDNA) is inherited as a protein-DNA complex (the nucleoid). We show that activation of the general amino acid response pathway in rho(+) and rho(-) petite cells results in an increased number of nucleoids without an increase in mtDNA copy number. In rho(-) cells, activation of the general amino acid response pathway results in increased intramolecular recombination between tandemly repeated sequences of rho(-) mtDNA to produce small, circular oligomers that are packaged into individual nucleoids, resulting in an approximately 10-fold increase in nucleoid number. The parsing of mtDNA into nucleoids due to general amino acid control requires Ilv5p, a mitochondrial protein that also functions in branched chain amino acid biosynthesis, and one or more factors required for mtDNA recombination. Two additional proteins known to function in mtDNA recombination, Abf2p and Mgt1p, are also required for parsing mtDNA into a larger number of nucleoids, although expression of these proteins is not under general amino acid control. Increased nucleoid number leads to increased mtDNA transmission, suggesting a mechanism to enhance mtDNA inheritance under amino acid starvation conditions.

A properly configured ring structure is critical for the function of the mitochondrial DNA recombination protein, Mgm101.

PubMed

Nardozzi, Jonathan D; Wang, Xiaowen; Mbantenkhu, MacMillan; Wilkens, Stephan; Chen, Xin Jie

2012-10-26

Mgm101 is a Rad52-type recombination protein of bacteriophage origin required for the repair and maintenance of mitochondrial DNA (mtDNA). It forms large oligomeric rings of ∼14-fold symmetry that catalyze the annealing of single-stranded DNAs in vitro. In this study, we investigated the structural elements that contribute to this distinctive higher order structural organization and examined its functional implications. A pair of vicinal cysteines, Cys-216 and Cys-217, was found to be essential for mtDNA maintenance. Mutations to the polar serine, the negatively charged aspartic and glutamic acids, and the hydrophobic amino acid alanine all destabilize mtDNA in vivo. The alanine mutants have an increased propensity of forming macroscopic filaments. In contrast, mutations to aspartic acid drastically destabilize the protein and result in unstructured aggregates with severely reduced DNA binding activity. Interestingly, the serine mutants partially disassemble the Mgm101 rings into smaller oligomers. In the case of the C216S mutant, a moderate increase in DNA binding activity was observed. By using small angle x-ray scattering analysis, we found that Mgm101 forms rings of ∼200 Å diameter in solution, consistent with the structure previously established by transmission electron microscopy. We also found that the C216A/C217A double mutant tends to form broken rings, which likely provide free ends for seeding the growth of the super-stable but functionally defective filaments. Taken together, our data underscore the importance of a delicately maintained ring structure critical for Mgm101 activity. We discuss a potential role of Cys-216 and Cys-217 in regulating Mgm101 function and the repair of damaged mtDNA under stress conditions.

Simultaneous In Vitro Characterisation of DNA Deaminase Function and Associated DNA Repair Pathways

PubMed Central

Franchini, Don-Marc; Incorvaia, Elisabetta; Rangam, Gopinath; Coker, Heather A.; Petersen-Mahrt, Svend K.

2013-01-01

During immunoglobulin (Ig) diversification, activation-induced deaminase (AID) initiates somatic hypermutation and class switch recombination by catalysing the conversion of cytosine to uracil. The synergy between AID and DNA repair pathways is fundamental for the introduction of mutations, however the molecular and biochemical mechanisms underlying this process are not fully elucidated. We describe a novel method to efficiently decipher the composition and activity of DNA repair pathways that are activated by AID-induced lesions. The in vitro resolution (IVR) assay combines AID based deamination and DNA repair activities from a cellular milieu in a single assay, thus avoiding synthetically created DNA-lesions or genetic-based readouts. Recombinant GAL4-AID fusion protein is targeted to a plasmid containing GAL4 binding sites, allowing for controlled cytosine deamination within a substrate plasmid. Subsequently, the Xenopus laevis egg extract provides a source of DNA repair proteins and functional repair pathways. Our results demonstrated that DNA repair pathways which are in vitro activated by AID-induced lesions are reminiscent of those found during AID-induced in vivo Ig diversification. The comparative ease of manipulation of this in vitro systems provides a new approach to dissect the complex DNA repair pathways acting on defined physiologically lesions, can be adapted to use with other DNA damaging proteins (e.g. APOBECs), and provide a means to develop and characterise pharmacological agents to inhibit these potentially oncogenic processes. PMID:24349193

Reverse genetics in high throughput: rapid generation of complete negative strand RNA virus cDNA clones and recombinant viruses thereof.

PubMed

Nolden, T; Pfaff, F; Nemitz, S; Freuling, C M; Höper, D; Müller, T; Finke, Stefan

2016-04-05

Reverse genetics approaches are indispensable tools for proof of concepts in virus replication and pathogenesis. For negative strand RNA viruses (NSVs) the limited number of infectious cDNA clones represents a bottleneck as clones are often generated from cell culture adapted or attenuated viruses, with limited potential for pathogenesis research. We developed a system in which cDNA copies of complete NSV genomes were directly cloned into reverse genetics vectors by linear-to-linear RedE/T recombination. Rapid cloning of multiple rabies virus (RABV) full length genomes and identification of clones identical to field virus consensus sequence confirmed the approache's reliability. Recombinant viruses were recovered from field virus cDNA clones. Similar growth kinetics of parental and recombinant viruses, preservation of field virus characters in cell type specific replication and virulence in the mouse model were confirmed. Reduced titers after reporter gene insertion indicated that the low level of field virus replication is affected by gene insertions. The flexibility of the strategy was demonstrated by cloning multiple copies of an orthobunyavirus L genome segment. This important step in reverse genetics technology development opens novel avenues for the analysis of virus variability combined with phenotypical characterization of recombinant viruses at a clonal level.

RAD51 interconnects between DNA replication, DNA repair and immunity.

PubMed

Bhattacharya, Souparno; Srinivasan, Kalayarasan; Abdisalaam, Salim; Su, Fengtao; Raj, Prithvi; Dozmorov, Igor; Mishra, Ritu; Wakeland, Edward K; Ghose, Subroto; Mukherjee, Shibani; Asaithamby, Aroumougame

2017-05-05

RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. In the absence of RAD51, the unprotected newly replicated genome is degraded by the exonuclease activity of MRE11, and the fragmented nascent DNA accumulates in the cytosol, initiating an innate immune response. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Colorimetric determination of DNase I activity with a DNA-methyl green substrate.

PubMed

Sinicropi, D; Baker, D L; Prince, W S; Shiffer, K; Shak, S

1994-11-01

A simple, high throughput, and precise assay was developed for quantification of deoxyribonuclease I (DNase; IUB 3.1.21.1) activity. The method was adapted from the procedure devised by Kurnick which employs a substrate comprised of highly polymerized native DNA complexed with methyl green. Hydrolysis of the DNA produced unbound methyl green and a decrease in the absorbance of the solution at 620 nm. By adjusting the time and temperature of the reaction, the assay permits quantification of DNase activity over a wide concentration range (0.4 to 8900 ng/ml). Samples and standards were added to the substrate in microtiter plates and were incubated for 1-24 h at 25-37 degrees C to achieve the desired assay range. The DNase activity of the samples was interpolated from a standard curve generated with Pulmozyme recombinant human deoxyribonuclease I (rhDNase). Interassay precision was less than 12% CV and recovery was within 100 +/- 11%. Activity determination by the DNA-methyl green method correlated well with that determined by the widely used "hyperchromicity" method originated by Kunitz, which is based on the increase in absorbance at 260 nm upon hydrolysis of DNA. The DNA-methyl green assay was simpler and more versatile than the hyperchromicity method and was used to characterize the activity of rhDNase and DNase isolated from human urine.

A complex of RAG-1 and RAG-2 proteins persists on DNA after single-strand cleavage at V(D)J recombination signal sequences.

PubMed Central

Grawunder, U; Lieber, M R

1997-01-01

The recombination activating gene (RAG) 1 and 2 proteins are required for initiation of V(D)J recombination in vivo and have been shown to be sufficient to introduce DNA double-strand breaks at recombination signal sequences (RSSs) in a cell-free assay in vitro. RSSs consist of a highly conserved palindromic heptamer that is separated from a slightly less conserved A/T-rich nonamer by either a 12 or 23 bp spacer of random sequence. Despite the high sequence specificity of RAG-mediated cleavage at RSSs, direct binding of the RAG proteins to these sequences has been difficult to demonstrate by standard methods. Even when this can be demonstrated, questions about the order of events for an individual RAG-RSS complex will require methods that monitor aspects of the complex during transitions from one step of the reaction to the next. Here we have used template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) in order to assess occupancy of the reaction intermediates by the RAG complex during the reaction. In addition, this approach allows analysis of the accessibility of end products of a RAG-catalyzed cleavage reaction for N nucleotide addition. The results indicate that RAG proteins form a long-lived complex with the RSS once the initial nick is generated, because the 3'-OH group at the nick remains obstructed for TdT-catalyzed N nucleotide addition. In contrast, the 3'-OH group generated at the signal end after completion of the cleavage reaction can be efficiently tailed by TdT, suggesting that the RAG proteins disassemble from the signal end after DNA double-strand cleavage has been completed. Therefore, a single RAG complex maintains occupancy from the first step (nick formation) to the second step (cleavage). In addition, the results suggest that N region diversity at V(D)J junctions within rearranged immunoglobulin and T cell receptor gene loci can only be introduced after the generation of RAG-catalyzed DNA double-strand breaks, i

DNA sequences, recombinant DNA molecules and processes for producing the A and B subunits of cholera toxin and preparations containing so-obtained subunit or subunits

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

Harford, N.; De Wilde, M.

1987-05-19

A recombinant DNA molecule is described comprising at least a portion coding for subunits A and B of cholera toxin, or a fragment or derivative of the portion wherein the fragment or derivative codes for a polypeptide have an activity which can induce an immune response to subunit A; can induce an immune response to subunit A and cause epithelial cell penetration and the enzymatic effect leading to net loss of fluid into the gut lumen; can bind to the membrane receptor for the B subunit of cholera toxin; can induce an immune response to subunit B; can induce anmore » immune response to subunit B and bind to the membrane receptor; or has a combination of the activities.« less

Infectious mutants of cassava latent virus generated in vivo from intact recombinant DNA clones containing single copies of the genome.

PubMed Central

Stanley, J; Townsend, R

1986-01-01

Intact recombinant DNAs containing single copies of either component of the cassava latent virus genome can elicit infection when mechanically inoculated to host plants in the presence of the appropriate second component. Characterisation of infectious mutant progeny viruses, by analysis of virus-specific supercoiled DNA intermediates, indicates that most if not all of the cloning vector has been deleted, achieved at least in some cases by intermolecular recombination in vivo between DNAs 1 and 2. Significant rearrangements within the intergenic region of DNA 2, predominantly external to the common region, can be tolerated without loss of infectivity suggesting a somewhat passive role in virus multiplication for the sequences in question. Although packaging constraints might impose limits on the amount of DNA within geminate particles, isolation of an infectious coat protein mutant defective in virion production suggests that packaging is not essential for systemic spread of the viral DNA. Images PMID:2875435

An alkaline thermostable recombinant Humicola grisea var. thermoidea cellobiohydrolase presents bifunctional (endo/exoglucanase) activity on cellulosic substrates.

PubMed

Oliveira, G S; Ulhoa, C J; Silveira, M H L; Andreaus, J; Silva-Pereira, I; Poças-Fonseca, M J; Faria, F P

2013-01-01

Humicola grisea var. thermoidea is a deuteromycete which secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. This study focused on the heterologous expression and recombinant enzyme analysis of the major secreted cellulase when the fungus is grown on sugarcane bagasse as the sole carbon source. Cellobiohydrolase 1.2 (CBH 1.2) cDNA was cloned in Pichia pastoris under control of the AOX1 promoter. Recombinant protein (rCBH1.2) was efficiently produced and secreted as a functional enzyme, presenting a molecular mass of 47 kDa. Maximum enzyme production was achieved at 96 h, in culture medium supplemented with 1.34 % urea and 1 % yeast extract and upon induction with 1 % methanol. Recombinant enzyme exhibited optimum activity at 60 °C and pH 8, and presented a remarkable thermostability, particularly at alkaline pH. Activity was evaluated on different cellulosic substrates (carboxymethyl cellulose, filter paper, microcrystalline cellulose and 4-para-nitrophenyl β-D-glucopyranoside). Interestingly, rCBH1.2 presented both exoglucanase and endoglucanase activities and mechanical agitation increased substrate hydrolysis. Results indicate that rCBH1.2 is a potential biocatalyst for applications in the textile industry or detergent formulation.

Cross-system excision of chaperone-mediated proteolysis in chaperone-assisted recombinant protein production

PubMed Central

Martínez-Alonso, Mónica; Villaverde, Antonio

2010-01-01

Main Escherichia coli cytosolic chaperones such as DnaK are key components of the control quality network designed to minimize the prevalence of polypeptides with aberrant conformations. This is achieved by both favoring refolding activities but also stimulating proteolytic degradation of folding reluctant species. This last activity is responsible for the decrease of the proteolytic stability of recombinant proteins when co-produced along with DnaK, where an increase in solubility might be associated to a decrease in protein yield. However, when DnaK and its co-chaperone DnaJ are co-produced in cultured insect cells or whole insect larvae (and expectedly, in other heterologous hosts), only positive, folding-related effects of these chaperones are observed, in absence of proteolysis-mediated reduction of recombinant protein yield. PMID:21326941

The homologous recombination machinery modulates the formation of RNA–DNA hybrids and associated chromosome instability

PubMed Central

Wahba, Lamia; Gore, Steven K; Koshland, Douglas

2013-01-01

Genome instability in yeast and mammals is caused by RNA–DNA hybrids that form as a result of defects in different aspects of RNA biogenesis. We report that in yeast mutants defective for transcription repression and RNA degradation, hybrid formation requires Rad51p and Rad52p. These proteins normally promote DNA–DNA strand exchange in homologous recombination. We suggest they also directly promote the DNA–RNA strand exchange necessary for hybrid formation since we observed accumulation of Rad51p at a model hybrid-forming locus. Furthermore, we provide evidence that Rad51p mediates hybridization of transcripts to homologous chromosomal loci distinct from their site of synthesis. This hybrid formation in trans amplifies the genome-destabilizing potential of RNA and broadens the exclusive co-transcriptional models that pervade the field. The deleterious hybrid-forming activity of Rad51p is counteracted by Srs2p, a known Rad51p antagonist. Thus Srs2p serves as a novel anti-hybrid mechanism in vivo. DOI: http://dx.doi.org/10.7554/eLife.00505.001 PMID:23795288

Induction of homologous recombination in Saccharomyces cerevisiae.

PubMed

Simon, J R; Moore, P D

1988-09-01

We have investigated the effects of UV irradiation of Saccharomyces cerevisiae in order to distinguish whether UV-induced recombination results from the induction of enzymes required for homologous recombination, or the production of substrate sites for recombination containing regions of DNA damage. We utilized split-dose experiments to investigate the induction of proteins required for survival, gene conversion, and mutation in a diploid strain of S. cerevisiae. We demonstrate that inducing doses of UV irradiation followed by a 6 h period of incubation render the cells resistant to challenge doses of UV irradiation. The effects of inducing and challenge doses of UV irradiation upon interchromosomal gene conversion and mutation are strictly additive. Using the yeast URA3 gene cloned in non-replicating single- and double-stranded plasmid vectors that integrate into chromosomal genes upon transformation, we show that UV irradiation of haploid yeast cells and homologous plasmid DNA sequences each stimulate homologous recombination approximately two-fold, and that these effects are additive. Non-specific DNA damage has little effect on the stimulation of homologous recombination, as shown by studies in which UV-irradiated heterologous DNA was included in transformation/recombination experiments. We further demonstrate that the effect of competing single- and double-stranded heterologous DNA sequences differs in UV-irradiated and unirradiated cells, suggesting an induction of recombinational machinery in UV-irradiated S. cerevisiae cells.

Armored DNA in recombinant Baculoviruses as controls in molecular genetic assays.

PubMed

Freystetter, Andrea; Paar, Christian; Stekel, Herbert; Berg, Jörg

2017-10-01

The widespread use of molecular PCR-based assays in analytical and clinical laboratories brings about the need for test-specific, stable, and reliable external controls (EC) as well as standards and internal amplification controls (IC), in order to arrive at consistent test results. In addition, there is also a growing need to produce and provide stable, well-characterized molecular controls for quality assurance programs. In this study, we describe a novel approach to generate armored double-stranded DNA controls, which are encapsulated in baculovirus (BV) particles of the species Autographa californica multiple nucleopolyhedrovirus. We used the well-known BacPAK™ Baculovirus Expression System (Takara-Clontech), removed the polyhedrin promoter used for protein expression, and generated recombinant BV-armored DNAs. The obtained BV-armored DNAs were readily extracted by standard clinical DNA extraction methods, showed favorable linearity and performance in our clinical PCR assays, were resistant to DNase I digestion, and exhibited marked stability in human plasma and serum. BV-armored DNA ought to be used as ECs, quantification standards, and ICs in molecular assays, with the latter application allowing for the entire monitoring of clinical molecular assays for sample adequacy. BV-armored DNA may also be used to produce double-stranded DNA reference materials for, e.g., quality assurance programs. The ease to produce BV-armored DNA should make this approach feasible for a broad spectrum of molecular applications. Finally, as BV-armored DNAs are non-infectious to mammals, they may be even more conveniently shipped than clinical specimen.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots.

PubMed

Baker, Christopher L; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M; Paigen, Kenneth

2015-09-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9+/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.

Multimer Formation Explains Allelic Suppression of PRDM9 Recombination Hotspots

PubMed Central

Baker, Christopher L.; Petkova, Pavlina; Walker, Michael; Flachs, Petr; Mihola, Ondrej; Trachtulec, Zdenek; Petkov, Petko M.; Paigen, Kenneth

2015-01-01

Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. In populations exhibiting high frequencies of heterozygosity, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is also dosage sensitive; Prdm9 +/- heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape. PMID:26368021

Targeting vector construction through recombineering.

PubMed

Malureanu, Liviu A

2011-01-01

Gene targeting in mouse embryonic stem cells is an essential, yet still very expensive and highly time-consuming, tool and method to study gene function at the organismal level or to create mouse models of human diseases. Conventional cloning-based methods have been largely used for generating targeting vectors, but are hampered by a number of limiting factors, including the variety and location of restriction enzymes in the gene locus of interest, the specific PCR amplification of repetitive DNA sequences, and cloning of large DNA fragments. Recombineering is a technique that exploits the highly efficient homologous recombination function encoded by λ phage in Escherichia coli. Bacteriophage-based recombination can recombine homologous sequences as short as 30-50 bases, allowing manipulations such as insertion, deletion, or mutation of virtually any genomic region. The large availability of mouse genomic bacterial artificial chromosome (BAC) libraries covering most of the genome facilitates the retrieval of genomic DNA sequences from the bacterial chromosomes through recombineering. This chapter describes a successfully applied protocol and aims to be a detailed guide through the steps of generation of targeting vectors through recombineering.

Therapeutic Recombinant Monoclonal Antibodies

ERIC Educational Resources Information Center

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

A broad survey of recombination in animal mitochondria.

PubMed

Piganeau, Gwenaël; Gardner, Michael; Eyre-Walker, Adam

2004-12-01

Recombination in mitochondrial DNA (mtDNA) remains a controversial topic. Here we present a survey of 279 animal mtDNA data sets, of which 12 were from asexual species. Using four separate tests, we show that there is widespread evidence of recombination; for one test as many as 14.2% of the data sets reject a model of clonal inheritance and in several data sets, including primates, the recombinants can be identified visually. We show that none of the tests give significant results for obligate clonal species (apomictic pathogens) and that the sexual species show significantly greater evidence of recombination than asexual species. For some data sets, such as Macaca nemestrina, additional data sets suggest that the recombinants are not artifacts. For others, it cannot be determined whether the recombinants are real or produced by laboratory error. Either way, the results have important implications for how mtDNA is sequenced and used.

The consequences of sequence erosion in the evolution of recombination hotspots.

PubMed

Tiemann-Boege, Irene; Schwarz, Theresa; Striedner, Yasmin; Heissl, Angelika

2017-12-19

Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans -acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion.This article is part of the themed issue 'Evolutionary causes and consequences of recombination rate variation in sexual organisms'. © 2017 The Authors.

The consequences of sequence erosion in the evolution of recombination hotspots

PubMed Central

Schwarz, Theresa; Heissl, Angelika

2017-01-01

Meiosis is initiated by a double-strand break (DSB) introduced in the DNA by a highly controlled process that is repaired by recombination. In many organisms, recombination occurs at specific and narrow regions of the genome, known as recombination hotspots, which overlap with regions enriched for DSBs. In recent years, it has been demonstrated that conversions and mutations resulting from the repair of DSBs lead to a rapid sequence evolution at recombination hotspots eroding target sites for DSBs. We still do not fully understand the effect of this erosion in the recombination activity, but evidence has shown that the binding of trans-acting factors like PRDM9 is affected. PRDM9 is a meiosis-specific, multi-domain protein that recognizes DNA target motifs by its zinc finger domain and directs DSBs to these target sites. Here we discuss the changes in affinity of PRDM9 to eroded recognition sequences, and explain how these changes in affinity of PRDM9 can affect recombination, leading sometimes to sterility in the context of hybrid crosses. We also present experimental data showing that DNA methylation reduces PRDM9 binding in vitro. Finally, we discuss PRDM9-independent hotspots, posing the question how these hotspots evolve and change with sequence erosion. This article is part of the themed issue ‘Evolutionary causes and consequences of recombination rate variation in sexual organisms’. PMID:29109225

Evolution via recombination: Cell-to-cell contact facilitates larger recombination events in Streptococcus pneumoniae.

PubMed

Cowley, Lauren A; Petersen, Fernanda C; Junges, Roger; Jimson D Jimenez, Med; Morrison, Donald A; Hanage, William P

2018-06-01

Homologous recombination in the genetic transformation model organism Streptococcus pneumoniae is thought to be important in the adaptation and evolution of this pathogen. While competent pneumococci are able to scavenge DNA added to laboratory cultures, large-scale transfers of multiple kb are rare under these conditions. We used whole genome sequencing (WGS) to map transfers in recombinants arising from contact of competent cells with non-competent 'target' cells, using strains with known genomes, distinguished by a total of ~16,000 SNPs. Experiments designed to explore the effect of environment on large scale recombination events used saturating purified donor DNA, short-term cell assemblages on Millipore filters, and mature biofilm mixed cultures. WGS of 22 recombinants for each environment mapped all SNPs that were identical between the recombinant and the donor but not the recipient. The mean recombination event size was found to be significantly larger in cell-to-cell contact cultures (4051 bp in filter assemblage and 3938 bp in biofilm co-culture versus 1815 bp with saturating DNA). Up to 5.8% of the genome was transferred, through 20 recombination events, to a single recipient, with the largest single event incorporating 29,971 bp. We also found that some recombination events are clustered, that these clusters are more likely to occur in cell-to-cell contact environments, and that they cause significantly increased linkage of genes as far apart as 60,000 bp. We conclude that pneumococcal evolution through homologous recombination is more likely to occur on a larger scale in environments that permit cell-to-cell contact.

Ig heavy chain class switch recombination: mechanism and regulation

PubMed Central

Stavnezer, Janet; Schrader, Carol E.

2014-01-01

Ig heavy chain class switching occurs rapidly after activation of mature naïve B cells, resulting in a switch from expressing IgM and IgD to expression of IgG, IgE, or IgA; this switch improves the ability of antibodies to remove the pathogen that induces the humoral immune response. Class switching occurs by a deletional recombination between two different switch (S) regions, each of which is associated with a heavy chain constant (CH) region gene. Class switch recombination (CSR) is instigated by activation-induced cytidine deaminase (AID), which converts cytosines in S regions to uracils. The uracils are subsequently removed by two DNA repair pathways, resulting in mutations, single-strand DNA breaks, and the double-strand breaks required for CSR. We discuss several aspects of CSR, including how CSR is induced, CSR in B-cell progenitors, the roles for transcription and chromosomal looping in CSR, and the roles of certain DNA repair enzymes in CSR. PMID:25411432

Characterization of the Caenorhabditis elegans HIM-6/BLM Helicase: Unwinding Recombination Intermediates

PubMed Central

Choi, Seoyoon; Lee, Hyunwoo; Ahn, Byungchan

2014-01-01

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates. PMID:25036527

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange*

PubMed Central

Borgogno, María V.; Monti, Mariela R.; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E.; Pezza, Roberto J.

2016-01-01

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3′ end of the initiating DNA strand have a small effect, whereas most mismatches near the 5′ end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. PMID:26709229

Conversion of Deletions during Recombination in Pneumococcal Transformation

PubMed Central

Lefevre, J. C.; Mostachfi, P.; Gasc, A. M.; Guillot, E.; Pasta, F.; Sicard, M.

1989-01-01

Genetic analysis of 16 deletions obtained in the amiA locus of pneumococcus is described. When present on donor DNA, all deletions increased drastically the frequency of wild-type recombinants in two-point crosses. This effect was maximal for deletions longer than 200 bases. It was reduced for heterologies shorter than 76 bases and did not exist for very short deletions. In three-point crosses in which the deletion was localized between two point mutations, we demonstrated that this excess of wild-type recombinants was the result of a genetic conversion. This conversion extended over several scores of bases outside the deletion. Conversion takes place during the heteroduplex stage of recombination. Therefore, in pneumococcal transformation, long heterologies participated in this heteroduplex configuration. As this conversion did not require an active DNA polymerase A gene it is proposed that the mechanism of conversion is not a DNA repair synthesis but involves breakage and ligation between DNA molecules. Conversion of deletions did not require the Hex system of correction of mismatched bases. It differs also from localized conversion. It appears that it is a process that evolved to correct errors of replication which lead to long heterologies and which are not eliminated by other systems. PMID:2599365

Regulation of the DNA damage response by DNA-PKcs inhibitory phosphorylation of ATM

PubMed Central

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T.

2017-01-01

SUMMARY Ataxia-Telangiectasia Mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. PMID:27939942

Regulation of the DNA Damage Response by DNA-PKcs Inhibitory Phosphorylation of ATM.

PubMed

Zhou, Yi; Lee, Ji-Hoon; Jiang, Wenxia; Crowe, Jennie L; Zha, Shan; Paull, Tanya T

2017-01-05

Ataxia-telangiectasia mutated (ATM) regulates the DNA damage response as well as DNA double-strand break repair through homologous recombination. Here we show that ATM is hyperactive when the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) is chemically inhibited or when the DNA-PKcs gene is deleted in human cells. Pre-incubation of ATM protein with active DNA-PKcs also significantly reduces ATM activity in vitro. We characterize several phosphorylation sites in ATM that are targets of DNA-PKcs and show that phospho-mimetic mutations at these residues significantly inhibit ATM activity and impair ATM signaling upon DNA damage. In contrast, phospho-blocking mutations at one cluster of sites increase the frequency of apoptosis during normal cell growth. DNA-PKcs, which is integral to the non-homologous end joining pathway, thus negatively regulates ATM activity through phosphorylation of ATM. These observations illuminate an important regulatory mechanism for ATM that also controls DNA repair pathway choice. Copyright © 2017 Elsevier Inc. All rights reserved.

BRIT1/MCPH1 is essential for mitotic and meiotic recombination DNA repair and maintaining genomic stability in mice.

PubMed

Liang, Yulong; Gao, Hong; Lin, Shiaw-Yih; Peng, Guang; Huang, Xingxu; Zhang, Pumin; Goss, John A; Brunicardi, Francis C; Multani, Asha S; Chang, Sandy; Li, Kaiyi

2010-01-22

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1(-/-) mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1(-/-) mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to gamma-irradiation. BRIT1(-/-) MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1(-/-) mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice.

p53 suppresses hyper-recombination by modulating BRCA1 function

PubMed Central

Dong, Chao; Zhang, Fengmei; Luo, Yue; Wang, Hui; Zhao, Xipeng; Guo, Gongshe; Powell, Simon N.; Feng, Zhihui

2015-01-01

Both p53 and BRCA1 are tumor suppressors and are involved in a number of cellular processes including cell cycle arrest, apoptosis, transcriptional regulation, and DNA damage repair. Some studies have suggested that the association of BRCA1 and p53 is required for transcriptional regulation of genes involved in cell replication and DNA repair pathways. However, the relationship between the two proteins in molecular mechanisms of DNA repair is still not clear. Therefore, we sought to determine whether there is a functional link between p53 and BRCA1 in DNA repair. Firstly, using a plasmid recombination substrate, pDR-GFP, integrated into the genome of breast cancer cell line MCF7, we have demonstrated that p53 suppressed Rad51-mediated hyper-recombinational repair by two independent cell models of HPV-E6 induced p53 inactivation and p53 knockdown assay. Our study further indicated that p53 mediated homologous recombination (HR) through inhibiting BRCA1 over-function via mechanism of transcription regulation in response to DNA repair. Since it was found p53 and BRCA1 existed in a protein complex, indicating both proteins may be associated at post-transcriptional level. Moreover, defective p53-induced hyper-recombination was associated with cell radioresistance and chromosomal stability, strongly supporting the involvement of p53 in the inhibition of hyper-recombination, which led to genetic stability and cellular function in response to DNA damage. In addition, it was found that p53 loss rescued BRCA1 deficiency via recovering HR and chromosomal stability, suggesting that p53 is also involved in the HR-inhibition independently of BRCA1. Thus, our data indicated that p53 was involved in inhibiting recombination by both BRCA1-dependent and -independent mechanisms, and there is a functional link between p53-suppression and BRCA1-promotion in regulation of HR activity at transcription level and possible post-transcription level. PMID:26162908

Recombinant DNA production of spider silk proteins

PubMed Central

Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

2013-01-01

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. PMID:24119078

Tolerance of DNA Mismatches in Dmc1 Recombinase-mediated DNA Strand Exchange.

PubMed

Borgogno, María V; Monti, Mariela R; Zhao, Weixing; Sung, Patrick; Argaraña, Carlos E; Pezza, Roberto J

2016-03-04

Recombination between homologous chromosomes is required for the faithful meiotic segregation of chromosomes and leads to the generation of genetic diversity. The conserved meiosis-specific Dmc1 recombinase catalyzes homologous recombination triggered by DNA double strand breaks through the exchange of parental DNA sequences. Although providing an efficient rate of DNA strand exchange between polymorphic alleles, Dmc1 must also guard against recombination between divergent sequences. How DNA mismatches affect Dmc1-mediated DNA strand exchange is not understood. We have used fluorescence resonance energy transfer to study the mechanism of Dmc1-mediated strand exchange between DNA oligonucleotides with different degrees of heterology. The efficiency of strand exchange is highly sensitive to the location, type, and distribution of mismatches. Mismatches near the 3' end of the initiating DNA strand have a small effect, whereas most mismatches near the 5' end impede strand exchange dramatically. The Hop2-Mnd1 protein complex stimulates Dmc1-catalyzed strand exchange on homologous DNA or containing a single mismatch. We observed that Dmc1 can reject divergent DNA sequences while bypassing a few mismatches in the DNA sequence. Our findings have important implications in understanding meiotic recombination. First, Dmc1 acts as an initial barrier for heterologous recombination, with the mismatch repair system providing a second level of proofreading, to ensure that ectopic sequences are not recombined. Second, Dmc1 stepping over infrequent mismatches is likely critical for allowing recombination between the polymorphic sequences of homologous chromosomes, thus contributing to gene conversion and genetic diversity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Novel mitochondrial gene content and gene arrangement indicate illegitimate inter-mtDNA recombination in the chigger mite, Leptotrombidium pallidum.

PubMed

Shao, Renfu; Mitani, Harumi; Barker, Stephen C; Takahashi, Mamoru; Fukunaga, Masahito

2005-06-01

To better understand the evolution of mitochondrial (mt) genomes in the Acari (mites and ticks), we sequenced the mt genome of the chigger mite, Leptotrombidium pallidum (Arthropoda: Acari: Acariformes). This genome is highly rearranged relative to that of the hypothetical ancestor of the arthropods and the other species of Acari studied. The mt genome of L. pallidum has two genes for large subunit rRNA, a pseudogene for small subunit rRNA, and four nearly identical large noncoding regions. Nineteen of the 22 tRNAs encoded by this genome apparently lack either a T-arm or a D-arm. Further, the mt genome of L. pallidum has two distantly separated sections with identical sequences but opposite orientations of transcription. This arrangement cannot be accounted for by homologous recombination or by previously known mechanisms of mt gene rearrangement. The most plausible explanation for the origin of this arrangement is illegitimate inter-mtDNA recombination, which has not been reported previously in animals. In light of the evidence from previous experiments on recombination in nuclear and mt genomes of animals, we propose a model of illegitimate inter-mtDNA recombination to account for the novel gene content and gene arrangement in the mt genome of L. pallidum.

Immunoglobulin class-switch recombination deficiencies.

PubMed

Durandy, Anne; Kracker, Sven

2012-07-30

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches.

TOPBP1Dpb11 plays a conserved role in homologous recombination DNA repair through the coordinated recruitment of 53BP1Rad9

PubMed Central

Sims, Jennie Rae; Freire, Raimundo

2017-01-01

Genome maintenance and cancer suppression require homologous recombination (HR) DNA repair. In yeast and mammals, the scaffold protein TOPBP1Dpb11 has been implicated in HR, although its precise function and mechanism of action remain elusive. In this study, we show that yeast Dpb11 plays an antagonistic role in recombination control through regulated protein interactions. Dpb11 mediates opposing roles in DNA end resection by coordinating both the stabilization and exclusion of Rad9 from DNA lesions. The Mec1 kinase promotes the pro-resection function of Dpb11 by mediating its interaction with the Slx4 scaffold. Human TOPBP1Dpb11 engages in interactions with the anti-resection factor 53BP1 and the pro-resection factor BRCA1, suggesting that TOPBP1 also mediates opposing functions in HR control. Hyperstabilization of the 53BP1–TOPBP1 interaction enhances the recruitment of 53BP1 to nuclear foci in the S phase, resulting in impaired HR and the accumulation of chromosomal aberrations. Our results support a model in which TOPBP1Dpb11 plays a conserved role in mediating a phosphoregulated circuitry for the control of recombinational DNA repair. PMID:28228534

DNA damage tolerance pathway involving DNA polymerase ι and the tumor suppressor p53 regulates DNA replication fork progression.

PubMed

Hampp, Stephanie; Kiessling, Tina; Buechle, Kerstin; Mansilla, Sabrina F; Thomale, Jürgen; Rall, Melanie; Ahn, Jinwoo; Pospiech, Helmut; Gottifredi, Vanesa; Wiesmüller, Lisa

2016-07-26

DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonuclease-deficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linker-induced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.

Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

PubMed

Lenden Hasse, Hélène; Lescale, Chloé; Bianchi, Joy J; Yu, Wei; Bedora-Faure, Marie; Deriano, Ludovic

2017-12-01

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Recombinant DNA production of spider silk proteins.

PubMed

Tokareva, Olena; Michalczechen-Lacerda, Valquíria A; Rech, Elíbio L; Kaplan, David L

2013-11-01

Spider dragline silk is considered to be the toughest biopolymer on Earth due to an extraordinary combination of strength and elasticity. Moreover, silks are biocompatible and biodegradable protein-based materials. Recent advances in genetic engineering make it possible to produce recombinant silks in heterologous hosts, opening up opportunities for large-scale production of recombinant silks for various biomedical and material science applications. We review the current strategies to produce recombinant spider silks. © 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

Unequal homologous recombination between tandemly arranged sequences stably incorporated into cultured rat cells.

PubMed Central

Stringer, J R; Kuhn, R M; Newman, J L; Meade, J C

1985-01-01

Cultured rat cells deficient in endogenous thymidine kinase activity (tk) were stably transformed with a recombination-indicator DNA substrate constructed in vitro by rearrangement of the herpes simplex virus tk gene sequences into a partially redundant permutation of the functional gene. The recombination-indicator DNA did not express tk, but was designed to allow formation of a functional tk gene via homologous recombination. A clonal cell line (519) was isolated that harbored several permuted herpes simplex virus tk genes. 519 cells spontaneously produced progeny that survived in medium containing hypoxanthine, aminopterin, and thymidine. Acquisition of resistance to hypoxanthine, aminopterin, and thymidine was accompanied by the rearrangement of the defective tk gene to functional configuration. The rearrangement apparently occurred by unequal exchange between one permuted tk gene and a replicated copy of itself. Recombination was between 500-base-pair tracts of DNA sequence homology that were separated by 3.4 kilobases. Exchanges occurred spontaneously at a frequency of approximately 5 X 10(-6) events per cell per generation. Recombination also mediated reversion to the tk- phenotype; however, the predominant mechanism by which cells escaped death in the presence of drugs rendered toxic by thymidine kinase was not recombination, but rather inactivation of the intact tk gene. Images PMID:3016511

An active recombinant cocoonase from the silkworm Bombyx mori: bleaching, degumming and sericin degrading activities.

PubMed

Unajak, Sasimanas; Aroonluke, Suradet; Promboon, Amornrat

2015-04-01

Cocoonase is a serine protease produced by silk moths and used for softening the cocoons so that they can escape. Degumming is one of the important steps in silk processing. This research aimed to produce an active recombinant Bombyx mori cocoonase (BmCoc) for the silk degumming process. A recombinant BmCoc was successfully expressed in a Pichia pastoris system. The purified enzyme showed specific activity of 227 U mg(-1) protein, 2.4-fold purification, 95% yield and a molecular weight of 26 kDa. The enzyme exhibited optimal temperature at 40 °C and optimal pH at 8, and showed thermal stability at 25-45 °C and pH stability at 5-9. The recombinant enzyme exhibited sericin degumming ability and color bleaching characteristics, and did not affect the fibroin fiber. The enzyme also degraded sericin substrate with a product size about 30-70 kDa. In this study, we successfully produced the active recombinant BmCoc in P. pastoris with promising functions for the Thai silk degumming process, which includes degumming, sericin degrading and color bleaching activities. Our data clearly indicated that the recombinant enzyme had proteolytic activity on sericin but not on fibroin proteins. The recombinant BmCoc has proven to be suitable for numerous applications in the silk industry. © 2014 Society of Chemical Industry.

A Delicate Balance Between Repair and Replication Factors Regulates Recombination Between Divergent DNA Sequences in Saccharomyces cerevisiae

PubMed Central

Chakraborty, Ujani; George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

2016-01-01

Single-strand annealing (SSA) is an important homologous recombination mechanism that repairs DNA double strand breaks (DSBs) occurring between closely spaced repeat sequences. During SSA, the DSB is acted upon by exonucleases to reveal complementary sequences that anneal and are then repaired through tail clipping, DNA synthesis, and ligation steps. In baker’s yeast, the Msh DNA mismatch recognition complex and the Sgs1 helicase act to suppress SSA between divergent sequences by binding to mismatches present in heteroduplex DNA intermediates and triggering a DNA unwinding mechanism known as heteroduplex rejection. Using baker’s yeast as a model, we have identified new factors and regulatory steps in heteroduplex rejection during SSA. First we showed that Top3-Rmi1, a topoisomerase complex that interacts with Sgs1, is required for heteroduplex rejection. Second, we found that the replication processivity clamp proliferating cell nuclear antigen (PCNA) is dispensable for heteroduplex rejection, but is important for repairing mismatches formed during SSA. Third, we showed that modest overexpression of Msh6 results in a significant increase in heteroduplex rejection; this increase is due to a compromise in Msh2-Msh3 function required for the clipping of 3′ tails. Thus 3′ tail clipping during SSA is a critical regulatory step in the repair vs. rejection decision; rejection is favored before the 3′ tails are clipped. Unexpectedly, Msh6 overexpression, through interactions with PCNA, disrupted heteroduplex rejection between divergent sequences in another recombination substrate. These observations illustrate the delicate balance that exists between repair and replication factors to optimize genome stability. PMID:26680658

Homologous Recombination and Translesion DNA Synthesis Play Critical Roles on Tolerating DNA Damage Caused by Trace Levels of Hexavalent Chromium

PubMed Central

Chen, Youjun; Zhou, Yi-Hui; Neo, Dayna; Clement, Jean; Takata, Minoru; Takeda, Shunichi; Sale, Julian; Wright, Fred A.; Swenberg, James A.; Nakamura, Jun

2016-01-01

Contamination of potentially carcinogenic hexavalent chromium (Cr(VI)) in the drinking water is a major public health concern worldwide. However, little information is available regarding the biological effects of a nanomoler amount of Cr(VI). Here, we investigated the genotoxic effects of Cr(VI) at nanomoler levels and their repair pathways. We found that DNA damage response analyzed based on differential toxicity of isogenic cells deficient in various DNA repair proteins is observed after a three-day incubation with K2CrO4 in REV1-deficient DT40 cells at 19.2 μg/L or higher as well as in TK6 cells deficient in polymerase delta subunit 3 (POLD3) at 9.8 μg/L or higher. The genotoxicity of Cr(VI) decreased ~3000 times when the incubation time was reduced from three days to ten minutes. TK mutation rate also significantly decreased from 6 day to 1 day exposure to Cr(VI). The DNA damage response analysis suggest that DNA repair pathways, including the homologous recombination and REV1- and POLD3-mediated error-prone translesion synthesis pathways, are critical for the cells to tolerate to DNA damage caused by trace amount of Cr(VI). PMID:27907204

Ring-shaped architecture of RecR: implications for its role in homologous recombinational DNA repair

PubMed Central

Lee, Byung Il; Kim, Kyoung Hoon; Park, Soo Jeong; Eom, Soo Hyun; Song, Hyun Kyu; Suh, Se Won

2004-01-01

RecR, together with RecF and RecO, facilitates RecA loading in the RecF pathway of homologous recombinational DNA repair in procaryotes . The human Rad52 protein is a functional counterpart of RecFOR. We present here the crystal structure of RecR from Deinococcus radiodurans (DR RecR). A monomer of DR RecR has a two-domain structure: the N-terminal domain with a helix–hairpin–helix (HhH) motif and the C-terminal domain with a Cys4 zinc-finger motif, a Toprim domain and a Walker B motif. Four such monomers form a ring-shaped tetramer of 222 symmetry with a central hole of 30−35 Å diameter. In the crystal, two tetramers are concatenated, implying that the RecR tetramer is capable of opening and closing. We also show that DR RecR binds to both dsDNA and ssDNA, and that its HhH motif is essential for DNA binding. PMID:15116069

Recombinant antibody mediated delivery of organelle-specific DNA pH sensors along endocytic pathways

NASA Astrophysics Data System (ADS)

Modi, Souvik; Halder, Saheli; Nizak, Clément; Krishnan, Yamuna

2013-12-01

DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing the sequence of the pH sensitive domain of the DNA sensor, we have been able to tune their pH sensitive regimes and create a family of DNA sensors spanning ranges from pH 4 to 7.6. To enable a generalizable targeting methodology, this new sensor design also incorporates a `handle' domain. We have identified, using a phage display screen, a set of three recombinant antibodies (scFv) that bind sequence specifically to the handle domain. Sequence analysis of these antibodies revealed several conserved residues that mediate specific interactions with the cognate DNA duplex. We also found that all three scFvs clustered into different branches indicating that their specificity arises from mutations in key residues. When one of these scFvs is fused to a membrane protein (furin) that traffics via the cell surface, the scFv-furin chimera binds the `handle' and ferries a family of DNA pH sensors along the furin endocytic pathway. Post endocytosis, all DNA nanodevices retain their functionality in cellulo and provide spatiotemporal pH maps of retrogradely trafficking furin inside living cells. This new molecular technology of DNA-scFv-protein chimeras can be used to site-specifically complex DNA nanostructures for bioanalytical applications.DNA has been used to build nanomachines with potential in cellulo and in vivo applications. However their different in cellulo applications are limited by the lack of generalizable strategies to deliver them to precise intracellular locations. Here we describe a new molecular design of DNA pH sensors with response times that are nearly 20 fold faster. Further, by changing

DNA Resection at Chromosome Breaks Promotes Genome Stability by Constraining Non-Allelic Homologous Recombination

PubMed Central

Koshland, Douglas

2012-01-01

DNA double-strand breaks impact genome stability by triggering many of the large-scale genome rearrangements associated with evolution and cancer. One of the first steps in repairing this damage is 5′→3′ resection beginning at the break site. Recently, tools have become available to study the consequences of not extensively resecting double-strand breaks. Here we examine the role of Sgs1- and Exo1-dependent resection on genome stability using a non-selective assay that we previously developed using diploid yeast. We find that Saccharomyces cerevisiae lacking Sgs1 and Exo1 retains a very efficient repair process that is highly mutagenic to genome structure. Specifically, 51% of cells lacking Sgs1 and Exo1 repair a double-strand break using repetitive sequences 12–48 kb distal from the initial break site, thereby generating a genome rearrangement. These Sgs1- and Exo1-independent rearrangements depend partially upon a Rad51-mediated homologous recombination pathway. Furthermore, without resection a robust cell cycle arrest is not activated, allowing a cell with a single double-strand break to divide before repair, potentially yielding multiple progeny each with a different rearrangement. This profusion of rearranged genomes suggests that cells tolerate any dangers associated with extensive resection to inhibit mutagenic pathways such as break-distal recombination. The activation of break-distal recipient repeats and amplification of broken chromosomes when resection is limited raise the possibility that genome regions that are difficult to resect may be hotspots for rearrangements. These results may also explain why mutations in resection machinery are associated with cancer. PMID:22479212

The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

PubMed

Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

2016-06-01

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo

PubMed Central

Powers, Natalie R.; Parvanov, Emil D.; Baker, Christopher L.; Walker, Michael; Petkov, Petko M.; Paigen, Kenneth

2016-01-01

In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

Isolation and purification of recombinant proteins, antibodies and plasmid DNA with hydroxyapatite chromatography.

PubMed

Hilbrig, Frank; Freitag, Ruth

2012-01-01

Hydroxyapatite and related stationary phases increasingly play a role in the downstream processing of high-value biological materials, such as recombinant proteins, therapeutic antibodies and pharmaceutical-grade plasmid DNA. Chromatographic hydroxyapatite is an inorganic, ceramic material identical in composition, if not in structure, to calcium phosphate found in human bones and teeth. The interaction of hydroxyapatite with biomacromolecules is complex and highly dynamic, which can make predicting performance difficult, but also allows the design of very selective isolation processes. This review discusses the currently commercially available chromatographic materials, different retention mechanisms supported by these materials and differential exploitation for the design of highly specific isolation procedures. The state of the art of antibody purification by hydroxy- and fluoroapatite is reviewed together with tested routines for method development and implementation. Finally, the isolation of plasmid DNA is discussed, since the purification of DNA therapeutics at a sufficiently large scale is an emerging need in bioprocess development and perhaps the area in bioseparation where apatite chromatography can make its most important contribution to date. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of European mtDNAs for recombination.

PubMed

Elson, J L; Andrews, R M; Chinnery, P F; Lightowlers, R N; Turnbull, D M; Howell, N

2001-01-01

The standard paradigm postulates that the human mitochondrial genome (mtDNA) is strictly maternally inherited and that, consequently, mtDNA lineages are clonal. As a result of mtDNA clonality, phylogenetic and population genetic analyses should therefore be free of the complexities imposed by biparental recombination. The use of mtDNA in analyses of human molecular evolution is contingent, in fact, on clonality, which is also a condition that is critical both for forensic studies and for understanding the transmission of pathogenic mtDNA mutations within families. This paradigm, however, has been challenged recently by Eyre-Walker and colleagues. Using two different tests, they have concluded that recombination has contributed to the distribution of mtDNA polymorphisms within the human population. We have assembled a database that comprises the complete sequences of 64 European and 2 African mtDNAs. When this set of sequences was analyzed using any of three measures of linkage disequilibrium, one of the tests of Eyre-Walker and colleagues, there was no evidence for mtDNA recombination. When their test for excess homoplasies was applied to our set of sequences, only a slight excess of homoplasies was observed. We discuss possible reasons that our results differ from those of Eyre-Walker and colleagues. When we take the various results together, our conclusion is that mtDNA recombination has not been sufficiently frequent during human evolution to overturn the standard paradigm.

Modulating Cellular Recombination Potential through Alterations in RecA Structure and Regulation

PubMed Central

Bakhlanova, Irina V.; Dudkina, Alexandra V.; Baitin, Dima M.; Knight, Kendall L.; Cox, Michael M.; Lanzov, Vladislav A.

2010-01-01

The wild type E. coli RecA protein is a recombinase platform with unrealized recombination potential. We have explored the factors affecting recombination during conjugation with a quantitative assay. Regulatory proteins that affect RecA function have the capacity to increase or decrease recombination frequencies by factors up to 6 fold. Autoinhibition by the RecA C-terminus can affect recombination frequency by factors up to 4 fold. The greatest changes in recombination frequency measured here are brought about by point mutations in the recA gene. RecA variants can increase recombination frequencies by more than 50 fold. The RecA protein thus possesses an inherently broad functional range. The RecA protein of Escherichia coli (EcRecA) is not optimized for recombination function. Instead, much of the recombination potential of EcRecA is structurally suppressed, probably reflecting cellular requirements. One point mutation in EcRecA with a particularly dramatic effect on recombination frequency, D112R, exhibits an enhanced capacity to load onto SSB-coated ssDNA, overcome the effects of regulatory proteins such as PsiB and RecX, and to pair homologous DNAs. Comparisons of key RecA protein mutants reveal two components to RecA recombination function – filament formation and the inherent DNA pairing activity of the formed filaments. PMID:21143322

Phenylbutyrate inhibits homologous recombination induced by camptothecin and methyl methanesulfonate.

PubMed

Kaiser, Gitte S; Germann, Susanne M; Westergaard, Tine; Lisby, Michael

2011-08-01

Homologous recombination is accompanied by extensive changes to chromatin organization at the site of DNA damage. Some of these changes are mediated through acetylation/deacetylation of histones. Here, we show that recombinational repair of DNA damage induced by the anti-cancer drug camptothecin (CPT) and the alkylating agent methyl methanesulfonate (MMS) is blocked by sodium phenylbutyrate (PBA) in the budding yeast Saccharomyces cerevisiae. In particular, PBA suppresses CPT- and MMS-induced genetic recombination as well as DNA double-strand break repair during mating-type interconversion. Treatment with PBA is accompanied by a dramatic reduction in histone H4 lysine 8 acetylation. Live cell imaging of homologous recombination proteins indicates that repair of CPT-induced DNA damage is redirected to a non-recombinogenic pathway in the presence of PBA without loss in cell viability. In contrast, the suppression of MMS-induced recombination by PBA is accompanied by a dramatic loss in cell viability. Taken together, our results demonstrate that PBA inhibits DNA damage-induced homologous recombination likely by mediating changes in chromatin acetylation. Moreover, the combination of PBA with genotoxic agents can lead to different cell fates depending on the type of DNA damage inflicted. 2011 Elsevier B.V. All rights reserved.

The over-expression of the β2 catalytic subunit of the proteasome decreases homologous recombination and impairs DNA double-strand break repair in human cells.

PubMed

Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

2011-01-01

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

The Over-expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double-Strand Break Repair in Human Cells

PubMed Central

Collavoli, Anita; Comelli, Laura; Cervelli, Tiziana; Galli, Alvaro

2011-01-01

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p. PMID:21660142

Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks.

PubMed

Tichy, Elisia D; Pillai, Resmi; Deng, Li; Liang, Li; Tischfield, Jay; Schwemberger, Sandy J; Babcock, George F; Stambrook, Peter J

2010-11-01

Embryonic stem (ES) cells give rise to all cell types of an organism. Since mutations at this embryonic stage would affect all cells and be detrimental to the overall health of an organism, robust mechanisms must exist to ensure that genomic integrity is maintained. To test this proposition, we compared the capacity of murine ES cells to repair DNA double-strand breaks with that of differentiated cells. Of the 2 major pathways that repair double-strand breaks, error-prone nonhomologous end joining (NHEJ) predominated in mouse embryonic fibroblasts, whereas the high fidelity homologous recombinational repair (HRR) predominated in ES cells. Microhomology-mediated end joining, an emerging repair pathway, persisted at low levels in all cell types examined. The levels of proteins involved in HRR and microhomology-mediated end joining were highly elevated in ES cells compared with mouse embryonic fibroblasts, whereas those for NHEJ were quite variable, with DNA Ligase IV expression low in ES cells. The half-life of DNA Ligase IV protein was also low in ES cells. Attempts to increase the abundance of DNA Ligase IV protein by overexpression or inhibition of its degradation, and thereby elevate NHEJ in ES cells, were unsuccessful. When ES cells were induced to differentiate, however, the level of DNA Ligase IV protein increased, as did the capacity to repair by NHEJ. The data suggest that preferential use of HRR rather than NHEJ may lend ES cells an additional layer of genomic protection and that the limited levels of DNA Ligase IV may account for the low level of NHEJ activity.

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

PubMed Central

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-01-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

Controlled Release from Recombinant Polymers

PubMed Central

Price, Robert; Poursaid, Azadeh; Ghandehari, Hamidreza

2014-01-01

Recombinant polymers provide a high degree of molecular definition for correlating structure with function in controlled release. The wide array of amino acids available as building blocks for these materials lend many advantages including biorecognition, biodegradability, potential biocompatibility, and control over mechanical properties among other attributes. Genetic engineering and DNA manipulation techniques enable the optimization of structure for precise control over spatial and temporal release. Unlike the majority of chemical synthetic strategies used, recombinant DNA technology has allowed for the production of monodisperse polymers with specifically defined sequences. Several classes of recombinant polymers have been used for controlled drug delivery. These include, but are not limited to, elastin-like, silk-like, and silk-elastinlike proteins, as well as emerging cationic polymers for gene delivery. In this article, progress and prospects of recombinant polymers used in controlled release will be reviewed. PMID:24956486

A nuclear mutation defective in mitochondrial recombination in yeast.

PubMed

Ling, F; Makishima, F; Morishima, N; Shibata, T

1995-08-15

Homologous recombination (crossing over and gene conversion) is generally essential for heritage and DNA repair, and occasionally causes DNA aberrations, in nuclei of eukaryotes. However, little is known about the roles of homologous recombination in the inheritance and stability of mitochondrial DNA which is continuously damaged by reactive oxygen species, by-products of respiration. Here, we report the first example of a nuclear recessive mutation which suggests an essential role for homologous recombination in the stable inheritance of mitochondrial DNA. For the detection of this class of mutants, we devised a novel procedure, 'mitochondrial crossing in haploid', which has enabled us to examine many mutant clones. Using this procedure, we examined mutants of Saccharomyces cerevisiae that showed an elevated UV induction of respiration-deficient mutations. We obtained a mutant that was defective in both the omega-intron homing and Endo.SceI-induced homologous gene conversion. We found that the mutant cells are temperature sensitive in the maintenance of mitochondrial DNA. A tetrad analysis indicated that elevated UV induction of respiration-deficient mutations, recombination deficiency and temperature sensitivity are all caused by a single nuclear mutation (mhr1) on chromosome XII. The pleiotropic characteristics of the mutant suggest an essential role for the MHR1 gene in DNA repair, recombination and the maintenance of DNA in mitochondria.

Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity

PubMed Central

Zhang, Jin; Ruhlman, Tracey A.; Sabir, Jamal S. M.; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K.

2016-01-01

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear–plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. PMID:26893456

The role of DNA double-strand breaks in spontaneous homologous recombination in S. cerevisiae.

PubMed

Lettier, Gaëlle; Feng, Qi; de Mayolo, Adriana Antúnez; Erdeniz, Naz; Reid, Robert J D; Lisby, Michael; Mortensen, Uffe H; Rothstein, Rodney

2006-11-10

Homologous recombination (HR) is a source of genomic instability and the loss of heterozygosity in mitotic cells. Since these events pose a severe health risk, it is important to understand the molecular events that cause spontaneous HR. In eukaryotes, high levels of HR are a normal feature of meiosis and result from the induction of a large number of DNA double-strand breaks (DSBs). By analogy, it is generally believed that the rare spontaneous mitotic HR events are due to repair of DNA DSBs that accidentally occur during mitotic growth. Here we provide the first direct evidence that most spontaneous mitotic HR in Saccharomyces cerevisiae is initiated by DNA lesions other than DSBs. Specifically, we describe a class of rad52 mutants that are fully proficient in inter- and intra-chromosomal mitotic HR, yet at the same time fail to repair DNA DSBs. The conclusions are drawn from genetic analyses, evaluation of the consequences of DSB repair failure at the DNA level, and examination of the cellular re-localization of Rad51 and mutant Rad52 proteins after introduction of specific DSBs. In further support of our conclusions, we show that, as in wild-type strains, UV-irradiation induces HR in these rad52 mutants, supporting the view that DNA nicks and single-stranded gaps, rather than DSBs, are major sources of spontaneous HR in mitotic yeast cells.

Annealing Vs. Invasion in Phage λ Recombination

PubMed Central

Stahl, M. M.; Thomason, L.; Poteete, A. R.; Tarkowski, T.; Kuzminov, A.; Stahl, F. W.

1997-01-01

Genetic recombination catalyzed by λ's Red pathway was studied in rec(+) and recA mutant bacteria by examining both intracellular λ DNA and mature progeny particles. Recombination of nonreplicating phage chromosomes was induced by double-strand breaks delivered at unique sites in vivo. In rec(+) cells, cutting only one chromosome gave nearly maximal stimulation of recombination; the recombinants formed contained relatively short hybrid regions, suggesting strand invasion. In contrast, in recA mutant cells, cutting the two parental chromosomes at non-allelic sites was required for maximal stimulation; the recombinants formed tended to be hybrid over the entire region between the two cuts, implying strand annealing. We conclude that, in the absence of RecA and the presence of non-allelic DNA ends, the Red pathway of λ catalyzes recombination primarily by annealing. PMID:9383045

The evolutionary turnover of recombination hot spots contributes to speciation in mice

PubMed Central

Smagulova, Fatima; Brick, Kevin; Pu, Yongmei; Camerini-Otero, R. Daniel; Petukhova, Galina V.

2016-01-01

Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation. PMID:26833728

Selections that isolate recombinant mitochondrial genomes in animals

PubMed Central

Ma, Hansong; O'Farrell, Patrick H

2015-01-01

Homologous recombination is widespread and catalyzes evolution. Nonetheless, its existence in animal mitochondrial DNA is questioned. We designed selections for recombination between co-resident mitochondrial genomes in various heteroplasmic Drosophila lines. In four experimental settings, recombinant genomes became the sole or dominant genome in the progeny. Thus, selection uncovers occurrence of homologous recombination in Drosophila mtDNA and documents its functional benefit. Double-strand breaks enhanced recombination in the germline and revealed somatic recombination. When the recombination partner was a diverged Drosophila melanogaster genome or a genome from a different species such as Drosophila yakuba, sequencing revealed long continuous stretches of exchange. In addition, the distribution of sequence polymorphisms in recombinants allowed us to map a selected trait to a particular region in the Drosophila mitochondrial genome. Thus, recombination can be harnessed to dissect function and evolution of mitochondrial genome. DOI: http://dx.doi.org/10.7554/eLife.07247.001 PMID:26237110

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

PubMed

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Mitochondrial Genome Rearrangements in Glomus Species Triggered by Homologous Recombination between Distinct mtDNA Haplotypes

PubMed Central

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms. AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence, were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigate dpo diversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity. We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants. PMID:23925788

Immunoglobulin class-switch recombination deficiencies

PubMed Central

2012-01-01

Immunoglobulin class-switch recombination deficiencies (Ig-CSR-Ds) are rare primary immunodeficiencies characterized by defective switched isotype (IgG/IgA/IgE) production. Depending on the molecular defect in question, the Ig-CSR-D may be combined with an impairment in somatic hypermutation (SHM). Some of the mechanisms underlying Ig-CSR and SHM have been described by studying natural mutants in humans. This approach has revealed that T cell-B cell interaction (resulting in CD40-mediated signaling), intrinsic B-cell mechanisms (activation-induced cytidine deaminase-induced DNA damage), and complex DNA repair machineries (including uracil-N-glycosylase and mismatch repair pathways) are all involved in class-switch recombination and SHM. However, several of the mechanisms required for full antibody maturation have yet to be defined. Elucidation of the molecular defects underlying the diverse set of Ig-CSR-Ds is essential for understanding Ig diversification and has prompted better definition of the clinical spectrum of diseases and the development of increasingly accurate diagnostic and therapeutic approaches. PMID:22894609

ATR suppresses endogenous DNA damage and allows completion of homologous recombination repair.

PubMed

Brown, Adam D; Sager, Brian W; Gorthi, Aparna; Tonapi, Sonal S; Brown, Eric J; Bishop, Alexander J R

2014-01-01

DNA replication fork stalling or collapse that arises from endogenous damage poses a serious threat to genome stability, but cells invoke an intricate signaling cascade referred to as the DNA damage response (DDR) to prevent such damage. The gene product ataxia telangiectasia and Rad3-related (ATR) responds primarily to replication stress by regulating cell cycle checkpoint control, yet it's role in DNA repair, particularly homologous recombination (HR), remains unclear. This is of particular interest since HR is one way in which replication restart can occur in the presence of a stalled or collapsed fork. Hypomorphic mutations in human ATR cause the rare autosomal-recessive disease Seckel syndrome, and complete loss of Atr in mice leads to embryonic lethality. We recently adapted the in vivo murine pink-eyed unstable (pun) assay for measuring HR frequency to be able to investigate the role of essential genes on HR using a conditional Cre/loxP system. Our system allows for the unique opportunity to test the effect of ATR loss on HR in somatic cells under physiological conditions. Using this system, we provide evidence that retinal pigment epithelium (RPE) cells lacking ATR have decreased density with abnormal morphology, a decreased frequency of HR and an increased level of chromosomal damage.

Genome-wide control of the distribution of meiotic recombination.

PubMed

Grey, Corinne; Baudat, Frédéric; de Massy, Bernard

2009-02-17

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

PubMed

Manfrini, Nicola; Trovesi, Camilla; Wery, Maxime; Martina, Marina; Cesena, Daniele; Descrimes, Marc; Morillon, Antonin; d'Adda di Fagagna, Fabrizio; Longhese, Maria Pia

2015-02-01

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. © 2014 The Authors.

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

PubMed

Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

2013-07-31

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

Methods of introducing nucleic acids into cellular DNA

DOEpatents

Lajoie, Marc J.; Gregg, Christopher J.; Mosberg, Joshua A.; Church, George M.

2017-06-27

A method of introducing a nucleic acid sequence into a cell is provided where the cell has impaired or inhibited or disrupted DnaG primase activity or impaired or inhibited or disrupted DnaB helicase activity, or larger or increased gaps or distance between Okazaki fragments or lowered or reduced frequency of Okazaki fragment initiation, or the cell has increased single stranded DNA (ssDNA) on the lagging strand of the replication fork including transforming the cell through recombination with a nucleic acid oligomer.

[Desensitization to human recombinant DNA insulin in an adolescent with insulin-dependent diabetes mellitus].

PubMed

Rosas Vargas, M A; Alvarez Amador, M; Alvarez Amador, L M; del Río Navarro, B E; Avila Castanón, L; Sienra Monge, J J

2001-01-01

Adverse reactions to drugs have increased in the last years, about 15% of all side effects are thought to be immune mediated according to the Coombs and Gell classification they can be type I (immediate) hypersensitivity, type II (cytotoxic) type III (immune complex mediated) or type IV (delay). Allergy to insulin is defined as an immunological response type I, and type II or III to exogenous insulin solutions occurring the 0.1% and 0.2% of the patients. A 13 year old female with a 4-year history of insulin-dependent diabetes mellitus who presented hypersensitivity against recombinant DNA (rDNA) insulin manifested with urticaria and itching. We used a premedication therapy without good response and impossibility to use alternative therapy for her metabolic control, so she needed desensitization with insulin. Skin prick testing with rapid insulin preparations 1:10 W/V dilution were positive. IgE antibodies to insulin weren't presented. IgE serum values were normal. We began the desensitization with a rapid 1:1000 UI insulin solution by intradermal route, than by subcutaneous route until reaching the accumulated doses necessary per day. During the process it appeared a papular rash and itching which were treated with an intravenous antihistaminic without troubles. The patient tolerated the desensitization procedure very well. For the past 14 months she has been treated uneventfully by subcutaneous administration of rDNA insulin. The desensitization against drugs is not a frequently process it only has to be used when it is impossible to substitute the treatment. Our patient showed probably hypersensitivity type 1 to insulin. However, we have to take into account the cytotoxic reaction caused by IgG or IgM antibodies or by immune complex. The desensitization finally was tolerated, 14 months after our patient accepts correctly her daily dose of human recombinant insulin.

Schizosaccharomyces pombe Retrotransposon Tf2 Mobilizes Primarily through Homologous cDNA Recombination

PubMed Central

Hoff, Eleanor F.; Levin, Henry L.; Boeke, Jef D.

1998-01-01

The Tf2 retrotransposon, found in the fission yeast Schizosaccharomyces pombe, is nearly identical to its sister element, Tf1, in its reverse transcriptase-RNase H and integrase domains but is very divergent in the gag domain, the protease, the 5′ untranslated region, and the U3 domain of the long terminal repeats. It has now been demonstrated that a neo-marked copy of Tf2 overexpressed from a heterologous promoter can mobilize into the S. pombe genome and produce true transposition events. However, the Tf2-neo mobilization frequency is 10- to 20-fold lower than that of Tf1-neo, and 70% of the Tf2-neo events are homologous recombination events generated independently of a functional Tf2 integrase. Thus, the Tf2 element is primarily dependent on homologous recombination with preexisting copies of Tf2 for its propagation. Finally, production of Tf2-neo proteins and cDNA was also analyzed; surprisingly, Tf2 was found to produce its reverse transcriptase as a single species in which it is fused to protease, unlike all other retroviruses and retrotransposons. PMID:9774697

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity*

PubMed Central

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.; Marqusee, Susan; Keck, James L.

2015-01-01

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. PMID:25903123

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity

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

Petzold, Christine; Marceau, Aimee H.; Miller, Katherine H.

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding sitemore » are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.« less

Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity.

PubMed

Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L

2015-06-05

Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Efficient sequence-specific isolation of DNA fragments and chromatin by in vitro enChIP technology using recombinant CRISPR ribonucleoproteins.

PubMed

Fujita, Toshitsugu; Yuno, Miyuki; Fujii, Hodaka

2016-04-01

The clustered regularly interspaced short palindromic repeats (CRISPR) system is widely used for various biological applications, including genome editing. We developed engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR to isolate target genomic regions from cells for their biochemical characterization. In this study, we developed 'in vitro enChIP' using recombinant CRISPR ribonucleoproteins (RNPs) to isolate target genomic regions. in vitro enChIP has the great advantage over conventional enChIP of not requiring expression of CRISPR complexes in cells. We first showed that in vitro enChIP using recombinant CRISPR RNPs can be used to isolate target DNA from mixtures of purified DNA in a sequence-specific manner. In addition, we showed that this technology can be used to efficiently isolate target genomic regions, while retaining their intracellular molecular interactions, with negligible contamination from irrelevant genomic regions. Thus, in vitro enChIP technology is of potential use for sequence-specific isolation of DNA, as well as for identification of molecules interacting with genomic regions of interest in vivo in combination with downstream analysis. © 2016 The Authors. Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Repair and recombination of X-irradiated plasmids in Xenopus laevis oocytes

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

Sweigert, S.E.; Carroll, D.

1990-11-01

Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detectedmore » directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.« less

Coevolution between Nuclear-Encoded DNA Replication, Recombination, and Repair Genes and Plastid Genome Complexity.

PubMed

Zhang, Jin; Ruhlman, Tracey A; Sabir, Jamal S M; Blazier, John Chris; Weng, Mao-Lun; Park, Seongjun; Jansen, Robert K

2016-02-17

Disruption of DNA replication, recombination, and repair (DNA-RRR) systems has been hypothesized to cause highly elevated nucleotide substitution rates and genome rearrangements in the plastids of angiosperms, but this theory remains untested. To investigate nuclear-plastid genome (plastome) coevolution in Geraniaceae, four different measures of plastome complexity (rearrangements, repeats, nucleotide insertions/deletions, and substitution rates) were evaluated along with substitution rates of 12 nuclear-encoded, plastid-targeted DNA-RRR genes from 27 Geraniales species. Significant correlations were detected for nonsynonymous (dN) but not synonymous (dS) substitution rates for three DNA-RRR genes (uvrB/C, why1, and gyrA) supporting a role for these genes in accelerated plastid genome evolution in Geraniaceae. Furthermore, correlation between dN of uvrB/C and plastome complexity suggests the presence of nucleotide excision repair system in plastids. Significant correlations were also detected between plastome complexity and 13 of the 90 nuclear-encoded organelle-targeted genes investigated. Comparisons revealed significant acceleration of dN in plastid-targeted genes of Geraniales relative to Brassicales suggesting this correlation may be an artifact of elevated rates in this gene set in Geraniaceae. Correlation between dN of plastid-targeted DNA-RRR genes and plastome complexity supports the hypothesis that the aberrant patterns in angiosperm plastome evolution could be caused by dysfunction in DNA-RRR systems. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Extended Minus-Strand DNA as Template for R-U5-Mediated Second-Strand Transfer in Recombinational Rescue of Primer Binding Site-Modified Retroviral Vectors

PubMed Central

Mikkelsen, Jacob Giehm; Lund, Anders H.; Dybkær, Karen; Duch, Mogens; Pedersen, Finn Skou

1998-01-01

We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439–1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed. PMID:9499117

Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

PubMed

Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

2013-12-16

Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

Overlapping activation-induced cytidine deaminase hotspot motifs in Ig class-switch recombination

PubMed Central

Han, Li; Masani, Shahnaz; Yu, Kefei

2011-01-01

Ig class-switch recombination (CSR) is directed by the long and repetitive switch regions and requires activation-induced cytidine deaminase (AID). One of the conserved switch-region sequence motifs (AGCT) is a preferred site for AID-mediated DNA-cytosine deamination. By using somatic gene targeting and recombinase-mediated cassette exchange, we established a cell line-based CSR assay that allows manipulation of switch sequences at the endogenous locus. We show that AGCT is only one of a family of four WGCW motifs in the switch region that can facilitate CSR. We go on to show that it is the overlap of AID hotspots at WGCW sites on the top and bottom strands that is critical. This finding leads to a much clearer model for the difference between CSR and somatic hypermutation. PMID:21709240

Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

PubMed

Boksha, I S; Lavrova, N V; Grishin, A V; Demidenko, A V; Lyashchuk, A M; Galushkina, Z M; Ovchinnikov, R S; Umyarov, A M; Avetisian, L R; Chernukha, M Iu; Shaginian, I A; Lunin, V G; Karyagina, A S

2016-05-01

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

cDNA cloning, functional expression and antifungal activities of a dimeric plant defensin SPE10 from Pachyrrhizus erosus seeds.

PubMed

Song, Xiaomin; Wang, Jing; Wu, Fang; Li, Xu; Teng, Maikun; Gong, Weimin

2005-01-01

SPE10 is an antifungal protein isolated from the seeds of Pachyrrhizus erosus. cDNA encoding a 47 amino acid peptide was cloned by RT-PCR and the gene sequence proved SPE10 to be a new member of plant defensin family. The synthetic cDNA with codons preferred in yeast was cloned into the pPIC9 plasmid directly in-frame with the secretion signal alpha-mating factor, and highly expressed in methylotrophic Pichia pastoris. Activity assays showed the recombinant SPE10 inhibited specifically the growth of several pathogenic fungi as native SPE10. Circular dichroism and fluorescence spectroscopy analysis indicated that the native and recombinant protein should have same folding, though there are eight cystein residues in the sequence. Several evidence suggested SPE10 should be the first dimeric plant defensin reported so far.

Prdm9 controls activation of mammalian recombination hotspots.

PubMed

Parvanov, Emil D; Petkov, Petko M; Paigen, Kenneth

2010-02-12

Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, ensures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse chromosome 17, which controls activation of recombination at multiple distant hotspots, has been mapped within a 181-kilobase interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is expressed in early meiosis and whose deficiency results in sterility in both sexes. Mus musculus exhibits five alleles of Prdm9; human populations exhibit two predominant alleles and multiple minor alleles. The identification of Prdm9 as a protein regulating mammalian recombination hotspots initiates molecular studies of this important biological control system.

Gene conversion at the gray locus of Sordaria fimicola: fit of the experimental data to a hybrid DNA model of recombination.

PubMed

Kalogeropoulos, A; Thuriaux, P

1985-03-01

A hybrid DNA (hDNA) model of recombination has been algebraically formulated, which allows the prediction of frequencies of postmeiotic segregation and conversion of a given allele and their probability of being associated with a crossing over. The model considered is essentially the "Aviemore model." In contrast to some other interpretations of recombination, it states that gene conversion can only result from the repair of heteroduplex hDNA, with postmeiotic segregation resulting from unrepaired heteroduplexes. The model also postulates that crossing over always occurs distally to the initiation site of the hDNA. Eleven types of conversion and postmeiotic segregation with or without associated crossover were considered. Their theoretical frequencies are given by 11 linear equations with ten variables, four describing heteroduplex repair, four giving the probability of hDNA formation and its topological properties and two giving the probability that crossing over occurs at the left or right of the converting allele. Using the experimental data of Kitani and coworkers on conversion at the six best studied gray alleles of Sordaria fimicola, we found that the model considered fit the data at a P level above or very close (allele h4) to the 5% level of sampling error provided that the hDNA is partly asymmetric. The best fitting solutions are such that the hDNA has an equal probability of being formed on either chromatid or, alternatively, that both DNA strands have the same probability of acting as the invading strand during hDNA formation. The two mismatches corresponding to a given allele are repaired with different efficiencies. Optimal solutions are found if one allows for repair to be more efficient on the asymmetric hDNA than on the symmetric one. In the case of allele g1, our data imply that the direction of repair is nonrandom with respect to the strand on which it occurs.

The evolutionary turnover of recombination hot spots contributes to speciation in mice.

PubMed

Smagulova, Fatima; Brick, Kevin; Pu, Yongmei; Camerini-Otero, R Daniel; Petukhova, Galina V

2016-02-01

Meiotic recombination is required for the segregation of homologous chromosomes and is essential for fertility. In most mammals, the DNA double-strand breaks (DSBs) that initiate meiotic recombination are directed to a subset of genomic loci (hot spots) by sequence-specific binding of the PRDM9 protein. Rapid evolution of the DNA-binding specificity of PRDM9 and gradual erosion of PRDM9-binding sites by gene conversion will alter the recombination landscape over time. To better understand the evolutionary turnover of recombination hot spots and its consequences, we mapped DSB hot spots in four major subspecies of Mus musculus with different Prdm9 alleles and in their F1 hybrids. We found that hot spot erosion governs the preferential usage of some Prdm9 alleles over others in hybrid mice and increases sequence diversity specifically at hot spots that become active in the hybrids. As crossovers are disfavored at such hot spots, we propose that sequence divergence generated by hot spot turnover may create an impediment for recombination in hybrids, potentially leading to reduced fertility and, eventually, speciation. Published by Cold Spring Harbor Laboratory Press.

Functions of the high mobility group protein, Abf2p, in mitochondrial DNA segregation, recombination and copy number in Saccharomyces cerevisiae.

PubMed

Zelenaya-Troitskaya, O; Newman, S M; Okamoto, K; Perlman, P S; Butow, R A

1998-04-01

Previous studies have established that the mitochondrial high mobility group (HMG) protein, Abf2p, of Saccharomyces cerevisiae influences the stability of wild-type (rho+) mitochondrial DNA (mtDNA) and plays an important role in mtDNA organization. Here we report new functions for Abf2p in mtDNA transactions. We find that in homozygous deltaabf2 crosses, the pattern of sorting of mtDNA and mitochondrial matrix protein is altered, and mtDNA recombination is suppressed relative to homozygous ABF2 crosses. Although Abf2p is known to be required for the maintenance of mtDNA in rho+ cells growing on rich dextrose medium, we find that it is not required for the maintenance of mtDNA in p cells grown on the same medium. The content of both rho+ and rho- mtDNAs is increased in cells by 50-150% by moderate (two- to threefold) increases in the ABF2 copy number, suggesting that Abf2p plays a role in mtDNA copy control. Overproduction of Abf2p by > or = 10-fold from an ABF2 gene placed under control of the GAL1 promoter, however, leads to a rapid loss of rho+ mtDNA and a quantitative conversion of rho+ cells to petites within two to four generations after a shift of the culture from glucose to galactose medium. Overexpression of Abf2p in rho- cells also leads to a loss of mtDNA, but at a slower rate than was observed for rho+ cells. The mtDNA instability phenotype is related to the DNA-binding properties of Abf2p because a mutant Abf2p that contains mutations in residues of both HMG box domains known to affect DNA binding in vitro, and that binds poorly to mtDNA in vivo, complements deltaabf2 cells only weakly and greatly lessens the effect of overproduction on mtDNA instability. In vivo binding was assessed by colocalization to mtDNA of fusions between mutant or wild-type Abf2p and green fluorescent protein. These findings are discussed in the context of a model relating mtDNA copy number control and stability to mtDNA recombination.

Activation of Dun1 in response to nuclear DNA instability accounts for the increase in mitochondrial point mutations in Rad27/FEN1 deficient S. cerevisiae.

PubMed

Kaniak-Golik, Aneta; Kuberska, Renata; Dzierzbicki, Piotr; Sledziewska-Gojska, Ewa

2017-01-01

Rad27/FEN1 nuclease that plays important roles in the maintenance of DNA stability in the nucleus has recently been shown to reside in mitochondria. Accordingly, it has been established that Rad27 deficiency causes increased mutagenesis, but decreased microsatellite instability and homologous recombination in mitochondria. Our current analysis of mutations leading to erythromycin resistance indicates that only some of them arise in mitochondrial DNA and that the GC→AT transition is a hallmark of the mitochondrial mutagenesis in rad27 null background. We also show that the mitochondrial mutator phenotype resulting from Rad27 deficiency entirely depends on the DNA damage checkpoint kinase Dun1. DUN1 inactivation suppresses the mitochondrial mutator phenotype caused by Rad27 deficiency and this suppression is eliminated at least in part by subsequent deletion of SML1 encoding a repressor of ribonucleotide reductase. We conclude that Rad27 deficiency causes a mitochondrial mutator phenotype via activation of DNA damage checkpoint kinase Dun1 and that a Dun1-mediated increase of dNTP pools contributes to this phenomenon. These results point to the nuclear DNA instability as the source of mitochondrial mutagenesis. Consistently, we show that mitochondrial mutations occurring more frequently in yeast devoid of Rrm3, a DNA helicase involved in rDNA replication, are also dependent on Dun1. In addition, we have established that overproduction of Exo1, which suppresses DNA damage sensitivity and replication stress in nuclei of Rad27 deficient cells, but does not enter mitochondria, suppresses the mitochondrial mutagenesis. Exo1 overproduction restores also a great part of allelic recombination and microsatellite instability in mitochondria of Rad27 deficient cells. In contrast, the overproduction of Exo1 does not influence mitochondrial direct-repeat mediated deletions in rad27 null background, pointing to this homologous recombination pathway as the direct target of Rad27

Breeding and identification of novel koji molds with high activity of acid protease by genome recombination between Aspergillus oryzae and Aspergillus niger.

PubMed

Xu, Defeng; Pan, Li; Zhao, Haifeng; Zhao, Mouming; Sun, Jiaxin; Liu, Dongmei

2011-09-01

Acid protease is essential for degradation of proteins during soy sauce fermentation. To breed more suitable koji molds with high activity of acid protease, interspecific genome recombination between A. oryzae and A. niger was performed. Through stabilization with d-camphor and haploidization with benomyl, several stable fusants with higher activity of acid protease were obtained, showing different degrees of improvement in acid protease activity compared with the parental strain A. oryzae. In addition, analyses of mycelial morphology, expression profiles of extracellular proteins, esterase isoenzyme profiles, and random amplified polymorphic DNA (RAPD) were applied to identify the fusants through their phenotypic and genetic relationships. Morphology analysis of the mycelial shape of fusants indicated a phenotype intermediate between A. oryzae and A. niger. The profiles of extracellular proteins and esterase isoenzyme electrophoresis showed the occurrence of genome recombination during or after protoplast fusion. The dendrogram constructed from RAPD data revealed great heterogeneity, and genetic dissimilarity indices showed there were considerable differences between the fusants and their parental strains. This investigation suggests that genome recombination is a powerful tool for improvement of food-grade industrial strains. Furthermore, the presented strain improvement procedure will be applicable for widespread use for other industrial strains.

The absence of oligonucleosomal DNA fragmentation during apoptosis of IMR-5 neuroblastoma cells: disappearance of the caspase-activated DNase.

PubMed

Yuste, V J; Bayascas, J R; Llecha, N; Sánchez-López, I; Boix, J; Comella, J X

2001-06-22

Caspase-activated DNase is responsible for the oligonucleosomal DNA degradation during apoptosis. DNA degradation is thought to be important for multicellular organisms to prevent oncogenic transformation or as a mechanism of viral defense. It has been reported that certain cells, including some neuroblastoma cell lines such as IMR-5, enter apoptosis without digesting DNA in such a way. We have analyzed the causes for the absence of DNA laddering in staurosporine-treated IMR-5 cells, and we have found that most of the molecular mechanisms controlling apoptosis are well preserved in this cell line. These include degradation of substrates for caspases, blockade of cell death by antiapoptotic genes such as Bcl-2 or Bcl-X(L), or normal levels and adequate activation of caspase-3. Moreover, these cells display normal levels of caspase-activated DNase and its inhibitory protein, inhibitor of caspase-activated DNase, and their cDNA sequences are identical to those reported previously. Nevertheless, IMR-5 cells lose caspase-activated DNase during apoptosis and recover their ability to degrade DNA when human recombinant caspase-activated DNase is overexpressed. Our results lead to the conclusion that caspase-activated DNase is processed during apoptosis of IMR-5 cells, making these cells a good model to study the relevance of this endonuclease in physiological or pathological conditions.

Site-specific DNA Inversion by Serine Recombinases

PubMed Central

2015-01-01

Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome

PubMed Central

2013-01-01

Background Phage-encoded serine integrases, such as φC31 integrase, are widely used for genome engineering. Fifteen such integrases have been described but their utility for genome engineering has not been compared in uniform assays. Results We have compared fifteen serine integrases for their utility for DNA manipulations in mammalian cells after first demonstrating that all were functional in E. coli. Chromosomal recombination reporters were used to show that seven integrases were active on chromosomally integrated DNA in human fibroblasts and mouse embryonic stem cells. Five of the remaining eight enzymes were active on extra-chromosomal substrates thereby demonstrating that the ability to mediate extra-chromosomal recombination is no guide to ability to mediate site-specific recombination on integrated DNA. All the integrases that were active on integrated DNA also promoted DNA integration reactions that were not mediated through conservative site-specific recombination or damaged the recombination sites but the extent of these aberrant reactions varied over at least an order of magnitude. Bxb1 integrase yielded approximately two-fold more recombinants and displayed about two fold less damage to the recombination sites than the next best recombinase; φC31 integrase. Conclusions We conclude that the Bxb1 and φC31 integrases are the reagents of choice for genome engineering in vertebrate cells and that DNA damage repair is a major limitation upon the utility of this class of site-specific recombinase. PMID:24139482

Metal binding proteins, recombinant host cells and methods

DOEpatents

Summers, Anne O.; Caguiat, Jonathan J.

2004-06-15

The present disclosure provides artificial heavy metal binding proteins termed chelons by the inventors. These chelons bind cadmium and/or mercuric ions with relatively high affinity. Also disclosed are coding sequences, recombinant DNA molecules and recombinant host cells comprising those recombinant DNA molecules for expression of the chelon proteins. In the recombinant host cells or transgenic plants, the chelons can be used to bind heavy metals taken up from contaminated soil, groundwater or irrigation water and to concentrate and sequester those ions. Recombinant enteric bacteria can be used within the gastrointestinal tracts of animals or humans exposed to toxic metal ions such as mercury and/or cadmium, where the chelon recombinantly expressed in chosen in accordance with the ion to be rededicated. Alternatively, the chelons can be immobilized to solid supports to bind and concentrate heavy metals from a contaminated aqueous medium including biological fluids.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice.

PubMed

Li, Ting; Liu, Bo; Chen, Chih Ying; Yang, Bing

2016-05-20

Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes of OsALS were obtained in T0 generation at the efficiency of 1.4%-6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T1 generation. The edited T1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms. Copyright © 2016 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31.

PubMed

Chappell, William H; Gautam, Dipendra; Ok, Suzan T; Johnson, Bryan A; Anacker, Daniel C; Moody, Cary A

2015-12-23

High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for productive viral

Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

PubMed Central

Chappell, William H.; Gautam, Dipendra; Ok, Suzan T.; Johnson, Bryan A.; Anacker, Daniel C.

2015-01-01

ABSTRACT High-risk human papillomavirus 31 (HPV31)-positive cells exhibit constitutive activation of the ATM-dependent DNA damage response (DDR), which is necessary for productive viral replication. In response to DNA double-strand breaks (DSBs), ATM activation leads to DNA repair through homologous recombination (HR), which requires the principal recombinase protein Rad51, as well as BRCA1. Previous studies from our lab demonstrated that Rad51 and BRCA1 are expressed at high levels in HPV31-positive cells and localize to sites of viral replication. These results suggest that HPV may utilize ATM activity to increase HR activity as a means to facilitate viral replication. In this study, we demonstrate that high-risk HPV E7 expression alone is sufficient for the increase in Rad51 and BRCA1 protein levels. We have found that this increase occurs, at least in part, at the level of transcription. Studies analyzing protein stability indicate that HPV may also protect Rad51 and BRCA1 from turnover, contributing to the overall increase in cellular levels. We also demonstrate that Rad51 is bound to HPV31 genomes, with binding increasing per viral genome upon productive replication. We have found that depletion of Rad51 and BRCA1, as well as inhibition of Rad51's recombinase activity, abrogates productive viral replication upon differentiation. Overall, these results indicate that Rad51 and BRCA1 are required for the process of HPV31 genome amplification and suggest that productive replication occurs in a manner dependent upon recombination. IMPORTANCE Productive replication of HPV31 requires activation of an ATM-dependent DNA damage response, though how ATM activity contributes to replication is unclear. Rad51 and BRCA1 play essential roles in repair of double-strand breaks, as well as the restart of stalled replication forks through homologous recombination (HR). Given that ATM activity is required to initiate HR repair, coupled with the requirement of Rad51 and BRCA1 for

Identification of defective illegitimate recombinational repair of oxidatively-induced DNA double-strand breaks in ataxia-telangiectasia cells

NASA Technical Reports Server (NTRS)

Dar, M. E.; Winters, T. A.; Jorgensen, T. J.

1997-01-01

Ataxia-telangiectasia (A-T) is an autosomal-recessive lethal human disease. Homozygotes suffer from a number of neurological disorders, as well as very high cancer incidence. Heterozygotes may also have a higher than normal risk of cancer, particularly for the breast. The gene responsible for the disease (ATM) has been cloned, but its role in mechanisms of the disease remain unknown. Cellular A-T phenotypes, such as radiosensitivity and genomic instability, suggest that a deficiency in the repair of DNA double-strand breaks (DSBs) may be the primary defect; however, overall levels of DSB rejoining appear normal. We used the shuttle vector, pZ189, containing an oxidatively-induced DSB, to compare the integrity of DSB rejoining in one normal and two A-T fibroblast cells lines. Mutation frequencies were two-fold higher in A-T cells, and the mutational spectrum was different. The majority of the mutations found in all three cell lines were deletions (44-63%). The DNA sequence analysis indicated that 17 of the 17 plasmids with deletion mutations in normal cells occurred between short direct-repeat sequences (removing one of the repeats plus the intervening sequences), implicating illegitimate recombination in DSB rejoining. The combined data from both A-T cell lines showed that 21 of 24 deletions did not involve direct-repeats sequences, implicating a defect in the illegitimate recombination pathway. These findings suggest that the A-T gene product may either directly participate in illegitimate recombination or modulate the pathway. Regardless, this defect is likely to be important to a mechanistic understanding of this lethal disease.

8-hydroxyquinoline inhibition of DNA synthesis and intragenic recombination during yeast meiosis.

PubMed

Mills, D

1978-06-14

Complete inhibition of sporulation was observed in two strains of Saccharomyces cerevisiae to which 8-hydroxyquinoline was added at a final concentration of 5 microgram/ml during the initial 4 to 6 h of sporulation. The cells were most sensitive to the inhibitor during 4 to 6 interval beginning at approximately 2 h (T2). Its addition during that interval resulted in 70 to 80% lethality in strain 4579 and about 40% in API at T24. When present from T0 onward, 5 microgram/ml of 8-hydroxyquinoline severely inhibited premeiotic DNA replication and reduced the frequency of intragenic recombination at the ade 2 and leu 2 loci by 70 and 100%, respectively, relative to control cultures which did not have the inhibitor present. During the period when the cells were most sensitive, the incorporation of 14C-leucine into protein and 14C-adenine into RNA was not inhibited nor was the polysome content affected. At 150 microgram/ml of inhibitor, incorporation of labeled precursors into RNA and protein were inhibited and the percentage of active ribosomes was reduced by 35% within 45 min, but neither transcription or translation appeared to be completely inhibited at this concentration of the inhibitor.

Opinion: uracil DNA glycosylase (UNG) plays distinct and non-canonical roles in somatic hypermutation and class switch recombination.

PubMed

Yousif, Ashraf S; Stanlie, Andre; Begum, Nasim A; Honjo, Tasuku

2014-10-01

Activation-induced cytidine deaminase (AID) is essential to class switch recombination (CSR) and somatic hypermutation (SHM). Uracil DNA glycosylase (UNG), a member of the base excision repair complex, is required for CSR. The role of UNG in CSR and SHM is extremely controversial. AID deficiency in mice abolishes both CSR and SHM, while UNG-deficient mice have drastically reduced CSR but augmented SHM raising a possibility of differential functions of UNG in CSR and SHM. Interestingly, UNG has been associated with a CSR-specific repair adapter protein Brd4, which interacts with acetyl histone 4, γH2AX and 53BP1 to promote non-homologous end joining during CSR. A non-canonical scaffold function of UNG, but not the catalytic activity, can be attributed to the recruitment of essential repair proteins associated with the error-free repair during SHM, and the end joining during CSR. © The Japanese Society for Immunology. 2014. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Assays for the determination of the activity of DNA nucleases based on the fluorometric properties of the YOYO dye.

PubMed

Fernández-Sierra, Mónica; Quiñones, Edwin

2015-03-15

Here we characterize the fluorescence of the YOYO dye as a tool for studying DNA-protein interactions in real time and present two continuous YOYO-based assays for sensitively monitoring the kinetics of DNA digestion by λ-exonuclease and the endonuclease EcoRV. The described assays rely on the different fluorescence intensities between single- and double-stranded DNA-YOYO complexes, allowing straightforward determination of nuclease activity and quantitative determination of reaction products. The assays were also employed to assess the effect of single-stranded DNA-binding proteins on the λ-exonuclease reaction kinetics, showing that the extreme thermostable single-stranded DNA-binding protein (ET-SSB) significantly reduced the reaction rate, while the recombination protein A (RecA) displayed no effect. Copyright © 2015 Elsevier Inc. All rights reserved.

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae.

PubMed

Saparbaev, M; Prakash, L; Prakash, S

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3 delta mutation has an effect on recombination similar to that of the rad1 delta and rad10 delta mutations. The msh2 delta mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integration of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAD1-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process.

Direct DNA binding by Brca1.

PubMed

Paull, T T; Cortez, D; Bowers, B; Elledge, S J; Gellert, M

2001-05-22

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Site-specific genetic recombination: hops, flips, and flops.

PubMed

Sadowski, P D

1993-06-01

Genetic recombination plays a key role in the life of organisms as diverse as bacteriophages and humans. Contrary to our idea that chromosomes are stable structures, studies of recombination over the past few decades have shown that in fact DNA replicons are remarkably plastic, undergoing frequent recombination-induced rearrangements. This review summarizes our recent knowledge of the biochemistry of the two major classes of site-specific recombination: 1) transpositional recombination, and 2) conservative site-specific recombination.

Regulation of Meiotic Recombination

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

Gregory p. Copenhaver

Meiotic recombination results in the heritable rearrangement of DNA, primarily through reciprocal exchange between homologous chromosome or gene conversion. In plants these events are critical for ensuring proper chromosome segregation, facilitating DNA repair and providing a basis for genetic diversity. Understanding this fundamental biological mechanism will directly facilitate trait mapping, conventional plant breeding, and development of genetic engineering techniques that will help support the responsible production and conversion of renewable resources for fuels, chemicals, and the conservation of energy (1-3). Substantial progress has been made in understanding the basal recombination machinery, much of which is conserved in organisms as diversemore » as yeast, plants and mammals (4, 5). Significantly less is known about the factors that regulate how often and where that basal machinery acts on higher eukaryotic chromosomes. One important mechanism for regulating the frequency and distribution of meiotic recombination is crossover interference - or the ability of one recombination event to influence nearby events. The MUS81 gene is thought to play an important role in regulating the influence of interference on crossing over. The immediate goals of this project are to use reverse genetics to identify mutants in two putative MUS81 homologs in the model plant Arabidopsis thaliana, characterize those mutants and initiate a novel forward genetic screen for additional regulators of meiotic recombination. The long-term goal of the project is to understand how meiotic recombination is regulated in higher eukaryotes with an emphasis on the molecular basis of crossover interference. The ability to monitor recombination in all four meiotic products (tetrad analysis) has been a powerful tool in the arsenal of yeast geneticists. Previously, the qrt mutant of Arabidopsis, which causes the four pollen products of male meiosis to remain attached, was developed as a

Heteroduplex DNA Position Defines the Roles of the Sgs1, Srs2, and Mph1 Helicases in Promoting Distinct Recombination Outcomes

PubMed Central

Mitchel, Katrina; Lehner, Kevin; Jinks-Robertson, Sue

2013-01-01

The contributions of the Sgs1, Mph1, and Srs2 DNA helicases during mitotic double-strand break (DSB) repair in yeast were investigated using a gap-repair assay. A diverged chromosomal substrate was used as a repair template for the gapped plasmid, allowing mismatch-containing heteroduplex DNA (hDNA) formed during recombination to be monitored. Overall DSB repair efficiencies and the proportions of crossovers (COs) versus noncrossovers (NCOs) were determined in wild-type and helicase-defective strains, allowing the efficiency of CO and NCO production in each background to be calculated. In addition, the products of individual NCO events were sequenced to determine the location of hDNA. Because hDNA position is expected to differ depending on whether a NCO is produced by synthesis-dependent-strand-annealing (SDSA) or through a Holliday junction (HJ)–containing intermediate, its position allows the underlying molecular mechanism to be inferred. Results demonstrate that each helicase reduces the proportion of CO recombinants, but that each does so in a fundamentally different way. Mph1 does not affect the overall efficiency of gap repair, and its loss alters the CO-NCO by promoting SDSA at the expense of HJ–containing intermediates. By contrast, Sgs1 and Srs2 are each required for efficient gap repair, strongly promoting NCO formation and having little effect on CO efficiency. hDNA analyses suggest that all three helicases promote SDSA, and that Sgs1 and Srs2 additionally dismantle HJ–containing intermediates. The hDNA data are consistent with the proposed role of Sgs1 in the dissolution of double HJs, and we propose that Srs2 dismantles nicked HJs. PMID:23516370

Sulforaphane induces DNA double strand breaks predominantly repaired by homologous recombination pathway in human cancer cells

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

Sekine-Suzuki, Emiko; Graduate School of Advanced Integration Science, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522; Yu, Dong

2008-12-12

Cytotoxicity and DNA double strand breaks (DSBs) were studied in HeLa cells treated with sulforaphane (SFN), a well-known chemo-preventive agent. Cell survival was impaired by SFN in a concentration and treatment time-dependent manner. Both constant field gel electrophoresis (CFGE) and {gamma}-H2AX assay unambiguously indicated formation of DSBs by SFN, reflecting the cell survival data. These DSBs were predominantly processed by homologous recombination repair (HRR), judging from the SFN concentration-dependent manner of Rad51 foci formation. On the other hand, the phosphorylation of DNA-PKcs, a key non-homologous end joining (NHEJ) protein, was not observed by SFN treatment, suggesting that NHEJ may notmore » be involved in DSBs induced by this chemical. G2/M arrest by SFN, a typical response for cells exposed to ionizing radiation was also observed. Our new data indicate the clear induction of DSBs by SFN and a useful anti-tumor aspect of SFN through the induction of DNA DSBs.« less

The Dynamic Mu Transpososome: MuB activation prevents disintegration

PubMed Central

Lemberg, Kathryn M.; Schweidenback, Caterina T. H.; Baker, Tania A.

2007-01-01

Summary DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration vs. disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the ‘joining’ configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and thus may provide a mechanism to antagonize formation of single-end transposition products. PMID:17988683

Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast.

PubMed

Li, Ping; Jin, Hui; Yu, Hong-Guo

2014-10-01

During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. © 2014 Li et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Asilomar moments: formative framings in recombinant DNA and solar climate engineering research.

PubMed

Schäfer, Stefan; Low, Sean

2014-12-28

We examine the claim that in governance for solar climate engineering research, and especially field tests, there is no need for external governance beyond existing mechanisms such as peer review and environmental impact assessments that aim to assess technically defined risks to the physical environment. By drawing on the historical debate on recombinant DNA research, we show that defining risks is not a technical question but a complex process of narrative formation. Governance emerges from within, and as a response to, narratives of what is at stake in a debate. In applying this finding to the case of climate engineering, we find that the emerging narrative differs starkly from the narrative that gave meaning to rDNA technology during its formative period, with important implications for governance. While the narrative of rDNA technology was closed down to narrowly focus on technical risks, that of climate engineering continues to open up and includes social, political and ethical issues. This suggests that, in order to be legitimate, governance must take into account this broad perception of what constitutes the relevant issues and risks of climate engineering, requiring governance that goes beyond existing mechanisms that focus on technical risks. Even small-scale field tests with negligible impacts on the physical environment warrant additional governance as they raise broader concerns that go beyond the immediate impacts of individual experiments. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

PubMed Central

Kim, Taejin; Chitteni-Pattu, Sindhu; Cox, Benjamin L.; Wood, Elizabeth A.; Sandler, Steven J.; Cox, Michael M.

2015-01-01

The recombination activity of Escherichia coli (E. coli) RecA protein reflects an evolutionary balance between the positive and potentially deleterious effects of recombination. We have perturbed that balance, generating RecA variants exhibiting improved recombination functionality via random mutagenesis followed by directed evolution for enhanced function in conjugation. A recA gene segment encoding a 59 residue segment of the protein (Val79-Ala137), encompassing an extensive subunit-subunit interface region, was subjected to degenerate oligonucleotide-mediated mutagenesis. An iterative selection process generated at least 18 recA gene variants capable of producing a higher yield of transconjugants. Three of the variant proteins, RecA I102L, RecA V79L and RecA E86G/C90G were characterized based on their prominence. Relative to wild type RecA, the selected RecA variants exhibited faster rates of ATP hydrolysis, more rapid displacement of SSB, decreased inhibition by the RecX regulator protein, and in general displayed a greater persistence on DNA. The enhancement in conjugational function comes at the price of a measurable RecA-mediated cellular growth deficiency. Persistent DNA binding represents a barrier to other processes of DNA metabolism in vivo. The growth deficiency is alleviated by expression of the functionally robust RecX protein from Neisseria gonorrhoeae. RecA filaments can be a barrier to processes like replication and transcription. RecA regulation by RecX protein is important in maintaining an optimal balance between recombination and other aspects of DNA metabolism. PMID:26047498

Validation of biological activity testing procedure of recombinant human interleukin-7.

PubMed

Lutsenko, T N; Kovalenko, M V; Galkin, O Yu

2017-01-01

Validation procedure for method of monitoring the biological activity of reсombinant human interleukin-7 has been developed and conducted according to the requirements of national and international recommendations. This method is based on the ability of recombinant human interleukin-7 to induce proliferation of T lymphocytes. It has been shown that to control the biological activity of recombinant human interleukin-7 peripheral blood mononuclear cells (PBMCs) derived from blood or cell lines can be used. Validation charac­teristics that should be determined depend on the method, type of product or object test/measurement and biological test systems used in research. The validation procedure for the method of control of biological activity of recombinant human interleukin-7 in peripheral blood mononuclear cells showed satisfactory results on all parameters tested such as specificity, accuracy, precision and linearity.

Recombinant HT{sub m4} gene, protein and assays

DOEpatents

Lim, B.; Adra, C.N.; Lelias, J.M.

1996-09-03

The invention relates to a recombinant DNA molecule which encodes a HT{sub m4} protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT{sub m4} protein and a recombinant HT{sub m4} protein. The invention also relates to a method for detecting the presence of a hereditary atopy. 2 figs.

Recombinant HT.sub.m4 gene, protein and assays

DOEpatents

Lim, Bing; Adra, Chaker N.; Lelias, Jean-Michel

1996-01-01

The invention relates to a recombinant DNA molecule which encodes a HT.sub.m4 protein, a transformed host cell which has been stably transfected with a DNA molecule which encodes a HT.sub.m4 protein and a recombinant HT.sub.m4 protein. The invention also relates to a method for detecting the presence of a hereditary atopy.

Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.

PubMed

Jeevan, A; McFarland, C T; Yoshimura, T; Skwor, T; Cho, H; Lasco, T; McMurray, D N

2006-01-01

Gamma interferon (IFN-gamma) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-gamma (gpIFN-gamma) and reported that BCG vaccination induced a significant increase in the IFN-gamma mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-gamma mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-gamma with a histidine tag at the N terminus (His-tagged rgpIFN-gamma) in Escherichia coli. rgpIFN-gamma was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-gamma. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-gamma was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-gamma did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-gamma induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-gamma has been successfully produced and characterized in our laboratory. The study of rgpIFN-gamma will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

Control of the yeast telomeric senescence survival pathways of recombination by the Mec1 and Mec3 DNA damage sensors and RPA

PubMed Central

Grandin, Nathalie; Charbonneau, Michel

2007-01-01

Saccharomyces cerevisiae telomerase-negative cells undergo homologous recombination on subtelomeric or TG1–3 telomeric sequences, thus allowing Type I or Type II post-senescence survival, respectively. Here, we find that the DNA damage sensors, Mec1, Mec3 and Rad24 control Type II recombination, while the Rad9 adaptor protein and the Rad53 and Chk1 effector kinases have no effect on survivor type selection. Therefore, the Mec1 and Mec3 checkpoint complexes control telomeric recombination independently of their roles in generating and amplifying the Mec1-Rad53-Chk1 kinase cascade. rfa1-t11 mutant cells, bearing a mutation in Replication Protein A (RPA) conferring a defect in recruiting Mec1-Ddc2, were also deficient in both types of telomeric recombination. Importantly, expression of an Rfa1-t11-Ddc2 hybrid fusion protein restored checkpoint-dependent arrest, but did not rescue defective telomeric recombination. Therefore, the Rfa1-t11-associated defect in telomeric recombination is not solely due to its failure to recruit Mec1. We have also isolated novel alleles of RFA1 that were deficient in Type I but not in Type II recombination and proficient in checkpoint control. Therefore, the checkpoint and recombination functions of RPA can be genetically separated, as can the RPA-mediated control of the two types of telomeric recombination. PMID:17202155

The mismatch repair and meiotic recombination endonuclease Mlh1-Mlh3 is activated by polymer formation and can cleave DNA substrates in trans.

PubMed

Manhart, Carol M; Ni, Xiaodan; White, Martin A; Ortega, Joaquin; Surtees, Jennifer A; Alani, Eric

2017-04-01

Crossing over between homologs is initiated in meiotic prophase by the formation of DNA double-strand breaks that occur throughout the genome. In the major interference-responsive crossover pathway in baker's yeast, these breaks are resected to form 3' single-strand tails that participate in a homology search, ultimately forming double Holliday junctions (dHJs) that primarily include both homologs. These dHJs are resolved by endonuclease activity to form exclusively crossovers, which are critical for proper homolog segregation in Meiosis I. Recent genetic, biochemical, and molecular studies in yeast are consistent with the hypothesis of Mlh1-Mlh3 DNA mismatch repair complex acting as the major endonuclease activity that resolves dHJs into crossovers. However, the mechanism by which the Mlh1-Mlh3 endonuclease is activated is unknown. Here, we provide evidence that Mlh1-Mlh3 does not behave like a structure-specific endonuclease but forms polymers required to generate nicks in DNA. This conclusion is supported by DNA binding studies performed with different-sized substrates that contain or lack polymerization barriers and endonuclease assays performed with varying ratios of endonuclease-deficient and endonuclease-proficient Mlh1-Mlh3. In addition, Mlh1-Mlh3 can generate religatable double-strand breaks and form an active nucleoprotein complex that can nick DNA substrates in trans. Together these observations argue that Mlh1-Mlh3 may not act like a canonical, RuvC-like Holliday junction resolvase and support a novel model in which Mlh1-Mlh3 is loaded onto DNA to form an activated polymer that cleaves DNA.

Complexity of genetic mechanisms conferring nonuniformity of recombination in maize.

PubMed

Pan, Qingchun; Deng, Min; Yan, Jianbing; Li, Lin

2017-04-26

Recombinations occur nonuniformly across the maize genome. To dissect the genetic mechanisms underlying the nonuniformity of recombination, we performed quantitative trait locus (QTL) mapping using recombinant inbred line populations. Genome-wide QTL scan identified hundreds of QTLs with both cis-prone and trans- effects for recombination number variation. To provide detailed insights into cis- factors associated with recombination variation, we examined the genomic features around recombination hot regions, including density of genes, DNA transposons, retrotransposons, and some specific motifs. Compared to recombination variation in whole genome, more QTLs were mapped for variations in recombination hot regions. The majority QTLs for recombination hot regions are trans-QTLs and co-localized with genes from the recombination pathway. We also found that recombination variation was positively associated with the presence of genes and DNA transposons, but negatively related to the presence of long terminal repeat retrotransposons. Additionally, 41 recombination hot regions were fine-mapped. The high-resolution genotyping of five randomly selected regions in two F 2 populations verified that they indeed have ultra-high recombination frequency, which is even higher than that of the well-known recombination hot regions sh1-bz and a1-sh2. Taken together, our results further our understanding of recombination variation in plants.

Biotechnology and genetic engineering in the new drug development. Part I. DNA technology and recombinant proteins.

PubMed

Stryjewska, Agnieszka; Kiepura, Katarzyna; Librowski, Tadeusz; Lochyński, Stanisław

2013-01-01

Pharmaceutical biotechnology has a long tradition and is rooted in the last century, first exemplified by penicillin and streptomycin as low molecular weight biosynthetic compounds. Today, pharmaceutical biotechnology still has its fundamentals in fermentation and bioprocessing, but the paradigmatic change affected by biotechnology and pharmaceutical sciences has led to an updated definition. The biotechnology revolution redrew the research, development, production and even marketing processes of drugs. Powerful new instruments and biotechnology related scientific disciplines (genomics, proteomics) make it possible to examine and exploit the behavior of proteins and molecules. Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. This technology, now approximately 25 years old, is becoming one of the most important technologies developed in the 20(th) century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances regarding rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we describe these processes.

Recombinational Cloning Using Gateway and In-Fusion Cloning Schemes

PubMed Central

Throop, Andrea L.; LaBaer, Joshua

2015-01-01

The comprehensive study of protein structure and function, or proteomics, depends on the obtainability of full-length cDNAs in species-specific expression vectors and subsequent functional analysis of the expressed protein. Recombinational cloning is a universal cloning technique based on site-specific recombination that is independent of the insert DNA sequence of interest, which differentiates this method from the classical restriction enzyme-based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression-ready clones using the most popular recombinational cloning technologies, including the commercially available Gateway® (Life Technologies) and In-Fusion® (Clontech) cloning technologies. PMID:25827088

Genomic correlates of recombination rate and its variability across eight recombination maps in the western honey bee (Apis mellifera L.).

PubMed

Ross, Caitlin R; DeFelice, Dominick S; Hunt, Greg J; Ihle, Kate E; Amdam, Gro V; Rueppell, Olav

2015-02-21

Meiotic recombination has traditionally been explained based on the structural requirement to stabilize homologous chromosome pairs to ensure their proper meiotic segregation. Competing hypotheses seek to explain the emerging findings of significant heterogeneity in recombination rates within and between genomes, but intraspecific comparisons of genome-wide recombination patterns are rare. The honey bee (Apis mellifera) exhibits the highest rate of genomic recombination among multicellular animals with about five cross-over events per chromatid. Here, we present a comparative analysis of recombination rates across eight genetic linkage maps of the honey bee genome to investigate which genomic sequence features are correlated with recombination rate and with its variation across the eight data sets, ranging in average marker spacing ranging from 1 Mbp to 120 kbp. Overall, we found that GC content explained best the variation in local recombination rate along chromosomes at the analyzed 100 kbp scale. In contrast, variation among the different maps was correlated to the abundance of microsatellites and several specific tri- and tetra-nucleotides. The combined evidence from eight medium-scale recombination maps of the honey bee genome suggests that recombination rate variation in this highly recombining genome might be due to the DNA configuration instead of distinct sequence motifs. However, more fine-scale analyses are needed. The empirical basis of eight differing genetic maps allowed for robust conclusions about the correlates of the local recombination rates and enabled the study of the relation between DNA features and variability in local recombination rates, which is particularly relevant in the honey bee genome with its exceptionally high recombination rate.

Requirement of mismatch repair genes MSH2 and MSH3 in the RAD1-RAD10 pathway of mitotic recombination in Saccharomyces cerevisiae

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

Saparbaev, M.; Prakash, L.; Prakash, S.

1996-03-01

The RAD1 and RAD10 genes of Saccharomyces cerevisiae are required for nucleotide excision repair and they also act in mitotic recombination. The Rad1-Rad10 complex has a single-stranded DNA endonuclease activity. Here, we show that the mismatch repair genes MSH2 and MSH3 function in mitotic recombination. For both his3 and his4 duplications, and for homologous integration of a linear DNA fragment into the genome, the msh3-A mutation has an effect on recombination similar to that of the rad1{Delta} and rad10{Delta} mutations. The msh2{Delta} mutation also reduces the rate of recombination of the his3 duplication and lowers the incidence of homologous integrationmore » of a linear DNA fragment. Epistasis analyses indicate that MSH2 and MSH3 function in the RAD1-RAD10 recombination pathway, and studies presented here suggest an involvement of the RAM-RAD10 pathway in reciprocal recombination. The possible roles of Msh2, Msh3, Rad1, and Rad10 proteins in genetic recombination are discussed. Coupling of mismatch binding proteins with the recombinational machinery could be important for ensuring genetic fidelity in the recombination process. 59 refs., 2 figs., 7 tabs.« less

RecA-Dependent DNA Repair Results in Increased Heteroplasmy of the Arabidopsis Mitochondrial Genome1[C][W

PubMed Central

Miller-Messmer, Marie; Kühn, Kristina; Bichara, Marc; Le Ret, Monique; Imbault, Patrice; Gualberto, José M.

2012-01-01

Plant mitochondria have very active DNA recombination activities that are responsible for its plastic structures and that should be involved in the repair of double-strand breaks in the mitochondrial genome. Little is still known on plant mitochondrial DNA repair, but repair by recombination is believed to be a major determinant in the rapid evolution of plant mitochondrial genomes. In flowering plants, mitochondria possess at least two eubacteria-type RecA proteins that should be core components of the mitochondrial repair mechanisms. We have performed functional analyses of the two Arabidopsis (Arabidopsis thaliana) mitochondrial RecAs (RECA2 and RECA3) to assess their potential roles in recombination-dependent repair. Heterologous expression in Escherichia coli revealed that RECA2 and RECA3 have overlapping as well as specific activities that allow them to partially complement bacterial repair pathways. RECA2 and RECA3 have similar patterns of expression, and mutants of either display the same molecular phenotypes of increased recombination between intermediate-size repeats, thus suggesting that they act in the same recombination pathways. However, RECA2 is essential past the seedling stage and should have additional important functions. Treatment of plants with several DNA-damaging drugs further showed that RECA3 is required for different recombination-dependent repair pathways that significantly contribute to plant fitness under stress. Replication repair of double-strand breaks results in the accumulation of crossovers that increase the heteroplasmic state of the mitochondrial DNA. It was shown that these are transmitted to the plant progeny, enhancing the potential for mitochondrial genome evolution. PMID:22415515