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This article is from Frontiers in Microbiology , volume 5 . Abstract Faster-cycling PCR formulations, protocols, and instruments have been developed to address the need for increased throughput and shorter turn-around times for PCR-based assays. Although run times can be cut by up to 50%, shorter cycle times have been correlated with lower detection sensitivity and increased variability. To address these concerns, we applied Compartmentalized Self Replication (CSR) to evolve faster-cycling mutants of Taq DNA polymerase. After five rounds of selection using progressively shorter PCR extension times, individual mutations identified in the fastest-cycling clones were randomly combined using ligation-based multi-site mutagenesis. The best-performing combinatorial mutants exhibit 35- to 90-fold higher affinity (lower Kd) for primed template and a moderate (2-fold) increase in extension rate compared to wild-type Taq. Further characterization revealed that CSR-selected mutations provide increased resistance to inhibitors, and most notably, enable direct amplification from up to 65% whole blood. We discuss the contribution of individual mutations to fast-cycling and blood-resistant phenotypes.

This article is from PLoS ONE , volume 9 . Abstract The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs.

This article is from PLoS ONE , volume 9 . Abstract The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs.

Germ line mutations in BRCA1 confer elevated risks in the development of familial breast and ovarian cancers (1) (2). BRCA1 encodes a 1863-amino acid protein with a highly conserved RING finger domain at the amino terminus and two BRCT repeats at the extreme carboxyl terminus. While most disease-associated mutations of BRCA1 are predicted to result in gross function of the protein, 5-10% of the cancer-predisposing mutations cause single amino acid substitutions (3), many of which are located in the RING domain or BRCT repeats. It is generally assumed that both types of mutations lead to loss of the biological functions of the protein, however, several genotype-phenotype correlation studies suggest that BRCA1 mutations at different locations of the gene may confer different BRCA1-dependent cancer risks.

Chromosomal rearrangements and changes in copy number at various genomic loci are hallmarks of cancer cells and may be very early steps in tumorigenesis. The origins of genomic insults are poorly understood and this proposal aims to characterize one potential source of genomic instability inappropriate DNA re-replication. In a normal eukaryotic cell cycle the chromosomal DNA of a cell is replicated once and only once during S phase to ensure that each daughter cell receives exactly one complement of genomic material. By perturbing the regulation of several proteins involved in replication initiation our laboratory has been able to conditionally induce varying amounts of re- replication in yeast cells. We have demonstrated that re-replication induces a rapid and significant decrease in cell viability and a cellular DNA damage response. We elected to focus our studies of genome instability on gene amplification because of its clinical importance in breast cancer. We have shown that re-replication is a potent inducer of gene amplification that generates structures similar to amplicons seen but poorly understood in tumors. The high frequency at which these amplification structures are generated is specific to re-replication as similar structures are not observed when S phase DNA replication is impaired or DNA is directly damaged. We thus propose that re- replication arising from loss of replication control is a potential source of the genomic instability important for tumorigenesis.

This article is from Nucleic Acids Research , volume 40 . Abstract The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2′-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using ‘click’ chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.

This article is from Frontiers in Microbiology , volume 5 . Abstract DNA replication initiation, which starts at specific chromosomal site (known as replication origins), is the key regulatory stage of chromosome replication. Archaea, the third domain of life, use a single or multiple origin(s) to initiate replication of their circular chromosomes. The basic structure of replication origins is conserved among archaea, typically including an AT-rich unwinding region flanked by several conserved repeats (origin recognition box, ORB) that are located adjacent to a replication initiator gene. Both the ORB sequence and the adjacent initiator gene are considerably diverse among different replication origins, while in silico and genetic analyses have indicated the specificity between the initiator genes and their cognate origins. These replicator–initiator pairings are reminiscent of the oriC-dnaA system in bacteria, and a model for the negative regulation of origin activity by a downstream cluster of ORB elements has been recently proposed in haloarchaea. Moreover, comparative genomic analyses have revealed that the mosaics of replicator-initiator pairings in archaeal chromosomes originated from the integration of extrachromosomal elements. This review summarizes the research progress in understanding of archaeal replication origins with particular focus on the utilization, control and evolution of multiple replication origins in haloarchaea.

This article is from Microbial Cell Factories , volume 12 . Abstract Precise regulation of DNA replication is necessary to ensure the inheritance of genetic features by daughter cells after each cell division. Therefore, determining how the regulatory processes operate to control DNA replication is crucial to our understanding and application to biotechnological processes. Contrary to early concepts of DNA replication, it appears that this process is operated by large, stationary nucleoprotein complexes, called replication factories, rather than by single enzymes trafficking along template molecules. Recent discoveries indicated that in bacterial cells two processes, central carbon metabolism (CCM) and transcription, significantly and specifically influence the control of DNA replication of various replicons. The impact of these discoveries on our understanding of the regulation of DNA synthesis is discussed in this review. It appears that CCM may influence DNA replication by either action of specific metabolites or moonlighting activities of some enzymes involved in this metabolic pathway. The role of transcription in the control of DNA replication may arise from either topological changes in nucleic acids which accompany RNA synthesis or direct interactions between replication and transcription machineries. Due to intriguing similarities between some prokaryotic and eukaryotic regulatory systems, possible implications of studies on regulation of microbial DNA replication on understanding such a process occurring in human cells are discussed.

This article is from Genome Biology and Evolution , volume 6 . Abstract The archaeal machinery responsible for DNA replication is largely homologous to that of eukaryotes and is clearly distinct from its bacterial counterpart. Moreover, it shows high diversity in the various archaeal lineages, including different sets of components, heterogeneous taxonomic distribution, and a large number of additional copies that are sometimes highly divergent. This has made the evolutionary history of this cellular system particularly challenging to dissect. Here, we have carried out an exhaustive identification of homologs of all major replication components in over 140 complete archaeal genomes. Phylogenomic analysis allowed assigning them to either a conserved and probably essential core of replication components that were mainly vertically inherited, or to a variable and highly divergent shell of extra copies that have likely arisen from integrative elements. This suggests that replication proteins are frequently exchanged between extrachromosomal elements and cellular genomes. Our study allowed clarifying the history that shaped this key cellular process (ancestral components, horizontal gene transfers, and gene losses), providing important evolutionary and functional information. Finally, our precise identification of core components permitted to show that the phylogenetic signal carried by DNA replication is highly consistent with that harbored by two other key informational machineries (translation and transcription), strengthening the existence of a robust organismal tree for the Archaea.

This article is from Genome Biology , volume 14 . Abstract Background: Eukaryotic DNA replication follows a specific temporal program, with some genomic regions consistently replicating earlier than others, yet what determines this program is largely unknown. Highly transcribed regions have been observed to replicate in early S-phase in all plant and animal species studied to date, but this relationship is thought to be absent from both budding yeast and fission yeast. No association between cell-cycle regulated transcription and replication timing has been reported for any species. Results: Here I show that in budding yeast, fission yeast, and human, the genes most highly transcribed during S-phase replicate early, whereas those repressed in S-phase replicate late. Transcription during other cell-cycle phases shows either the opposite correlation with replication timing, or no relation. The relationship is strongest near late-firing origins of replication, which is not consistent with a previously proposed model—that replication timing may affect transcription—and instead suggests a potential mechanism involving the recruitment of limiting replication initiation factors during S-phase. Conclusions: These results suggest that S-phase transcription may be an important determinant of DNA replication timing across eukaryotes, which may explain the well-established association between transcription and replication timing.

This article is from Genome Announcements , volume 2 . Abstract Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients.

This article is from Genome Announcements , volume 2 . Abstract Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients.

This article is from Genome Announcements , volume 2 . Abstract Epidemiological data point to the involvement of a cow milk factor in the etiology of multiple sclerosis (MS). Eleven circular DNA molecules closely related to transmissible spongiform encephalopathy (TSE)-associated isolate Sphinx 1.76 were isolated from healthy cattle serum, cow milk, and serum and brain tissue from MS patients.

This article is from Scientific Reports , volume 4 . Abstract Di-(2-ethylhexyl)-phthalate (DEHP) is a ubiquitously used endocrine disruptor.There is widespread exposure to DEHP in the general population which has raised substantial public concern due to its potential detrimental health effects. It is particularly pertinent to investigate the molecular mechanisms of its testicular toxicity which are largely unknown. By feeding male rats DEHP for 2 weeks, rat spermatogenesis became disrupted, resulting in a decreased number of spermatocytes and spermatids. Since rapidly dividing tissues appeared to be particularly vulnerable to DEHP toxicity we investigated the effect of DEHP on DNA replication. Intriguingly, DEHP appeared to inhibit DNA replication as evidenced by results of fiber tract analysis. This led to induction of the mitochondrial apoptotic pathways and increased ROS production. Furthermore, the toxicity of DEHP led to respiratory chain defects and attenuation of ATP level probably brought about by hyperPARylation and undermined SIRT1 activity. Our findings reveal a previously unknown mitochondrial dysfunction in DEHP-induced testicular toxicity and highlight the importance of SIRT1 in male reproduction.

This article is from Frontiers in Microbiology , volume 5 . Abstract Three DNA polymerases of the B family function at the replication fork in eukaryotic cells: DNA polymerases α, δ, and ε. DNA polymerase α, an heterotetramer composed of two primase subunits and two polymerase subunits, initiates replication. DNA polymerases δ and ε elongate the primers generated by pol α. The DNA polymerase from bacteriophage RB69 has served as a model for eukaryotic B family polymerases for some time. The recent crystal structures of pol δ, α, and ε revealed similarities but also a number of unexpected differences between the eukaryotic polymerases and their bacteriophage counterpart, and also among the three yeast polymerases. This review will focus on their shared structural elements as well as the features that are unique to each of these polymerases.

This article is from PLoS ONE , volume 9 . Abstract We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.

This article is from PLoS ONE , volume 9 . Abstract We reported here the use of T4 bacteriophage β-glucosyltransferase (T4 β-GT) for the facile synthesis of base J-containing oligodeoxyribonucleotides (ODNs). We found that the enzyme could catalyze the glucosylation of 5-hydroxymethyl-2-deoxyuridine (5hmU) in both single- and double-stranded ODNs, though the latter reaction occurred only when 5hmU was mispaired with a guanine. In addition, base J blocked moderately DNA replication, but it did not induce mutations during replication in human cells.

This article is from Journal of Visualized Experiments : JoVE . Abstract Maintenance of replication fork stability is of utmost importance for dividing cells to preserve viability and prevent disease. The processes involved not only ensure faithful genome duplication in the face of endogenous and exogenous DNA damage but also prevent genomic instability, a recognized causative factor in tumor development.Here, we describe a simple and cost-effective fluorescence microscopy-based method to visualize DNA replication in the avian B-cell line DT40. This cell line provides a powerful tool to investigate protein function in vivo by reverse genetics in vertebrate cells1. DNA fiber fluorography in DT40 cells lacking a specific gene allows one to elucidate the function of this gene product in DNA replication and genome stability. Traditional methods to analyze replication fork dynamics in vertebrate cells rely on measuring the overall rate of DNA synthesis in a population of pulse-labeled cells. This is a quantitative approach and does not allow for qualitative analysis of parameters that influence DNA synthesis. In contrast, the rate of movement of active forks can be followed directly when using the DNA fiber technique2-4. In this approach, nascent DNA is labeled in vivo by incorporation of halogenated nucleotides (Fig 1A). Subsequently, individual fibers are stretched onto a microscope slide, and the labeled DNA replication tracts are stained with specific antibodies and visualized by fluorescence microscopy (Fig 1B). Initiation of replication as well as fork directionality is determined by the consecutive use of two differently modified analogues. Furthermore, the dual-labeling approach allows for quantitative analysis of parameters that influence DNA synthesis during the S-phase, i.e. replication structures such as ongoing and stalled forks, replication origin density as well as fork terminations. Finally, the experimental procedure can be accomplished within a day, and requires only general laboratory equipment and a fluorescence microscope.

This article is from BMC Plant Biology , volume 13 . Abstract Background: Mammalian BLM helicase is involved in DNA replication, DNA repair and homologous recombination (HR). These DNA transactions are associated tightly with cell division and are important for maintaining genome stability. However, unlike in mammals, cell division in higher plants is restricted mainly to the meristem, thus genome maintenance at the meristem is critical. The counterpart of BLM in Arabidopsis (AtRecQ4A) has been identified and its role in HR and in the response to DNA damage has been confirmed. However, the function of AtRecQ4A in the meristem during replication stress has not yet been well elucidated. Results: We isolated the BLM counterpart gene OsRecQl4 from rice and analyzed its function using a reverse genetics approach. Osrecql4 mutant plants showed hypersensitivity to DNA damaging agents and enhanced frequency of HR compared to wild-type (WT) plants. We further analyzed the effect of aphidicolin—an inhibitor of S-phase progression via its inhibitory effect on DNA polymerases—on genome stability in the root meristem in osrecql4 mutant plants and corresponding WT plants. The following effects were observed upon aphidicolin treatment: a) comet assay showed induction of DNA double-strand breaks (DSBs) in mutant plants, b) TUNEL assay showed enhanced DNA breaks at the root meristem in mutant plants, c) a recombination reporter showed enhanced HR frequency in mutant calli, d) propidium iodide (PI) staining of root tips revealed an increased incidence of cell death in the meristem of mutant plants. Conclusions: These results demonstrate that the aphidicolin-sensitive phenotype of osrecql4 mutants was in part due to induced DSBs and cell death, and that OsRecQl4 plays an important role as a caretaker, maintaining genome stability during DNA replication stress in the rice meristem.

This article is from Genes & Development , volume 28 . Abstract Recent evidence points to a role for Rif1 (Rap1-interacting factor) in DNA replication and genomic stability; however, the mechanism by which Rif1 functions has remained elusive. Hiraga el al. now report that budding yeast Rif1 controls DNA replication genome-wide and describe how Rif1 opposes Dbf4-dependent kinase (DDK) function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. PP1 interaction sites are evolutionarily conserved within the Rif1 sequence; thus, the authors propose that replication control by Rif1 through PP1 is a conserved mechanism.

DNA dependent RNA polymerase core subunits abb' conserved residues at frequencies most closely matched with codons at stage 10.4-11.1 in code evolution. An excess of acidic residues (stage 2 additions) lowered this estimate, but not by more than 2.3 stages. With 1 tryptophan (stage 14 addition) in 529 conserved residues, abb' significantly under-represented this amino acid, consistent with a cut-off in its residue profile before completion of the genetic code. Residue profiles in FEN-1 homologs and DNA topoisomerase I-5' placed their origin at stage 11-13. Prokaryote septation protein, FtsZ, arose earlier, between stage 8-11. Proteolipid in an ATP-driven proton pump served as a marker for cell membrane formation. It indicated this event took place near stage 7. Cell division, DNA replication and transcription were inferred to have originated in a protocell antecedent of the last common ancestor. Late-forming residue profiles characterized RNA dependent RNA replicase, DNA polymerase, reverse transcriptase and ribonucleotide reductase. This suggests some early processes, including RNA replication and deoxynucleotide synthesis, once depended on catalysts not found in extant residue sequences. Early formation of topoisomerase I, and enzymes that synthesize and trim RNA-DNA hybrids was viewed as evidence for a mixed duplex, with linear RNA and DNA strands, in the transition from an RNA to DNA genome.

DNA Replication

The report summarizes investigations of various enzymes in host and trypanosomes, the effects of trypanocidal drugs on enzyme systems isolated from trypanosomes, and the structure and transcription ability of purified kinetoplast DNA. Primary results and conclusions are: (1) Little homology exists between C. fasciculata purified kinetoplast and nuclear DNA; (2) The trypanocidal drugs berenil, ethidium bromide, antrycide, and suramin inhibit E. coli DNA and RNA polymerases; (3) Suramin is a potent inhibitor of E. coli RNA polymerase; (4) Isolated mitochondria have RNA polymerase activity; (5) Action spectral evidence has conclusively demonstrated the presence of cytochrome o and cytochrome aa3 in T. mega, B. culicis and L. tarentolae.

The report summarizes investigations of various enzymes in host and trypanosomes, the effects of trypanocidal drugs on enzyme systems isolated from trypanosomes, and the structure and transcription ability of purified kinetoplast DNA. Primary results and conclusions are: (1) Little homology exists between C. fasciculata purified kinetoplast and nuclear DNA; (2) The trypanocidal drugs berenil, ethidium bromide, antrycide, and suramin inhibit E. coli DNA and RNA polymerases; (3) Suramin is a potent inhibitor of E. coli RNA polymerase; (4) Isolated mitochondria have RNA polymerase activity; (5) Action spectral evidence has conclusively demonstrated the presence of cytochrome o and cytochrome aa3 in T. mega, B. culicis and L. tarentolae.

DNA and RNA translation, transcription and replication processes. Uploaded by Gerard Arthus into the Public Domain under the Creative Commons License.

At least 5 trillion cell divisions are required for a fertilized egg to develop into an adult human, resulting in the production of more than 20 trillion meters of DNA! And yet, with only two exceptions, the genome is replicated once and only once each time a cell divides. How is this feat accomplished? What happens when errors occur? This book addresses these questions by presenting a thorough analysis of the proteins and sequence of events that govern DNA replication in all eukaryotic cells and by revealing linkages between DNA replication, cell proliferation, human disease, and targeted therapeutics. For example, at least 160 different proteins are involved in replicating the human genome, and 80 genetic diseases result either from mutations in these proteins or from errors in DNA replication or repair. In addition, more than 40 diseases result from replication of DNA viruses, and at least 14 therapeutic drugs are targeted to DNA replication proteins. Not only will this book provide a rich source of information for researchers, medical doctors, and teachers, but it will also stimulate thinking about the relevance of DNA replication to human diseases. For additional information on this subject, chapters from the out of print CSHL Press monograph  DNA Replication in Eukaryotic Cells  can be downloaded  here .

At least 5 trillion cell divisions are required for a fertilized egg to develop into an adult human, resulting in the production of more than 20 trillion meters of DNA! And yet, with only two exceptions, the genome is replicated once and only once each time a cell divides. How is this feat accomplished? What happens when errors occur? This book addresses these questions by presenting a thorough analysis of the proteins and sequence of events that govern DNA replication in all eukaryotic cells and by revealing linkages between DNA replication, cell proliferation, human disease, and targeted therapeutics. For example, at least 160 different proteins are involved in replicating the human genome, and 80 genetic diseases result either from mutations in these proteins or from errors in DNA replication or repair. In addition, more than 40 diseases result from replication of DNA viruses, and at least 14 therapeutic drugs are targeted to DNA replication proteins. Not only will this book provide a rich source of information for researchers, medical doctors, and teachers, but it will also stimulate thinking about the relevance of DNA replication to human diseases. For additional information on this subject, chapters from the out of print CSHL Press monograph  DNA Replication in Eukaryotic Cells  can be downloaded  here .

Theta Model of DNA Replication

This article is from Nucleic Acids Research , volume 40 . Abstract OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted replication origin sites. The original database collated and curated Saccharomyces cerevisiae origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated origin mapping studies, and to include the collation of replication origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/.

This article is from Nucleic Acids Research , volume 40 . Abstract DNA replication initiation proteins (Reps) are subjected to degradation by cellular proteases. We investigated how the formation of nucleoprotein complex, involving Rep and a protease, affects Rep degradation. All known Escherichia coli AAA+ cytosolic proteases and the replication initiation protein TrfA of the broad-host-range plasmid RK2 were used. Our results revealed that DNA influences the degradation process and that the observed effects are opposite and protease specific. In the case of ClpXP and ClpYQ proteases, DNA abolishes proteolysis, while in the case of ClpAP and Lon proteases it stimulates the process. ClpX and ClpY cannot interact with DNA-bound TrfA, while the ClpAP and Lon activities are enhanced by the formation of nucleoprotein complexes involving both the protease and TrfA. Lon has to interact with TrfA before contacting DNA, or this interaction can occur with TrfA already bound to DNA. The TrfA degradation by Lon can be carried out only on DNA. The absence of Lon results with higher stability of TrfA in the cell.

DNA replication

Chapter 12-2 DNA Replication by MrBiology360

This article is from The Journal of Cell Biology , volume 195 . Abstract A number of different genetic backgrounds and growth conditions bypass DNA replication defects caused by the absence of yeast Hsk1 kinase, demonstrating the plasticity of the eukaryotic DNA replication program.

This article is from Nucleic Acids Research , volume 41 . Abstract The eukaryotic DNA replication initiation factor Mcm10 is essential for both replisome assembly and function. Human Mcm10 has two DNA-binding domains, the conserved internal domain (ID) and the C-terminal domain (CTD), which is specific to metazoans. SIRT1 is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that belongs to the sirtuin family. It is conserved from yeast to human and participates in cellular controls of metabolism, longevity, gene expression and genomic stability. Here we report that human Mcm10 is an acetylated protein regulated by SIRT1, which binds and deacetylates Mcm10 both in vivo and in vitro, and modulates Mcm10 stability and ability to bind DNA. Mcm10 and SIRT1 appear to act synergistically for DNA replication fork initiation. Furthermore, we show that the two DNA-binding domains of Mcm10 are modulated in distinct fashion by acetylation/deacetylation, suggesting an integrated regulation mechanism. Overall, our study highlights the importance of protein acetylation for DNA replication initiation and progression, and suggests that SIRT1 may mediate a crosstalk between cellular circuits controlling metabolism and DNA synthesis.

This article is from BMC Research Notes , volume 6 . Abstract Background: Fragile X Syndrome (FXS) and its associated disorders are caused by the expansion of the CGG repeat in the 5’ untranslated region of the fragile X mental retardation 1 (FMR1) gene, with disease classification based on the number of CGG repeats. The mechanisms of repeat expansion are dependent on the presence of cis elements and the absence of trans factors both of which are not mutually exclusive and contribute to repeat instability. Expansions associated with trans factors are due to the haploinsuffient or reduced expression of several DNA repair/metabolizing proteins. The reduction of expression in trans factors has been primarily conducted in animal models without substantial examination of many of these expansion mechanisms and trans factors in humans. Results: To understand the trans factors and pathways associated with trinucleotide repeat expansion we have analyzed two microarray datasets which characterized the transcript expression in patients with FXS and in controls. Conclusion: We observed significant down regulation of DNA damage/repair pathway transcripts. This observation was consistent in both datasets, which used different populations. Within these datasets, several transcripts overlapped in the direction of association and fold change. Further characterization of these genes will be critical to understand their role in trinucleotide repeat instability in FXS.

This article is from Cell Death & Disease , volume 5 . Abstract The disruption of DNA replication in cells triggers checkpoint responses that slow-down S-phase progression and protect replication fork integrity. These checkpoints are also determinants of cell fate and can help maintain cell viability or trigger cell death pathways. CHK1 has a pivotal role in such S-phase responses. It helps maintain fork integrity during replication stress and protects cells from several catastrophic fates including premature mitosis, premature chromosome condensation and apoptosis. Here we investigated the role of CHK1 in protecting cancer cells from premature mitosis and apoptosis. We show that premature mitosis (characterized by the induction of histone H3 phosphorylation, aberrant chromatin condensation, and persistent RPA foci in arrested S-phase cells) is induced in p53-deficient tumour cells depleted of CHK1 when DNA synthesis is disrupted. These events are accompanied by an activation of Aurora kinase B in S-phase cells that is essential for histone H3 Ser10 phosphorylation. Histone H3 phosphorylation precedes the induction of apoptosis in p53−/− tumour cell lines but does not appear to be required for this fate as an Aurora kinase inhibitor suppresses phosphorylation of both Aurora B and histone H3 but has little effect on cell death. In contrast, only a small fraction of p53+/+ tumour cells shows this premature mitotic response, although they undergo a more rapid and robust apoptotic response. Taken together, our results suggest a novel role for CHK1 in the control of Aurora B activation during DNA replication stress and support the idea that premature mitosis is a distinct cell fate triggered by the disruption of DNA replication when CHK1 function is suppressed.

This article is from PLoS ONE , volume 9 . Abstract Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24–36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

This article is from Nucleic Acids Research , volume 39 . Abstract The DNA of patients taking immunosuppressive and anti-inflammatory thiopurines contains 6-thioguanine (6-TG) and their skin is hypersensitive to ultraviolet A (UVA) radiation. DNA 6-TG absorbs UVA and generates reactive oxygen species that damage DNA and proteins. Here, we show that the DNA damage includes covalent DNA–protein crosslinks. An oligonucleotide containing a single 6-TG is photochemically crosslinked to cysteine-containing oligopeptides by low doses of UVA. Crosslinking is significantly more efficient if guanine sulphonate (GSO3)—an oxidized 6-TG and a previously identified UVA photoproduct—replaces 6-TG, suggesting that GSO3 is an important reaction intermediate. Crosslinking occurs via oligopeptide sulphydryl and free amino groups. The oligonucleotide–oligopeptide adducts are heat stable but are partially reversed by reducing treatments. UVA irradiation of human cells containing DNA 6-TG induces extensive heat- and reducing agent-resistant covalent DNA–protein crosslinks and diminishes the recovery of some DNA repair and replication proteins from nuclear extracts. DNA–protein crosslinked material has an altered buoyant density and can be purified by banding in cesium chloride (CsCl) gradients. PCNA, the MSH2 mismatch repair protein and the XPA nucleotide excision repair (NER) factor are among the proteins detectable in the DNA-crosslinked material. These findings suggest that the 6-TG/UVA combination might compromise DNA repair by sequestering essential proteins.

General Biology Lecture

This article is from Nucleic Acids Research , volume 42 . Abstract Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDNA.

Current work in our lab has shown that a synthetic structure consisting of M13 ssDNA with one annealed oligonucteotide (called 1-80) is capable of activating the checkpoint in a replication independent manner, confirming our model that primed ssDNA is the checkpoint activating signal (see above). To determine if the amount of ssDNA following a primer affects the level of Chkl phosphorylation, an oligonucleotide will be annealed to a smaller ssDNA plasmid. This structure and the original structure will be added to Xenopus egg extract in equal amounts and the levels of Chkl phosphorylation will be compared. If ssDNA following a primer-template junction amplifies the checkpoint response, then the original M13 1-80 structure will yield higher levels of Chkl phosphorylation than the smaller plasmid. We are also developing a protocol for analyzing proteins bound to ssDNA using a peptide-nucleic acid (PNA) sequence. This will allow us to determine protein binding requirements during checkpoint activation using the 1-80 structure.

BRCA1 is the major breast cancer susceptibility gene. It forms heterodimers with BARD1. Inactivation of either gene results in identical phenotypes suggesting that these proteins function primarily as a complex. BRCA1 deficiencies are associated with cellular phenotypes consistent with a DNA replication defect. We wished to test the hypothesis that BRCA1/BARD1 function during DNA replication supporting DNA transactions at replication forks. We are using cell-free extracts derived from Xenopus laevis eggs that support: 1. Semi-conservative, cell-cycle regulated DNA replication; 2. Many facets of the DNA damage response. Our key accomplishments were to generate specific antibodies against Xenopus BARD1 and BRCA1. We also demonstrate that the complex assembles to chromatin in a DNA replication-dependent manner. Finally, we show that BRCA1/BARD1 loading to chromatin does not dramatically increases following DNA damage, suggesting that it might be relocalized within chromatin compartments.

Hereditary predisposition to breast cancer is associate with mutations in the BRCA1 gene. BRCA1 functions as a tumor suppressor that is activated in response to DNA damage and the loss of normal BRCA1 activity leads to an increase in chromosome instability. BRCA1 is thought to respond to DNA damage incurred during S phase, but it is not clear precisely how BRCA1 recognizes DNA damage or transmits effector signals to delay cell cycle progression and allow efficient repair of damaged DNA. In this proposal, we present preliminary data that BRCA1 functions in a DNA checkpoint response for the origin of Epstein-Barr Virus DNA replication (Ori P). OriP replicates once and only once per cell cycle in synchrony with the cellular genome, and is therefore considered a valid model system for cellular replication origins. Importantly,, chromosomal replication proteins, including the origin recognition complex (ORC) and the licensing helicase complex (MCMs) associate with OriP. We have recently found that BRCA1 can be recruited to OriP in response to DNA damaging agents. We propose to study the mechanism of BRCA1 recruitment to OriP, its dependence on DNA- damage induced post-translational modifications, and to investigate its function at OriP in DNA replication and plasmid maintenance. We propose that these studies will provide valuable information concerning the function of OriP at replication origins and in the control of DNA replication initiation and genome stability.

Several recent studies have indicated that decreased levels of the MCM2-7 DNA replication proteins can lead to genomic instability (GIN) and cancer formation. Interestingly, genetic or RNAi-mediated depletion of one MCM has been demonstrated to cause decreases in other MCMs, presumably as a consequence of MCM heterohexamer destabilization. In the first year of my training grant, my research results show that in cells bearing only the Mcm4Chaos3 cancer susceptibility allele, the cause for reduced MCM protein levels is related to decreased Mcm2-7 and 10 mRNA. Despite being present at levels far exceeding that required for DNA replication under normal circumstances, we found that heterozygosity for 2 or more different MCMs caused genomic instability, and in the cases of MCM2, MCM6 and MCM7, synthetic lethality in conjunction with Mcm4Chaos3 homozygosity. These data suggest that proper stoichemistry of MCM components is carefully regulated, and that relatively minor disregulation or destabilization of MCM levels can have serious consequences for survival or cancer susceptibility in whole animals.

A DNA polymerase (DNAP) replicates a template DNA strand. It also exploits the template as the track for its own motor-like mechanical movement. In the polymerase mode it elongates the nascent DNA by one nucleotide in each step. But, whenever it commits an error by misincorporating an incorrect nucleotide, it can switch to an exonuclease mode. In the latter mode it excises the wrong nucleotide before switching back to its polymerase mode. We develop a stochastic kinetic model of DNA replication that mimics an {\it in-vitro} experiment where a single-stranded DNA, subjected to a mechanical tension $F$, is converted to a double-stranded DNA by a single DNAP. The $F$-dependence of the average rate of replication, which depends on the rates of both polymerase and exonuclease activities of the DNAP, is in good qualitative agreement with the corresponding experimental results. We introduce 9 novel distinct {\it conditional dwell times} of a DNAP. Using the methods of first-passage times, we also derive the exact analytical expressions for the probability distributions of these conditional dwell times. The predicted $F$-dependence of these distributions are, in principle, accessible to single-molecule experiments.

Presence of precise organelle DNA in mitochondria and chloroplasts became recognized over 3 years ago, proliferation of chloroplast DNA was first validated by the means of Chun et al, illustration of nuclear manipulation of the human mitochondrial genome chloroplast gene transcription managed transcription of cpDNA genes via the means of various factors from the nuclear basis, the number one elements affecting the transcription of cpDNA genes are NEP polymerase and non-intermediate subunits of PEP polymerase, where we explain the mechanism transporting barrel proteins from the outer mitochondrial membrane (OMM) through the TOM complex, and associated with chaperones TIM small cells within the IMS side and inserted into the OMM via sorting means and meeting equipment (SAM), we additionally annotate the chloroplast genome genes for some proteins required for the transcription and translation of encoded genes and, at the extreme, genes for photosynthesis, the locus of these repeats determines the site of unpaired reproduction the short (SSC) and extended unpaired reproductive site (LSC) in the chloroplast genome, leuco = white; plast = living) are colorless plastids that are identified in embryonic and germ cells.

MITOCHONDRIAL DNA DOUBLE STRAND BREAKS-IN REPLICATION AND IN REPAIR

This article is from Current Drug Targets , volume 13 . Abstract New antibiotics with novel modes of action are required to combat the growing threat posed by multi-drug resistant bacteria. Over the last decade, genome sequencing and other high-throughput techniques have provided tremendous insight into the molecular processes underlying cellular functions in a wide range of bacterial species. We can now use these data to assess the degree of conservation of certain aspects of bacterial physiology, to help choose the best cellular targets for development of new broad-spectrum antibacterials.DNA replication is a conserved and essential process, and the large number of proteins that interact to replicate DNA in bacteria are distinct from those in eukaryotes and archaea; yet none of the antibiotics in current clinical use acts directly on the replication machinery. Bacterial DNA synthesis thus appears to be an underexploited drug target. However, before this system can be targeted for drug design, it is important to understand which parts are conserved and which are not, as this will have implications for the spectrum of activity of any new inhibitors against bacterial species, as well as the potential for development of drug resistance. In this review we assess similarities and differences in replication components and mechanisms across the bacteria, highlight current progress towards the discovery of novel replication inhibitors, and suggest those aspects of the replication machinery that have the greatest potential as drug targets.

This article is from Nucleic Acids Research , volume 40 . Abstract Direct cellular DNA damage may lead to genome destabilization in unexposed, bystander, cells sharing the same milieu with directly damaged cells by means of the bystander effect. One proposed mechanism involves double strand break (DSB) formation in S phase cells at sites of single strand lesions in the DNA of replication complexes, which has a more open structure compared with neighboring DNA. The DNA in transcription complexes also has a more open structure, and hence may be susceptible to bystander DSB formation from single strand lesions. To examine whether transcription predisposes non-replicating cells to bystander effect-induced DNA DSBs, we examined two types of primary cells that exhibit high levels of transcription in the absence of replication, rat neurons and human lymphocytes. We found that non-replicating bystander cells with high transcription rates exhibited substantial levels of DNA DSBs, as monitored by γ-H2AX foci formation. Additionally, as reported in proliferating cells, TGF-β and NO were found to mimic bystander effects in cell populations lacking DNA synthesis. These results indicate that cell vulnerability to bystander DSB damage may result from transcription as well as replication. The findings offer insights into which tissues may be vulnerable to bystander genomic destabilization in vivo.

This article is from PLoS ONE , volume 9 . Abstract The TAR DNA binding protein (TDP-43) was originally identified as a host cell factor binding to the HIV-1 LTR and thereby suppressing HIV-1 transcription and gene expression (Ou et al., J.Virol. 1995, 69(6):3584). TDP-43 is a global regulator of transcription, can influence RNA metabolism in many different ways and is ubiquitously expressed. Thus, TDP-43 could be a major factor restricting HIV-1 replication at the level of LTR transcription and gene expression. These facts prompted us to revisit the role of TDP-43 for HIV-1 replication. We utilized established HIV-1 cell culture systems as well as primary cell models and performed a comprehensive analysis of TDP-43 function and investigated its putative impact on HIV-1 gene expression. In HIV-1 infected cells TDP-43 was neither degraded nor sequestered from the nucleus. Furthermore, TDP-43 overexpression as well as siRNA mediated knockdown did not affect HIV-1 gene expression and virus production in T cells and macrophages. In summary, our experiments argue against a restricting role of TDP-43 during HIV-1 replication in immune cells.