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RADX Promotes Genome Stability and Modulates Chemosensitivity by Regulating RAD51 at Replication Forks.
Dungrawala H, Bhat KP, Le Meur R, Chazin WJ, Ding X, Sharan SK, Wessel SR, Sathe AA, Zhao R, Cortez D
(2017) Mol Cell 67: 374-386.e5
MeSH Terms: A549 Cells, Animals, BRCA2 Protein, CRISPR-Cas Systems, DNA Breaks, Double-Stranded, DNA Repair, DNA, Neoplasm, Dose-Response Relationship, Drug, Drug Resistance, Neoplasm, Gene Expression Regulation, Neoplastic, Genomic Instability, HEK293 Cells, Humans, Mice, Models, Molecular, Mutation, Neoplasms, Poly(ADP-ribose) Polymerase Inhibitors, Protein Binding, RNA Interference, Rad51 Recombinase, Replication Origin, Transfection
Show Abstract · Added March 24, 2018
RAD51 promotes homology-directed repair (HDR), replication fork reversal, and stalled fork protection. Defects in these functions cause genomic instability and tumorigenesis but also generate hypersensitivity to cancer therapeutics. Here we describe the identification of RADX as an RPA-like, single-strand DNA binding protein. RADX is recruited to replication forks, where it prevents fork collapse by regulating RAD51. When RADX is inactivated, excessive RAD51 activity slows replication elongation and causes double-strand breaks. In cancer cells lacking BRCA2, RADX deletion restores fork protection without restoring HDR. Furthermore, RADX inactivation confers chemotherapy and PARP inhibitor resistance to cancer cells with reduced BRCA2/RAD51 pathway function. By antagonizing RAD51 at forks, RADX allows cells to maintain a high capacity for HDR while ensuring that replication functions of RAD51 are properly regulated. Thus, RADX is essential to achieve the proper balance of RAD51 activity to maintain genome stability.
Copyright © 2017 Elsevier Inc. All rights reserved.
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23 MeSH Terms
Substrate-selective repair and restart of replication forks by DNA translocases.
Bétous R, Couch FB, Mason AC, Eichman BF, Manosas M, Cortez D
(2013) Cell Rep 3: 1958-69
MeSH Terms: Animals, Baculoviridae, DNA, DNA Damage, DNA Helicases, DNA Repair, DNA Replication, HEK293 Cells, Humans, Insecta, Protein Binding, Replication Origin, Templates, Genetic
Show Abstract · Added March 5, 2014
Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.
Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.
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13 MeSH Terms
Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences.
Valenzuela MS, Chen Y, Davis S, Yang F, Walker RL, Bilke S, Lueders J, Martin MM, Aladjem MI, Massion PP, Meltzer PS
(2011) PLoS One 6: e17308
MeSH Terms: 5' Flanking Region, Base Sequence, Binding Sites, Cell Line, Chromatin, Conserved Sequence, DNA Replication, DNA, Intergenic, Evolution, Molecular, Gene Expression, Humans, Replication Origin
Show Abstract · Added March 10, 2014
BACKGROUND - Replication of mammalian genomes requires the activation of thousands of origins which are both spatially and temporally regulated by as yet unknown mechanisms. At the most fundamental level, our knowledge about the distribution pattern of origins in each of the chromosomes, among different cell types, and whether the physiological state of the cells alters this distribution is at present very limited.
METHODOLOGY/PRINCIPAL FINDINGS - We have used standard λ-exonuclease resistant nascent DNA preparations in the size range of 0.7-1.5 kb obtained from the breast cancer cell line MCF-7 hybridized to a custom tiling array containing 50-60 nt probes evenly distributed among genic and non-genic regions covering about 1% of the human genome. A similar DNA preparation was used for high-throughput DNA sequencing. Array experiments were also performed with DNA obtained from BT-474 and H520 cell lines. By determining the sites showing nascent DNA enrichment, we have localized several thousand origins of DNA replication. Our major findings are: (a) both array and DNA sequencing assay methods produced essentially the same origin distribution profile; (b) origin distribution is largely conserved (>70%) in all cell lines tested; (c) origins are enriched at the 5'ends of expressed genes and at evolutionarily conserved intergenic sequences; and (d) ChIP on chip experiments in MCF-7 showed an enrichment of H3K4Me3 and RNA Polymerase II chromatin binding sites at origins of DNA replication.
CONCLUSIONS/SIGNIFICANCE - Our results suggest that the program for origin activation is largely conserved among different cell types. Also, our work supports recent studies connecting transcription initiation with replication, and in addition suggests that evolutionarily conserved intergenic sequences have the potential to participate in origin selection. Overall, our observations suggest that replication origin selection is a stochastic process significantly dependent upon local accessibility to replication factors.
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12 MeSH Terms
Checkpoint signaling from a single DNA interstrand crosslink.
Ben-Yehoyada M, Wang LC, Kozekov ID, Rizzo CJ, Gottesman ME, Gautier J
(2009) Mol Cell 35: 704-15
MeSH Terms: Alkylating Agents, Animals, Ataxia Telangiectasia Mutated Proteins, Cell Cycle, Cell Cycle Proteins, Cell Proliferation, Checkpoint Kinase 1, DNA, DNA Damage, DNA Helicases, DNA Repair, DNA Replication, DNA-Directed DNA Polymerase, Fanconi Anemia Complementation Group A Protein, Fanconi Anemia Complementation Group D2 Protein, HeLa Cells, Humans, Nucleic Acid Conformation, Protein Kinases, Protein-Serine-Threonine Kinases, Recombinant Proteins, Replication Origin, Replication Protein A, Signal Transduction, Time Factors, Transfection, Xenopus Proteins, Xenopus laevis
Show Abstract · Added January 7, 2016
DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as mitomycin C and cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited, because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from, and repairing of a single site-specific ICL in cell-free extract derived from Xenopus eggs and in mammalian cells. Notably, we demonstrate that ICLs trigger a checkpoint response independently of origin-initiated DNA replication and uncoupling of DNA polymerase and DNA helicase. The Fanconi anemia pathway acts upstream of RPA-ATR-Chk1 to generate the ICL signal. The system also repairs ICLs in a reaction that involves extensive, error-free DNA synthesis. Repair occurs by both origin-dependent and origin-independent mechanisms. Our data suggest that cell sensitivity to crosslinking agents results from both checkpoint and DNA repair defects.
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28 MeSH Terms
New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria.
Ioannidis P, Dunning Hotopp JC, Sapountzis P, Siozios S, Tsiamis G, Bordenstein SR, Baldo L, Werren JH, Bourtzis K
(2007) BMC Genomics 8: 182
MeSH Terms: Bacterial Proteins, Base Sequence, Binding Sites, DNA Replication, DNA-Binding Proteins, Integration Host Factors, Replication Origin, Transcription Factors, Wolbachia
Show Abstract · Added February 8, 2016
BACKGROUND - The annotated genomes of two closely related strains of the intracellular bacterium Wolbachia pipientis have been reported without the identifications of the putative origin of replication (ori). Identifying the ori of these bacteria and related alpha-Proteobacteria as well as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria.
RESULTS - Using features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication (ori) regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE and COG1253 protein (CBS domain protein). Comparisons of the putative ori region among related Wolbachia strains showed higher conservation of bases within binding sites.
CONCLUSION - The sequences of the ori regions described here are only similar among closely related bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes, suggest that several of these obligate intracellular bacteria may have altered replication mechanisms. Based on these analyses, criteria are set forth for identifying the ori region in genome sequencing projects.
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9 MeSH Terms
Function of the ATR N-terminal domain revealed by an ATM/ATR chimera.
Chen X, Zhao R, Glick GG, Cortez D
(2007) Exp Cell Res 313: 1667-74
MeSH Terms: Adaptor Proteins, Signal Transducing, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Cell Line, DNA Breaks, Double-Stranded, DNA Breaks, Single-Stranded, DNA Damage, DNA, Single-Stranded, DNA-Binding Proteins, Exodeoxyribonucleases, Humans, Nuclear Proteins, Phosphoproteins, Phosphorylation, Protein Structure, Tertiary, Protein-Serine-Threonine Kinases, Recombinant Fusion Proteins, Replication Origin, Replication Protein A, Tumor Suppressor Proteins
Show Abstract · Added March 5, 2014
The ATM and ATR kinases function at the apex of checkpoint signaling pathways. These kinases share significant sequence similarity, phosphorylate many of the same substrates, and have overlapping roles in initiating cell cycle checkpoints. However, they sense DNA damage through distinct mechanisms. ATR primarily senses single stranded DNA (ssDNA) through its interaction with ATRIP, and ATM senses double strand breaks through its interaction with Nbs1. We determined that the N-terminus of ATR contains a domain that binds ATRIP. Attaching this domain to ATM allowed the fusion protein (ATM*) to bind ATRIP and associate with RPA-coated ssDNA. ATM* also gained the ability to localize efficiently to stalled replication forks as well as double strand breaks. Despite having normal kinase activity when tested in vitro and being phosphorylated on S1981 in vivo, ATM* is defective in checkpoint signaling and does not complement cellular deficiencies in either ATM or ATR. These data indicate that the N-terminus of ATR is sufficient to bind ATRIP and to promote localization to sites of replication stress.
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20 MeSH Terms
Structural mechanism of RPA loading on DNA during activation of a simple pre-replication complex.
Jiang X, Klimovich V, Arunkumar AI, Hysinger EB, Wang Y, Ott RD, Guler GD, Weiner B, Chazin WJ, Fanning E
(2006) EMBO J 25: 5516-26
MeSH Terms: Antigens, Polyomavirus Transforming, Binding Sites, DNA Replication, DNA, Single-Stranded, Humans, Magnetic Resonance Spectroscopy, Protein Interaction Mapping, Protein Structure, Quaternary, Protein Structure, Tertiary, Replication Origin, Replication Protein A, Static Electricity
Show Abstract · Added December 10, 2013
We report that during activation of the simian virus 40 (SV40) pre-replication complex, SV40 T antigen (Tag) helicase actively loads replication protein A (RPA) on emerging single-stranded DNA (ssDNA). This novel loading process requires physical interaction of Tag origin DNA-binding domain (OBD) with the RPA high-affinity ssDNA-binding domains (RPA70AB). Heteronuclear NMR chemical shift mapping revealed that Tag-OBD binds to RPA70AB at a site distal from the ssDNA-binding sites and that RPA70AB, Tag-OBD, and an 8-nucleotide ssDNA form a stable ternary complex. Intact RPA and Tag also interact stably in the presence of an 8-mer, but Tag dissociates from the complex when RPA binds to longer oligonucleotides. Together, our results imply that an allosteric change in RPA quaternary structure completes the loading reaction. A mechanistic model is proposed in which the ternary complex is a key intermediate that directly couples origin DNA unwinding to RPA loading on emerging ssDNA.
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12 MeSH Terms
CLB5-dependent activation of late replication origins in S. cerevisiae.
Donaldson AD, Raghuraman MK, Friedman KL, Cross FR, Brewer BJ, Fangman WL
(1998) Mol Cell 2: 173-82
MeSH Terms: Chromosomes, Fungal, Cyclin B, Cyclins, DNA Replication, DNA, Fungal, Fungal Proteins, Kinetics, Models, Biological, Mutation, Replication Origin, S Phase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 27, 2014
Replication origins in chromosomes are activated at specific times during the S phase. We show that the B-type cyclins are required for proper execution of this temporal program. clb5 cells activate early origins but not late origins, explaining the previously described long clb5 S phase. Origin firing appears normal in cIb6 mutants. In clb5 clb6 double mutant cells, the late origin firing defect is suppressed, accounting for the normal duration of the phase despite its delayed onset. Therefore, Clb5p promotes the timely activation of early and late origins, but Clb6p can activate only early origins. In clb5 clb6 mutants, the other B-type cyclins (Clb1-4p) promote an S phase during which both early and late replication origins fire.
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13 MeSH Terms
Replication profile of Saccharomyces cerevisiae chromosome VI.
Friedman KL, Brewer BJ, Fangman WL
(1997) Genes Cells 2: 667-78
MeSH Terms: Cell Cycle, Chromosome Mapping, Chromosomes, Fungal, DNA Replication, DNA, Fungal, Genome, Fungal, Methylation, Polymerase Chain Reaction, Replication Origin, S Phase, Saccharomyces cerevisiae, Telomere, Time Factors
Show Abstract · Added March 27, 2014
BACKGROUND - An understanding of the replication programme at the genome level will require the identification and characterization of origins of replication through large, contiguous regions of DNA. As a step toward this goal, origin efficiencies and replication times were determined for 10 ARSs spanning most of the 270 kilobase (kb) chromosome VI of Saccharomyces cerevisiae.
RESULTS - Chromosome VI shows a wide variation in the percentage of cell cycles in which different replication origins are utilized. Most of the origins are activated in only a fraction of cells, suggesting that the pattern of origin usage on chromosome VI varies greatly within the cell population. The replication times of fragments containing chromosome VI origins show a temporal pattern that has been recognized on other chromosomes--the telomeres replicate late in S phase, while the central region of the chromosome replicates early.
CONCLUSIONS - As demonstrated here for chromosome VI, analysis of the direction of replication fork movement along a chromosome and determination of replication time by measuring a period of hemimethylation may provide an efficient means of surveying origin activity over large regions of the genome.
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13 MeSH Terms
Multiple determinants controlling activation of yeast replication origins late in S phase.
Friedman KL, Diller JD, Ferguson BM, Nyland SV, Brewer BJ, Fangman WL
(1996) Genes Dev 10: 1595-607
MeSH Terms: Base Sequence, Chromosome Walking, Chromosomes, Fungal, Cloning, Molecular, DNA Replication, DNA, Fungal, Models, Genetic, Molecular Sequence Data, Plasmids, Replication Origin, Restriction Mapping, S Phase, Saccharomyces cerevisiae, Sequence Analysis, DNA
Show Abstract · Added March 27, 2014
Analysis of a 131-kb segment of the left arm of yeast chromosome XIV beginning 157 kb from the telomere reveals four highly active origins of replication that initiate replication late in S phase. Previous work has shown that telomeres act as determinants for late origin activation. However, at least two of the chromosome XIV origins maintain their late activation time when located on large circular plasmids, indicating that late replication is independent of telomeres. Analysis of the replication time of plasmid derivatives containing varying amounts of chromosome XIV DNA show that a minimum of three chromosomal elements, distinct from each tested origin, contribute to late activation time. These late determinants are functionally equivalent, because duplication of one set of contributing sequences can compensate for the removal of another set. Furthermore, insertion of an origin that is normally early activated into this domain results in a shift to late activation, suggesting that the chromosome XIV origins are not unique in their ability to respond to the late determinants.
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14 MeSH Terms