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author:("freon, marine")
1.  The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1 
PLoS Biology  2014;12(10):e1001968.
A molecular switch for times of replication stress - Chromatin Assembly Factor 1 helps to protect DNA during recombination-mediated template-switching, favoring the rescue of stalled replication forks by both beneficial and detrimental homologous recombination events.
At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.
Author Summary
Obstacles to the progression of DNA replication forks can result in genome rearrangements that are often observed in cancer cells and genomic disorders. Homologous recombination is a mechanism of restarting stalled replication fork that involves synthesis of the new DNA strands switching templates to a second (allelic) copy of the DNA sequence. However, the new strands can also occasionally recombine with nonallelic repeats (distinct regions of the genome that resemble the correct one) and thereby cause the inappropriate fusion of normally distant DNA segments; this is known as faulty template switching. The chromatin assembly factor 1 (CAF-1) is already known to be involved in depositing nucleosomes on DNA during DNA replication and repair. We have found that CAF-1 is also involved in the recombination-mediated template switch pathway in response to replication stress. Using both genetic and physical assays that allow the different steps of template switch to be analyzed, we reveal that CAF-1 protects recombination intermediates from disassembly by the RecQ-type helicase Rqh1, the homologue of BLM (people with mutations that affect BLM have Bloom's syndrome, an inherited predisposition to genome instability and cancer). Consequently, the likelihood of faulty template switch is controlled by the antagonistic activities of CAF-1 and Rqh1. We thus identified an evolutionarily conserved interplay between CAF-1 and RecQ-type helicases that helps to maintain genome stability in the face of replication stress.
doi:10.1371/journal.pbio.1001968
PMCID: PMC4196752  PMID: 25313826
2.  Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone 
PLoS Genetics  2012;8(10):e1002976.
Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.
Author Summary
The appropriate transmission of genetic material during successive cell divisions requires the accurate duplication and segregation of parental DNA. The semi-conservative replication of chromosomes during S-phase is highly accurate and prevents accumulation of deleterious mutations. However, during each round of duplication, there are many impediments to the replication fork machinery that may hinder faithful chromosome duplication. Homologous recombination is a universal mechanism involved in the rescue of replication forks by rebuilding a replication apparatus at the fork (by mechanisms that are not yet understood). However, recombination can jeopardize genome stability because it allows genetic exchanges between homologous repeated sequences dispersed through the genome. In this study, we employ a fission yeast-based arrest of a single replication fork to investigate the consequences of replication fork arrest for genome stability. We report that a single blocked fork favours genomic deletions, translocations, and mutations; and this instability occurs during fork recovery by recombination. We also report that a single arrested fork that resumes its progression by recombination is prone to causing replication slippage mediated by micro-homology. We propose that deletions/duplications observed in human cancer cells suffering from replication stress can be viewed as scars left by error-prone replication forks restarted by recombination.
doi:10.1371/journal.pgen.1002976
PMCID: PMC3475662  PMID: 23093942
3.  A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability 
Molecular and Cellular Biology  2004;24(12):5130-5143.
The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.
doi:10.1128/MCB.24.12.5130-5143.2004
PMCID: PMC419856  PMID: 15169880

Results 1-3 (3)