Final Report Summary - REPSUBREP (Regulatory mechanisms coordinating replication integrity and intra-S repair)
DNA lesions compromise DNA synthesis, inducing fork stalling and discontinuities in the replicated chromosomes. This problem is largely prevented by the activation of DNA damage tolerance (DDT) mechanisms, which ensure the complete duplication of the genome by facilitating gap-filling and replication fork recovery. All organisms are endowed with two main modes of DDT. One mode is in principle error-free and involves a switch of templates (template switching, TS), from the damaged one to a homologous template, usually the sister chromatid. However, faulty TS may also arise during replication, leading to complex genomic rearrangements. The other DDT mode is error-prone and uses trans-lesion synthesis polymerases, capable of replicating across DNA lesions, but also introducing mutations. While the DDT mode has substantial implications for genome stability, the regulatory mechanisms underpinning the selection/usage of recombination-mediated DDT early during replication, as well as the mechanism and factors mediating this process remain still poorly understood. Investigating the molecular mechanism of TS and how it is regulated in the context of DDT and genomic replication represented the specific focus of the REPSUBREP project.
We previously found that TS is manifested via the formation of recombination-dependent sister chromatid junctions (SCJs). However, two genetically distinct recombination-mediated damage-bypass modes proceed via SCJ formation: TS – which involves the joint action between homologous recombination (HR) and error-free postreplicative repair (PRR) proteins such as Rad18, Rad5, Mms2-Ubc13 – and a salvage recombination pathway that still involves HR activities, but operates independently of error-free PRR. In the framework of the REPSUBREP project, we found that TS and the salvage pathway of recombination are differentially regulated in relation to the timing of DNA replication, with TS being the preferred pathway early during replication. We uncovered that genome architectural transitions coupled with early stages of replication and involving the molecular DNA bender, Hmo1/HMGB, set the stage for the error-free DDT pathway and prevent mutagenic DDT. Based on genetic, biochemical and structural features of TS intermediates that we identified in the course of the REPSUBREP project, we showed that error-free TS occurs predominantly behind replication forks at DNA gaps left behind moving replication forks. In conditions of limited re-priming, we found evidence for abnormal replication fork architecture and faulty annealing events that are the underlying cause for aberrant recombination and mutagenic DDT, and coincidently cause chromatin structural defects. The findings related to the work on TS also allowed us to formulate a recombination model accounting for mitotic recombination triggered by single stranded DNA or DNA gaps, thought to be the main driver of mitotic recombination. We also identified that structures resembling double Holliday Junctions (dHJs), proposed also as intermediates for double strand break repair, but so far only visualized in meiosis, are major intermediates of TS. The TS intermediates are generally dissolved by the RecQ helicase, Sgs1, whose ortholog in human cells, BLM, is mutated in the cancer-prone Bloom syndrome, in cooperation with Top3. In this process, the highly conserved Structural maintenance of chromosomes (SMC) complex Smc5/6-mediated SUMOylation and Esc2 participate to facilitate Sgs1-Top3-mediated dissolution and to set the chromatin stage by promoting chromatin association or turnover of specific repair factors. We found that alternate resolution of TS intermediates by endonucleases such as Mus81-Mms4 can lead to deleterious sister chromatid exchanges, a phenotype abnormally increased in sgs1/BLM or damage checkpoint mutants. We showed that the temporal regulation of Mus81-Mms4 nuclease – orchestrated by replication/damage checkpoint and CDK kinases, is crucial for genome integrity.
We previously found that TS is manifested via the formation of recombination-dependent sister chromatid junctions (SCJs). However, two genetically distinct recombination-mediated damage-bypass modes proceed via SCJ formation: TS – which involves the joint action between homologous recombination (HR) and error-free postreplicative repair (PRR) proteins such as Rad18, Rad5, Mms2-Ubc13 – and a salvage recombination pathway that still involves HR activities, but operates independently of error-free PRR. In the framework of the REPSUBREP project, we found that TS and the salvage pathway of recombination are differentially regulated in relation to the timing of DNA replication, with TS being the preferred pathway early during replication. We uncovered that genome architectural transitions coupled with early stages of replication and involving the molecular DNA bender, Hmo1/HMGB, set the stage for the error-free DDT pathway and prevent mutagenic DDT. Based on genetic, biochemical and structural features of TS intermediates that we identified in the course of the REPSUBREP project, we showed that error-free TS occurs predominantly behind replication forks at DNA gaps left behind moving replication forks. In conditions of limited re-priming, we found evidence for abnormal replication fork architecture and faulty annealing events that are the underlying cause for aberrant recombination and mutagenic DDT, and coincidently cause chromatin structural defects. The findings related to the work on TS also allowed us to formulate a recombination model accounting for mitotic recombination triggered by single stranded DNA or DNA gaps, thought to be the main driver of mitotic recombination. We also identified that structures resembling double Holliday Junctions (dHJs), proposed also as intermediates for double strand break repair, but so far only visualized in meiosis, are major intermediates of TS. The TS intermediates are generally dissolved by the RecQ helicase, Sgs1, whose ortholog in human cells, BLM, is mutated in the cancer-prone Bloom syndrome, in cooperation with Top3. In this process, the highly conserved Structural maintenance of chromosomes (SMC) complex Smc5/6-mediated SUMOylation and Esc2 participate to facilitate Sgs1-Top3-mediated dissolution and to set the chromatin stage by promoting chromatin association or turnover of specific repair factors. We found that alternate resolution of TS intermediates by endonucleases such as Mus81-Mms4 can lead to deleterious sister chromatid exchanges, a phenotype abnormally increased in sgs1/BLM or damage checkpoint mutants. We showed that the temporal regulation of Mus81-Mms4 nuclease – orchestrated by replication/damage checkpoint and CDK kinases, is crucial for genome integrity.