Homologous recombination (HR) plays an essential role in maintaining stability of genetic information, and even a minor deficiency in HR leads to severe diseases including cancer. Recombination repairs DNA double- strand breaks (DSBs) that occur spontaneously, or are induced by chemicals or irradiation such as used in cancer therapy. Nearly all we know about recombination processes comes from studies of two-ended double strand breaks (DSBs) induced by endonucleases (e.g. I-SceI, HO). However, it is well established that spontaneous chromosomal breaks are predominantly single-ended DSBs (seDSBs), as they arise during DNA replication when a replication fork runs into a nick. In bacteria that contain a single replication origin per genome, broken forks are repaired by Pri proteins capable of reloading the replisome at any genomic location. However, Pri proteins are not conserved, and the mechanism of broken fork repair in eukaryotes remains undefined. Our long-term goal is to understand the molecular mechanisms and regulation of DSB repair including broken replication fork repair, and to understand how deficiencies in these processes affect genomic instability. The objective of this project is to define the mechanistic features of Broken Fork Repair (BFR), which is the most common, yet poorly understood, type of DSB repair. We propose that eukaryotes repair broken replication forks using a combination of the structure-specific nuclease Mus81/Mms4 and a converging fork initiated at the next active or damage-activated origin. We further propose that this mechanism restricts the usage of highly mutagenic DNA synthesis via the well-characterized Break Induced Replication (BIR) process. The central question is whether eukaryotes are able to reestablish replication forks at the site of fork breakage as demonstrated in bacteria. What are the genetic requirements for broken fork repair and how do they differ from mutagenic BIR? What is the fate of replisome proteins at broken forks? These questions will be addressed in the yeast model organism Saccharomyces cerevisiae, where all replication origins are annotated and Flp recombinase-induced broken fork assays are available. We will define whether functional forks can be reestablished and whether dormant origins are activated in the vicinity of the broken fork using the hydrolytic end sequencing (HydEn-seq) method. The stability of the replisome after fork breakage will be studied using chromatin immunoprecipitation. We will also study the role and regulation of structure-specific nucleases in the repair of broken forks. The most common types of genomic rearrangements that occur during BFR and BIR stem from template switches and half crossovers. We will identify the genetic requirements for these events. At the conclusion of this project we expect to: (i) provide new molecular tools to study BFR, (ii) delineate the major mechanism of BFR, and (iii) uncover mechanisms that prevent mutagenic BIR, which is believed to account for a significant fraction of genomic rearrangements associated with human disease. Our work strives to define conserved pathways for the maintenance of chromosome integrity and has strong relevance to human health.
This proposal aims to understand the molecular mechanisms and regulation of DNA recombination, an essential DNA repair pathway preventing genome instability and cancer. Both the mechanism and proteins mediating DNA repair processes are conserved in evolution, so our proposed research in the model organism is highly relevant to our understanding of the DNA repair processes in humans.
Showing the most recent 10 out of 29 publications
