MECHANISMS OF ERROR PRONE REPAIR OF DNA BREAKS

Microhomology-mediated end joining (MMEJ) is an evolutionary conserved pathway to repair DNA doublestrand breaks (DSBs) by annealing small stretches (2-20 bps) of overlapping sequence (microhomology; MH)flanking the break site. By design, MMEJ is highly mutagenic because it always results in the deletion of oneof the MH and the inter-MH sequences. MMEJ also frequently leads to chromosomal rearrangements due toits ability to engage in promiscuous end joining. Moreover, we found that MMEJ is hypermutagenic, andaccumulates mutations flanking the repair junctions up to several kilobases from the break. Despite theserisks, MMEJ is widely adopted in cells as an alternative option to high fidelity DSB repair for a host of biologicalevents including copy number variations, immune system development, and telomere fusions. Most recently,MMEJ has also been implicated in pathogenic chromosomal translocations and is emerging as a promisingtherapeutic target in several types of cancers. Together these observations underscore the importance ofMMEJ as a genome destabilizer and warrant further studies to define genetic and biochemical mechanismsof MMEJ and its regulation under various environmental and metabolic conditions. The purpose of thisapplication is to resolve several fundamental questions on the physiological and pathological roles of MMEJcommon to all eukaryotic cells.One of the key unresolved questions in MMEJ is how cells recognize and select specific MHs for annealingand the effect of position and the number of mismatches in MH on this process. We also do not know whatare the factors catalyzing MH annealing and how cells regulate repair choice to limit unwanted MMEJ andsuppress repair-associated chromosomal instability. Lastly, we will examine why aging triggers changes inrepair pathway choice and how MMEJ activity contributes to age related chromosomal instability in yeast cells.Our preliminary results already unraveled several interesting rules and parameters in MH annealing andidentified end processing factors that are likely involved in this reaction. We also developed a novel reporterto monitor repair choice that for the first time includes MMEJ as one of the DSB repair options, and to evaluatethe roles of DNA damage response and chromatin remodelers in the decision making process. The outcomesof this investigation will reveal the underlying mechanism of key steps in MMEJ and functional interactionsbetween DSB repair pathways to sustain chromosomal integrity in response to various environmental andphysiological cues. The results will also help establish precise contributions of MMEJ in many genomemodification events and predict the frequency and types of MMEJ products at random sequences in geneediting and at-risk genome sequences.