The role of chromatin remodeling factors in DNA double strand break repair

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7. PROJECT SUMMARY/ABSTRACT Accurate DNA double strand break (DSB) repair is important to maintain chromosomal stability. In the absence of DSB repair, translocations, deletions, duplications and inversions of chromosomal DNA may occur that can drive tumorigenesis. Homologous recombination (HR) is the major mechanism for DSB repair and is highly conserved across eukaryotes from yeast to human. HR is generally error-free because it copies any missing information from an undamaged homologue or sister chromosome template. However, not all HR is error-free. A number of conditions, that includes replication stress from spontaneous, chemical or physical DNA damage, can activate error-prone recombination pathways that leads to intra-chromosomal deletions (ICDs) as well as other chromosome rearrangements. The central HR gene RAD52 participates in both error-free and error-prone repair. How RAD52 choses between these opposing pathways is poorly understood. It is our central hypothesis that chromatin remodeling factors are responsible for guiding this choice. Previous work utilizing a two-hybrid screen in the fission yeast Schizosaccharomyces pombe identified a physical interaction between RAD52 with the histone acetyltransferase KAT5 and the histone chaperone HIRA. KAT5 (also known as TIP60 in humans; Mst1 in yeast) is required to target chromatin for remodeling as well as DNA damage checkpoint activation. HIRA (Hip1 in yeast) functions in establishing centromeric chromatin, targeted transcriptional repression and replication-independent histone assembly. Our preliminary data demonstrate that KAT5 channels DSBs toward error-free repair while HIRA directs DSBs toward error-prone repair that results in ICDs. The goal of this proposal is to understand how the physical interactions and chromatin remodeling properties of KAT5 and HIRA impact the decision by RAD52 to function in error-free versus error-prone repair. We propose two Specific Aims: In Aim 1 we will perform comprehensive protein modeling to predict specific residues involved in the interactions between KAT5, HIRA and RAD52. These residues will be altered and examined for repair pathway choice using well-defined chromosomal recombination assays. In Aim 2 we will examine the effect of KAT5, HIRA and RAD52 mutations found in human tumors for their ability to influence error-free and/or error-prone repair pathway choice. These studies will determine the role of chromatin remodeling in accurate DNA damage repair that is required to prevent genetic alterations that leads to cancer.