Role of Chromatin and ATP-dependent Remodeling on DNA DSB Processing

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DESCRIPTION (provided by applicant): Role of Chromatin and ATP-dependent Remodeling on DNA Double-Strand Break Processing The repair of DNA double strand breaks (DSBs) is known to be a critical process in the maintenance of genomic integrity. DSBs can arise from both environmental agents (ionizing radiation, mutagenic chemicals, etc.) and/or endogenous sources (oxygen radicals, collapsed DNA replication forks, or programmed cellular processes such as meiosis). Defects in this repair pathway can lead to mutations such as gene translocations and gross chromosomal rearrangements which can cause cancer. While recent studies have shed light on some of the processes of DSB repair, the exact mechanism has yet to be elucidated. Furthermore, despite characterization of processing enzymes involved in DSB repair, the role of chromatin in this process is currently ill-defined. One prevalent theory suggests that the loss of histones and/or nucleosomes is required for the processing of DSBs. In support of this theory, previous studies have indicated that recruitment of Ino80, a chromatin remodeling complex, is required for proper DSB repair. Ino80 recruitment is dependent on histone H2AX phosphorylation and is believed to facilitate DSB repair through possible alteration of the structure of chromatin adjacent to breaks. Our preliminary results indicate that Ino80 recruitment itself may be dependent on DSB processing. This project will examine the genetic requirements to Ino80 recruitment to an induced DSB at a single specific locus. This analysis will be performed in the presence and absence of key processing enzymes to more clearly delineate the requirements for Ino80 recruitment. To determine the effect of chromatin on DSB repair, an in vitro system using purified DSB processing enzymes will be employed. This recently-developed biochemical assay allows for revelation of the minimum enzymatic components necessary for resection of DNA in vitro. While previous assays achieved successful resection with purified processing enzymes, only naked DNA was used as a substrate. This newly developed resection assay will be utilized to examine nucleosomal templates, which are more reflective of the environment of genomic DNA in vivo. This investigation is designed to shed light on the genetic and molecular mechanism of DNA DSB repair, specifically the role of chromatin and chromatin remodeling enzymes. Specifically it will ask: What is the effect of nucleosomes on DSB resection and what enzymatic processes are required to overcome this barrier? By identifying the enzymatic processes and order of recruitment required to overcome the barrier of chromatin, the understanding of DSB repair will be furthered. This characterization of chromatin and ATP- dependent remodelers during DSB repair will allow expedition of the development of pharmacologic agents for the treatment of cancer by possibly targeting chromatin remodeling at DSBs to promote repair through homologous recombination. PUBLIC HEALTH RELEVANCE: This proposal describes research that is focused on how chromosome structure affects repair of DNA breaks, and how the normal cellular machinery affects this structure, influencing DNA repair. Specifically, we propose studies on chromatin during DNA resection which is required for homologous recombination. Repair of DNA through homologous recombination is one of the critical pathways in the maintenance of genomic integrity which has been shown to influence several pathological human disorders such as age related diseases and certain types of cancer.