The goal of this application is to understand how DNA replication stress contributes to DNA rearrangements involved in cancer initiation and progression using mouse B lymphocytes as a model system. Lymphocytes are particularly prone to replication damage as they undergo massive bursts of proliferation throughout development and in response to antigen stimulation, initiating the programmed DNA mutation and rearrangement processes of somatic hypermutation (SH) and class switch recombination (CSR). While the enzymes responsible for generating the DNA lesions initiating programmed DNA rearrangements have been shown to play an important role in oncogenic translocation, little is known how replication stress contributes to this process. To identify novel fragile loci arising from replication stress, we mapped sites of DNA damage occurring in early S phase using the drug hydroxyurea (HU). By monitoring the coordinated recruitment of the single strand binding protein RPA, the DNA damage response marker g-H2AX, and the homologous recombination (HR) proteins Brca1 and Smc5 using chromatin precipitation followed by massive parallel sequencing (ChIP-Seq) in response to HU, we identified preferred genomic loci designated as early replicating fragile sites (ERFSs) that are susceptible to premature fork collapse. We confirmed that these sites are indeed hypersensitive to replication-associated DNA stress, exhibiting increased DNA breaks and rearrangements in response to either inhibition of the checkpoint kinase ATR or c-Myc oncogene overexpression. Importantly, the ERFS we identified are involved in translocation events observed in human lymphomas. Further characterization showed that ERFSs are not distributed randomly throughout the genome, but associate preferentially with transcriptionally active clusters. These results have led to the hypothesis that mis- regulation of replication initiation or transcriptional activity induces genomc instability by altering the frequency and location of replication initiation sites. To test this hypothesis, I propose to study the molecular factors that predispose specific genomic loci to replication stress in two ways: 1) examine molecular factors contributing to transcription-coupled replication stress, and 2) characterize how oncogene overexpression induces replication-mediated DNA damage. Repair of replication damage can result in insertions, deletions and complex rearrangement events, reminiscent to those observed in cancer. Furthermore, replication damage may also produce secondary mutations in cancer cells, inducing mutations that promote adaptation and chemoresistance. These studies will enhance our fundamental understanding on how replication-induced DNA damage contributes to tumor development and further rearrangement and evolution of the cancer genome.
DNA damage arises from both exogenous and endogenous sources, and DNA replication is a potent source of endogenous DNA damage. The inability to repair DNA damage dramatically increases the risk for developing cancer. By understanding how DNA replication induces DNA damage we will improve our ability to prevent, diagnose and treat cancer.
