Project Summary/Abstract Our overall goal is to gain a better understanding of mechanisms underlying the DNA damage response (DDR) and maintenance of genomic integrity. We will study the function of the CtIP tumor suppressor protein, conserved in all eukaryotes. Specifically, we will investigate the role of CtIP in DNA double-strand break (DSB) end processing, and ATR activation, and its function in the process of 5'->3' resection at a DSB. Our preliminary studies in the Xenopus egg extract indicate that CtIP acts downstream of ATM in response to DSBs, and promotes DSB 5' end resection to generate ssDNA, triggering ATR and checkpoint activation. We will use the Xenopus extract and human cell lines, coupled with biochemical studies, to gain mechanistic understanding of the checkpoint role of CtIP and the process of DSB 5' end resection. In particular, we will ask if CtIP is important for processing DSBs with blocked ends, and if CtIP has intrinsic nuclease activity required for 5'->3' resection. We will determine the functional relationships between CtIP, ATM and the Mre11-Rad50- Nbs1 complex in resection. We have discovered a conserved class of RING finger E3 ubiquitin ligases that specifically target SUMOylated proteins. RNF4 family proteins (S. pombe Rfp1/Rfp2-Slx8, or human RNF4) recognize SUMOylated proteins via an N-terminal SUMO-interacting motif (SIM), and ubiquitinate SUMOylated proteins in vitro, dependent on the SIM and RING domains. RNF4 orthologues are implicated in maintenance of genomic integrity in several organisms. To elucidate the physiological roles of RNF4 family proteins, we will identify in vivo targets for RNF4 family proteins using GST-SIM and tagged-SUMO to affinity purify SUMOylated proteins from yeast and human cells, followed by validation and functional characterization of identified proteins as RNF4 targets in vivo and in vitro. We will test candidate proteins implicated by genetic interactions with Rfp1/2 in S. pombe and through physical interaction with RNF4 as targets, including TRIM family proteins. We will determine the significance of the unique structural elements in the multi-SIM and RING domains of RNF4 family proteins in target recognition, and test the model that they recognize poly- SUMOylated proteins. Overall, we expect to learn how RNF4 family proteins function in the DNA damage response and in maintaining genomic integrity. The DNA damage response pathway involves activation of a series of protein kinases, including ATM, ATR, and Chk1/Chk2. To learn more about the network of events downstream of DNA damage, we will use SILAC labeling, phosphoprotein/peptide enrichment, and quantitative mass spectrometry to identify and functionally characterize novel protein phosphorylation sites in mammalian cells induced by DNA damage. For this purpose, we will use a synchronized HeLa cell system where DNA damage is induced with MMS; samples taken at various times and doses of MMS will be analyzed by mass spectrometry for novel phosphorylation events, whose role in the DDR will subsequently be determined.

Public Health Relevance The studies proposed in this application seek to elucidate at the molecular level how cells respond to DNA damage and maintain a stable genome, which are key to understanding how cells safeguard against the development of cancer-causing mutations. Since many cancer therapeutics kill cancer cells by eliciting DNA damage, our studies will also provide insights into the sensitivity of cancer cells to these agents, and suggest ways to improve their use.