The administration of therapeutic agents that cause DNA damage is a major approach to eliminate cancer in patients. The lack of complete tumor response following treatment with DNA crosslinking agents like cisplatin continues to be a persistent clinical problem. The long term goal of this project is to better understand how DNA damage response pathways cooperate to remove crosslinks that bind two opposite DNA strands and promote resistance to these lesions. The major objective of this proposal is to determine the molecular mechanisms by which the translesion DNA polymerases REV1 and DNA polymerase zeta (Pol?) facilitate DNA interstrand crosslink (ICL) repair. REV1 or Pol?-deficient cells display similar DNA repair deficiencies that are characteristic of cells derived frm Fanconi anemia (FA) patients, including profound hypersensitivity to ICL- generating drugs and the accumulation of chromosomal aberrations. Here we present evidence that the FA effector proteins, FANCI and FANCD2, specifically co-immunoprecipitate with REV1 or REV3 (the catalytic subunit of Pol?) and together increase the efficiency of homologous recombination (HR) repair. We will test the central hypothesis that proteins belonging to the FA pathway cooperate with REV1and Pol? to facilitate DNA repair. It has long been hypothesized that REV1, REV3 and REV7 (the Pol? accessory subunit) work together to perform translesion DNA synthesis (TLS) and DNA repair, however the functional significance of each component in the complex is poorly understood.
In Aim 1, we will create mutant versions of REV1 and REV3 that cannot interact with REV7 or the POLD2 subunit of DNA polymerase delta, which binds to REV3. We will determine which components of the complex are essential for DNA repair and chemoresistance.
In Aim 2, we will characterize the interactions between the FANCI/FANCD2 heterodimer and the REV1/Pol? complex. We will examine whether FANCI/FANCD2 and REV1/Pol? cooperate to facilitate DNA repair and maintain genomic stability.
In Aim 3, we will examine whether additional FA proteins associate with the REV1/Pol? complex and characterize three additional proteins we identified in REV1 or REV3 immunoprecipitates: DNA polymerase Nu the MSH2 mismatch repair protein, and the SLX4 endonuclease scaffold protein. We will determine whether these proteins directly bind to the REV1/Pol? complex and cooperate to resolve ICLs in DNA. This proposal is innovative since it will develop new models for understanding how the FA pathway interacts with translesion DNA synthesis polymerases and potentially identify new protein-protein interactions to explore for small molecule inhibitor development designed to disrupt the function of this complex. This proposal is significant because the proposed studies will further characterize the complex protein-protein interactions within the Fanconi anemia pathway and broaden our knowledge of the mechanisms by which cells remove the cytotoxic DNA lesions created by many clinically relevant cancer drugs. Increasing our understanding of these mechanisms may identify new strategies for overcoming tumor resistance to chemotherapeutic agents.
The proposed research will further characterize the complex pathways by which cells respond to and resist the therapeutic effectiveness of commonly used anticancer agents through efficient DNA repair of DNA breaks and interstrand crosslinks. These studies will investigate whether two translesion DNA polymerases, REV1 and Polz, cooperate with a family of proteins that when mutated and rendered dysfunctional cause the human disease Fanconi anemia (FA). This research will increase our knowledge of how translesion DNA polymerases may interact with a subset of the 16 and growing FA genes that when mutated lead to a clinical syndrome associated with hypersensitivity to interstrand crosslinks in DNA, congenital defects, bone marrow failure, and cancer susceptibility.
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|Sharma, Shilpy; Helchowski, Corey M; Canman, Christine E (2013) The roles of DNA polymerase ýý and the Y family DNA polymerases in promoting or preventing genome instability. Mutat Res 743-744:97-110|
|Ladd, Brendon; Ackroyd, Jeffrey J; Hicks, J Kevin et al. (2013) Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity. DNA Repair (Amst) 12:1114-21|
|Tian, Fen; Sharma, Shilpy; Zou, Jianqiu et al. (2013) BRCA1 promotes the ubiquitination of PCNA and recruitment of translesion polymerases in response to replication blockade. Proc Natl Acad Sci U S A 110:13558-63|
|Sharma, Shilpy; Canman, Christine E (2012) REV1 and DNA polymerase zeta in DNA interstrand crosslink repair. Environ Mol Mutagen 53:725-40|
|Sharma, Shilpy; Shah, Nicholas A; Joiner, Ariell M et al. (2012) DNA polymerase ? is a major determinant of resistance to platinum-based chemotherapeutic agents. Mol Pharmacol 81:778-87|
|Sharma, Shilpy; Hicks, J Kevin; Chute, Colleen L et al. (2012) REV1 and polymerase ? facilitate homologous recombination repair. Nucleic Acids Res 40:682-91|
|Hicks, J Kevin; Chute, Colleen L; Paulsen, Michelle T et al. (2010) Differential roles for DNA polymerases eta, zeta, and REV1 in lesion bypass of intrastrand versus interstrand DNA cross-links. Mol Cell Biol 30:1217-30|
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