DNA polymerase delta (Pol d) is a key enzyme that is essential for eukaryotic chromosomal DNA replication. Mammalian Pol d consists of four subunits, all of which are required for its full function in vitro. This proposal is based on the novel discovery that levels of the human Pol d p12 subunit are dramatically depleted after DNA damage by UV and other genotoxic agents that activate the ATR/Chk1 mediated S-phase checkpoint. We have shown that this results in the conversion of Pol d from a tetramer into a trimer, Pol d3. The biochemical properties of pol d3 will be compared to those of the parent enzyme. We will examine its kinetic properties, its abilities to bypass template lesions, and its abilities to act as a proof reading enzyme. Our working hypothesis is that the conversion to Pol d3 prevents Pol d from bypassing template lesions, thereby allowing repair processes to take place. The spatiotemporal aspects of the localization of Pol d3 to DNA damage foci will be studied by immunofluorescence microscopy and laser scanning cytometry after UV damage and compared to the recruitment of other DNA damage proteins. The role of ubiquitination in the depletion of pol d will be studied. The identity of the ubiquitination system will be determined using several different approaches, including siRNA knockdown of candidate ubiquitination proteins, identification of p12 binding proteins, and isolation of E3 ligases that act to ubiquitinate p12 using in vitro assays.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES014737-05
Application #
8197897
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Mcallister, Kimberly A
Project Start
2007-12-01
Project End
2013-08-07
Budget Start
2011-12-01
Budget End
2013-08-07
Support Year
5
Fiscal Year
2012
Total Cost
$331,151
Indirect Cost
$122,880
Name
New York Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041907486
City
Valhalla
State
NY
Country
United States
Zip Code
10595
Lee, Marietta Y W T; Zhang, Sufang; Lin, Szu Hua Sharon et al. (2014) The tail that wags the dog: p12, the smallest subunit of DNA polymerase ýý, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression. Cell Cycle 13:23-31
Lin, Szu Hua Sharon; Wang, Xiaoxiao; Zhang, Sufang et al. (2013) Dynamics of enzymatic interactions during short flap human Okazaki fragment processing by two forms of human DNA polymerase ýý. DNA Repair (Amst) 12:922-35
Nakao, Seiki; Zhang, Sufang; Vaara, Markku et al. (2013) Efficient long DNA gap-filling in a mammalian cell-free system: a potential new in vitro DNA replication assay. Biochimie 95:320-8
Zhang, Sufang; Zhou, Yajing; Sarkeshik, Ali et al. (2013) Identification of RNF8 as a ubiquitin ligase involved in targeting the p12 subunit of DNA polymerase ? for degradation in response to DNA damage. J Biol Chem 288:2941-50
Wong, Agnes; Zhang, Sufang; Mordue, Dana et al. (2013) PDIP38 is translocated to the spliceosomes/nuclear speckles in response to UV-induced DNA damage and is required for UV-induced alternative splicing of MDM2. Cell Cycle 12:3184-93
Zhang, Sufang; Zhao, Hong; Darzynkiewicz, Zbiegniew et al. (2013) A novel function of CRL4(Cdt2): regulation of the subunit structure of DNA polymerase ýý in response to DNA damage and during the S phase. J Biol Chem 288:29550-61
Hile, Suzanne E; Wang, Xiaoxiao; Lee, Marietta Y W T et al. (2012) Beyond translesion synthesis: polymerase ? fidelity as a potential determinant of microsatellite stability. Nucleic Acids Res 40:1636-47
Rahmeh, Amal A; Zhou, Yajing; Xie, Bin et al. (2012) Phosphorylation of the p68 subunit of Pol ? acts as a molecular switch to regulate its interaction with PCNA. Biochemistry 51:416-24
Zhou, Yajing; Chen, Huiqing; Li, Xiao et al. (2011) Production of recombinant human DNA polymerase delta in a Bombyx mori bioreactor. PLoS One 6:e22224
Meng, Xiao; Zhou, Yajing; Lee, Ernest Y C et al. (2010) The p12 subunit of human polymerase delta modulates the rate and fidelity of DNA synthesis. Biochemistry 49:3545-54

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