Abstract

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.

  Funding Agency

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

  Institution

Name
New York Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041907486
City
Valhalla
State
NY
Country
United States
Zip Code
10595

  Publications

Peddu, Chandana; Zhang, Sufang; Zhao, Hong et al. (2018) Phosphorylation Alters the Properties of Pol ?: Implications for Translesion Synthesis. iScience 6:52-67
Lee, Marietta Y W T; Wang, Xiaoxiao; Zhang, Sufang et al. (2017) Regulation and Modulation of Human DNA Polymerase ? Activity and Function. Genes (Basel) 8:
Gowda, A S Prakasha; Lee, Marietta; Spratt, Thomas E (2017) N2 -Substituted 2'-Deoxyguanosine Triphosphate Derivatives as Selective Substrates for Human DNA Polymerase ?. Angew Chem Int Ed Engl 56:2628-2631
Burkovics, Peter; Dome, Lili; Juhasz, Szilvia et al. (2016) The PCNA-associated protein PARI negatively regulates homologous recombination via the inhibition of DNA repair synthesis. Nucleic Acids Res 44:3176-89
Huehls, Amelia M; Huntoon, Catherine J; Joshi, Poorval M et al. (2016) Genomically Incorporated 5-Fluorouracil that Escapes UNG-Initiated Base Excision Repair Blocks DNA Replication and Activates Homologous Recombination. Mol Pharmacol 89:53-62
Wang, Xiaoxiao; Zhang, Sufang; Zheng, Rong et al. (2016) PDIP46 (DNA polymerase ? interacting protein 46) is an activating factor for human DNA polymerase ?. Oncotarget 7:6294-313
Darzynkiewicz, Zbigniew; Zhao, Hong; Zhang, Sufang et al. (2015) Initiation and termination of DNA replication during S phase in relation to cyclins D1, E and A, p21WAF1, Cdt1 and the p12 subunit of DNA polymerase ? revealed in individual cells by cytometry. Oncotarget 6:11735-50
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 ?, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression. Cell Cycle 13:23-31
Zhao, Hong; Zhang, Sufang; Xu, Dazhong et al. (2014) Expression of the p12 subunit of human DNA polymerase ? (Pol ?), CDK inhibitor p21(WAF1), Cdt1, cyclin A, PCNA and Ki-67 in relation to DNA replication in individual cells. Cell Cycle 13:3529-40
Zhang, Sufang; Zhao, Hong; Darzynkiewicz, Zbiegniew et al. (2013) A novel function of CRL4(Cdt2): regulation of the subunit structure of DNA polymerase ? in response to DNA damage and during the S phase. J Biol Chem 288:29550-61

Showing the most recent 10 out of 25 publications