The complete and accurate replication of the genome is a central process in biology. This involves multiple processes as well as complex regulatory mechanisms that allow the temporally coordinated duplication of the chromosomes during each cell cycle. Pol ? plays a crucial role in DNA replication but in addition also is a major participant in DNA repair processes. A knowledge of the properties of human Pol ?, its regulation and the integration of its functions by protein-protein interactions with other components of the replisome and with DNA repair machinery is thus central to our understanding of human DNA replication and repair. Far less is known about human Pol ? than its more extensively studied counterpart in budding yeast, an important point because they differ in subunit composition. Our goals are directed toward dissecting the roles of the p12 and p68 subunits in Pol ? function. Our research is driven by the hypothesis that p12 directly affects the catalytic functions of the polymerase and exonuclease sites of Pol ?, while p68 plays a role as a scaffold for protein-protein interactions.
In Aim 1 we take advantage of recent advances we have made in the expression of recombinant Pol ? and its subassemblies to make a rigorous biochemical comparisons of the core enzyme (p125/p50), core + p12, core + p68 and the holoenzyme.
In Aim 2 we focus on determining the roles by identifying the sites of p12 that allow it to bind to the p125 and p50 subunits of the core enzyme, and the analysis of their individual effects We also will analyze the subunit interactions of Pol ? and PCNA.
In Aim 3 we focus on the functions of p68, with the view that it acts a scaffold that mediates interactions of Pol ? with other proteins. We will investigate several hypotheses regarding the mechanisms by which phosphorylation-dephosphorylation regulates protein-protein interactions of p68, and also its role as a targeting protein that recruits protein phosphatase-1. Our studies could contribute to the understanding of cancer etiology, since it is more than likely that alterations in Pol ? could contribute to genomic instability.

Public Health Relevance

Human Pol ? is one of the key enzymes that are involved in the duplication and repair of chromosomal DNA. Thus, its functions are highly important in replicating DNA without errors, as well as in repairing errors, so that mutations can be avoided. Our research is directed toward understanding how the different subunits of this enzyme contribute to the function of the catalytic core. These studies are crucial to understanding how it is able to perform DNA replication accurately, and how it interacts with other proteins that comprise the replication and repair machinery. Our work will also provide insights as to how Pol ? functions are regulated. There is currently a large gap in our knowledge of the human Pol ? enzyme, and our work is important to understanding how alterations in its functions could contribute to increased mutations in human cells, and to the etiology of cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Hagan, Ann A
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New York Medical College
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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
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
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
Walsh, Erin; Wang, Xiaoxiao; Lee, Marietta Y et al. (2013) Mechanism of replicative DNA polymerase delta pausing and a potential role for DNA polymerase kappa in common fragile site replication. J Mol Biol 425:232-43
Clausen, Anders R; Zhang, Sufang; Burgers, Peter M et al. (2013) Ribonucleotide incorporation, proofreading and bypass by human DNA polymerase ?. DNA Repair (Amst) 12:121-7
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
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

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