Human DNA polymerase delta (Pol ?) is a central enzyme in the replication of human chromosomal DNA and its repair. Pol ? thus has important roles in maintaining genomic integrity. We have established that the Pol ? holoenzyme (Pol ?4) is converted into a 3-subunit enzyme, Pol ?3, by the loss of its p12 subunit after cellular UV irradiation. Our central hypothesis is that Pol ?3 has altered properties which contribute to the cellular defense against genomic damage. The goals of this project are directed toward a) elucidating the properties of Pol ?3 and Pol ?4 in Okazaki fragment maturation and in facilitating translesion synthesis, and b) characterizing the cellular and molecular basis of p12 degradation by the E3 ubiquitin ligases RNF8 and CRL4Cdt2 and how they integrate p12 degradation into the cellular networks of DNA damage responses.
Aim 1 addresses the abilities of Pol ?3 and Pol ?4 to perform Okazaki fragment processing in cooperation with Fen1, as well as the impact of ubiquitination of PCNA on this process. Model substrate templates will be used to test the hypotheses that Pol ?3 is well adapted to participate in Okazaki fragment processing, and that formation of ub- PCNA during DNA damage inhibits this process.
Aim 2 addresses the mechanism and role of p12 degradation in translesion synthesis, and will test a novel hypothesis for the mechanisms of switching between Pol h and Pol ? on ub-PCNA. This hypothesis takes into account the fact that ub-PCNA is a hexavalent molecule on which both Pol ? and Pol h exhibit multivalent interactions that drive the recruitment and switching process. We will use rigorous pre-steady state kinetic analysis to quantitatively assess their switching rates. These studies will include other translesion polymerases that exhibit similar domain structures as Pol h to determine the generality of this mechanism.
Aim 3 addresses our major discovery that p12 is a substrate of the CRL4Cdt2 E3 ligase. Here we will characterize in a rigorous manner it's in vivo role in regulating p12 degradation in response to UV damage, as well as in cell cycle progression. The consequences of depletion or overexpression of p12 on UV sensitivity and cell growth will be investigated to elucidate the cellular functions of p12.
Aim 4 addresses the role of RNF8 which targets p12 for degradation, and in addition, ubiquitinates PCNA to regulate translesion synthesis. Here we will determine the other components of RNF8 pathway that are needed for p12 degradation, and test the hypothesis that RNF8 integrates p12 degradation into several DNA damage signaling networks. This will focus on the currently unknown role of IR in triggering p12 degradation and its effect on HR. The goals of this project on the novel response to DNA damage caused by UV or chemical agents are directly related to the goals of the NIEHS. Impairments in this DNA damage response may increase genomic instability which is a key event in carcinogenesis.

Public Health Relevance

The goals of this work are to study a novel mechanism that represents a response of human cells to DNA damage. This mechanism involves the loss of the p12 subunit of DNA polymerase delta, an enzyme central to the synthesis of the human genome. The studies will characterize how the modified enzyme functions in DNA replication and repair, and help to protect the cell against DNA damage. The studies are relevant to public health goals that seek to understand and develop treatments against the effects of UV and other DNA damaging agents from the environment.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
2R01ES014737-06A1
Application #
8580329
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Mcallister, Kimberly A
Project Start
2006-04-01
Project End
2018-04-30
Budget Start
2013-08-08
Budget End
2014-04-30
Support Year
6
Fiscal Year
2013
Total Cost
$362,250
Indirect Cost
$137,250
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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