Abstract 1281: POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells.

Abstract

Abstract Methyl methanesulfonate (MMS) induces mutations in a nonlinear (hockey-stick-shaped) dose-response curve in DT40 cells, about 55% of which are located at A:T sites with a high frequency of A to T mutations. We hypothesized that these transversion mutations are due to base excision repair (BER) intermediates, such as abasic sites and single strand breaks derived from N3-methyladenine. One candidate polymerase to bypass abasic sites is polymerase delta (POLD). POLD is responsible for the lagging strand during DNA replication. POLD is referred to as a high fidelity DNA polymerase because of its ability to discriminate deoxynucleotides and its 3’->5’ exo-nuclease activity. One of the POLD subunits, POLD3, is important to recruit PCNA binding to the POLD core enzyme and is implied to have critical roles for translesion DNA synthesis. In yeast, cells deficient in POLD3 homologous gene POL32 are sensitive to DNA damaging agents and resistant to mutations caused by mutagens, such as MMS and UV. Here we generated POLD3 knock-down (KD) cells via lentivirus based shRNA in three different human cell lines. All POLD3 KD cells grow slower than the control cells, suggesting POLD3 function tolerates endogenous DNA lesions. Furthermore, we found that POLD3 KD cells were sensitive to various genotoxic agents including MMS and potassium chromate. To further investigate the role of POLD3 in the DNA damage response, we introduced a series of deletion mutations of POLD3 in POLD3 deficient cells. We found that both the POLD2 binding domain and the middle linker domain are critical for the activity of POLD3 while the C-terminal PCNA binding domain is dispensable. While there is little homology between human POLD3 and yeast POL32, function of these two proteins appears to be quite similar in structure biology analysis. Our results also suggest that POLD3 plays important roles to bypass DNA lesions caused by endogenous and various exogenous genotoxic agents. Citation Format: Xu Tian, James Swenberg, Jun Nakamura. POLD3 is required for DNA damage response to endogenous and exogenous DNA damage in human cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1281. doi:10.1158/1538-7445.AM2013-1281