DNA Polymerase η, the Product of the Xeroderma Pigmentosum Variant Gene and a Target of p53, Modulates the DNA Damage Checkpoint and p53 Activation

Abstract

DNA polymerase η (PolH) is the product of the xeroderma pigmentosum variant (XPV) gene and a well-characterized Y-family DNA polymerase for translesion synthesis. Cells derived from XPV patients are unable to faithfully bypass UV photoproducts and DNA adducts and thus acquire genetic mutations. Here, we found that PolH can be up-regulated by DNA breaks induced by ionizing radiation or chemotherapeutic agents, and knockdown of PolH gives cells resistance to apoptosis induced by DNA breaks in multiple cell lines and cell types in a p53-dependent manner. To explore the underlying mechanism, we examined p53 activation upon DNA breaks and found that p53 activation is impaired in PolH knockdown cells and PolH-null primary fibroblasts. Importantly, reconstitution of PolH into PolH knockdown cells restores p53 activation. Moreover, we provide evidence that, upon DNA breaks, PolH is partially colocalized with phosphorylated ATM at γ-H2AX foci and knockdown of PolH impairs ATM to phosphorylate Chk2 and p53. However, upon DNA damage by UV, PolH knockdown cells exhibit two opposing temporal responses: at the early stage, knockdown of PolH suppresses p53 activation and gives cells resistance to UV-induced apoptosis in a p53-dependent manner; at the late stage, knockdown of PolH suppresses DNA repair, leading to sustained activation of p53 and increased susceptibility to apoptosis in both a p53-dependent and a p53-independent manner. Taken together, we found that PolH has a novel role in the DNA damage checkpoint and that a p53 target can modulate the DNA damage response and subsequently regulate p53 activation.

View correction statement:

DNA Polymerase η, the Product of the Xeroderma Pigmentosum Variant Gene and a Target of p53, Modulates the DNA Damage Checkpoint and p53 Activation

We thank Cyrus Vaziri (Boston University) for the PolH cDNA construct and Wensheng Yan for the RKO tet-on cell line and Kelly Harms for the MCF-7 tet-on cell line.

This work was supported in part by an NIH grant (CA076069) from the National Cancer Institute and the Department of Defense Prostate Cancer Research Program under award number W81XWH-04-1-0079.

Views and opinions of, and endorsements by, the authors do not reflect those of the U.S. Army or the Department of Defense.