Phosphorylation of Nucleotide Excision Repair Factor Xeroderma Pigmentosum Group A by Ataxia Telangiectasia Mutated and Rad3-Related–Dependent Checkpoint Pathway Promotes Cell Survival in Response to UV Irradiation

Abstract

DNA damage triggers complex cellular responses in eukaryotic cells, including initiation of DNA repair and activation of cell cycle checkpoints. In addition to inducing cell cycle arrest, checkpoint also has been suggested to modulate a variety of other cellular processes in response to DNA damage. In this study, we present evidence showing that the cellular function of xeroderma pigmentosum group A (XPA), a major nucleotide excision repair (NER) factor, could be modulated by checkpoint kinase ataxia-telangiectasia mutated and Rad3-related (ATR) in response to UV irradiation. We observed the apparent interaction and colocalization of XPA with ATR in response to UV irradiation. We showed that XPA was a substrate for in vitro phosphorylation by phosphatidylinositol-3-kinase-related kinase family kinases whereas in cells XPA was phosphorylated in an ATR-dependent manner and stimulated by UV irradiation. The Ser196 of XPA was identified as a biologically significant residue to be phosphorylated in vivo. The XPA-deficient cells complemented with XPA-S196A mutant, in which Ser196 was substituted with an alanine, displayed significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Moreover, substitution of Ser196 with aspartic acid for mimicking the phosphorylation of XPA increased the cell survival to UV irradiation. Taken together, our results revealed a potential physical and functional link between NER and the ATRdependent checkpoint pathway in human cells and suggested that the ATR checkpoint pathway could modulate the cellular activity of NER through phosphorylation

ATR-dependent checkpoint modulates XPA nuclear import in response to UV irradiation

“…Given the central role of ATR/ATM kinases in the entire DNA damage response network, it is of particular interest to examine whether these kinases could directly regulate the cellular NER activity in response to UV damage. Previously, we have identified XPA as a phosphorylation substrate for checkpoint kinase ATR in response to UV irradiation (Wu et al, 2006). Here we found that XPA undergoes a dramatic cytoplasm-tonucleus translocation upon treatment of cells with UV, and this event also regulated by ATR.…”

“…Phosphorylation of XPA is unlikely to mediate this process as these two events did not correlate with each other in the time-course experiments (Wu et al, 2006). Given that ATR interacted significantly more efficiently and colocalized to DNA damage sites with XPA in cells upon UV irradiation (as indicated by the nuclear foci) ( Figure 5) (Wu et al, 2006), it is possible that formation of the ATR-XPA complex at the damage sites on chromatin may significantly reduce the concentration of free XPA in the nucleus. This may consequently disrupt the balanced XPA concentration across the nuclear membrane separating the nucleus from the cytoplasm.…”

“…Nuclear extracts were analysed by Western blotting probed with anti-XPA, anti-ATR and anti-ATM. Asterisk represents the phosphorylated form of XPA, which is also wortmannin and caffeine sensitive (Wu et al, 2006). (b) The UV-induced XPA nuclear accumulation was quantified using densitometry.…”

“…When activated, these serine/ threonine kinases phosphorylate and activate downstream checkpoint factors in DNA damage response network and eventually lead to the cell cycle arrest (Kim et al, 1999;Kastan and Lim, 2000;O'Neill et al, 2000). Recent evidences have indicated that checkpoint pathways may also modulate DNA repair processes, through regulation of phosphorylation and intracellular redistribution of DNA repair proteins (Zhou and Elledge, 2000;Barr et al, 2003;Feng et al, 2004;Wu et al, 2006).…”

Other Proteins Interacting with XP Proteins

“…XPA also has been shown to be phosphorylated by ATR in a UV-irradiation dependent manner and abrogation of this event diminished cell survival against UV treatment, although the underlying mechanism is still unknown. 38 XPA interaction with XAB2 (XPA-binding protein 2) was identified by yeast-two hybrid screening. 34 XAB2 interacts with a variety of proteins including RNA Pol II and is active in mRNA splicing.…”