Abstract
Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the single-strand DNA break repair pathway. Phosphorylated XRCC1 stimulates PNKP by binding to its forkhead-associated (FHA) domain, whereas nonphosphorylated XRCC1 stimulates PNKP by interacting with the PNKP catalytic domain. Here, we have further studied the interactions between these two proteins, including two variants of XRCC1 (R194W and R280H) arising from single-nucleotide polymorphisms (SNPs) that have been associated with elevated cancer risk in some reports. We observed that interaction of the PNKP FHA domain with phosphorylated XRCC1 extends beyond the immediate, well-characterized phosphorylated region of XRCC1 (residues 515-526). We also found that an XRCC1 fragment, comprising residues 166-436, binds tightly to PNKP and DNA and efficiently activates PNKP's kinase activity. However, interaction of either of the SNP-derived variants of this fragment with PNKP was considerably weaker, and their stimulation of PNKP was severely reduced, although they still could bind DNA effectively. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were equally efficient at localizing to the damaged DNA. These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA.
Highlights
Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the singlestrand DNA break repair pathway
It is generally considered that interaction between PNKP and XRCC1 is mediated by the binding of CK2-phosphorylated XRCC1 protein to the FHA domain of PNKP [5, 15], it is clear that XRCC1 in its nonphosphorylated form can interact with the catalytic domain of PNKP, thereby stimulating PNKP activity [9, 10, 12]
XRCC1 is regarded as a scaffolding protein capable of interacting with several proteins participating in single-strand break repair
Summary
Polynucleotide kinase/phosphatase (PNKP) and X-ray repair cross-complementing 1 (XRCC1) are key proteins in the singlestrand DNA break repair pathway. Laser microirradiation revealed reduced recruitment of PNKP to damaged DNA in cells expressing either XRCC1 variant compared with PNKP recruitment in cells expressing WT XRCC1 even though WT and variant XRCC1s were efficient at localizing to the damaged DNA These findings suggest that the elevated risk of cancer associated with these XRCC1 SNPs reported in some studies may be due in part to the reduced ability of these XRCC1 variants to recruit PNKP to damaged DNA. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. To gain further insight into the mechanism of activation of PNKP by XRCC1, we have examined the interactions between different regions of XRCC1 with PNKP and DNA For this purpose, we utilized several XRCC1 fragments (Fig. 1) including the following: (i) the extended BRCT2 domain of XRCC1 (EB2) from residues 511 to 633, because this region possesses a cluster of CK2 phosphorylation sites implicated in
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