XRCC4 plays a crucial role in the nonhomologous end joining (NHEJ) pathway of DNA double-strand break repair acting as a scaffold protein that recruits other NHEJ proteins to doublestrand breaks. Phosphorylation of XRCC4 by protein kinase CK2 promotes a high affinity interaction with the forkhead-associated domain of the end-processing enzyme polynucleotide kinase/phosphatase (PNKP). Here we reveal that unphosphorylated XRCC4 also interacts with PNKP through a lower affinity interaction site within the catalytic domain and that this interaction stimulates the turnover of PNKP. Unexpectedly, CK2-phosphorylated XRCC4 inhibited PNKP activity. Moreover, the XRCC4⅐DNA ligase IV complex also stimulated PNKP enzyme turnover, and this effect was independent of the phosphorylation of XRCC4 at threonine 233. Our results reveal that CK2-mediated phosphorylation of XRCC4 can have different effects on PNKP activity, with implications for the roles of XRCC4 and PNKP in NHEJ.Efficient repair of DNA double-strand breaks (DSBs) 3 is critical for the maintenance of genome stability. In mammalian cells, nonhomologous end joining (NHEJ) is the major pathway that repairs these DSBs (1, 2). Although many of the individual components involved in the NHEJ repair pathway are well established, the dynamics of the repair pathway remains poorly understood. One approach to achieving a better understanding of the step-by-step choreography of each enzymatic process, including the nature of the binding of repair proteins to their DNA substrates and to each other, is to use a detailed quantitative approach in which specific protein-protein and protein-DNA interactions are not just identified qualitatively but are accurately quantified, giving an estimation of their respective affinities.XRCC4 is regarded as a scaffold protein that recruits other proteins to DSBs (1). Of note, XRCC4 interacts with and catalytically stimulates the activity of DNA ligase IV (3, 4) to carry out the final step in the NHEJ pathway, joining the DNA ends (5). More recently, XRCC4 has been shown to interact with polynucleotide kinase/phosphatase (PNKP) (6, 7), a bifunctional enzyme that phosphorylates 5Ј-OH termini and dephosphorylates 3Ј-phosphate termini (8 -11), thereby providing the correct chemical end groups required for DNA ligation by DNA ligase IV. Because XRCC4 is an efficient substrate for DNA-PK in vitro (12, 13), most studies have focused on the impact of DNA-PK-mediated phosphorylation on XRCC4 function (3,14). However, phosphorylation of XRCC4 by DNA-PK cannot account for all of its functions. For instance, DNA-PK-dependent phosphorylation of XRCC4 does not appear to play a role in mediating resistance to ionizing radiation or V(D)J recombination (3, 14). On the other hand, phosphorylation by protein kinase CK2 mediates the interaction of XRCC4 with the forkhead-associated (FHA) domain of PNKP and thereby stimulates DNA ligation (7). An examination of the crystal structure of a short XRCC4 phospho-peptide bound to the FHA domain of PNKP indicated that t...
Human polynucleotide kinase/phosphatase (PNKP) is a dual specificity 5′-DNA kinase/3′-DNA phosphatase, with roles in base excision repair, DNA single-strand break repair and non-homologous end joining (NHEJ); yet precisely how PNKP functions in the repair of DNA double strand breaks (DSBs) remains unclear. We demonstrate that PNKP is phosphorylated by the DNA-dependent protein kinase (DNA-PK) and ataxia-telangiectasia mutated (ATM) in vitro. The major phosphorylation site for both kinases was serine 114, with serine 126 being a minor site. Ionizing radiation (IR)-induced phosphorylation of cellular PNKP on S114 was ATM dependent, whereas phosphorylation of PNKP on S126 required both ATM and DNA-PK. Inactivation of DNA-PK and/or ATM led to reduced PNKP at DNA damage sites in vivo. Cells expressing PNKP with alanine or aspartic acid at serines 114 and 126 were modestly radiosensitive and IR enhanced the association of PNKP with XRCC4 and DNA ligase IV; however, this interaction was not affected by mutation of PNKP phosphorylation sites. Purified PNKP protein with mutation of serines 114 and 126 had decreased DNA kinase and DNA phosphatase activities and reduced affinity for DNA in vitro. Together, our results reveal that IR-induced phosphorylation of PNKP by ATM and DNA-PK regulates PNKP function at DSBs.
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