The human DNA repair protein, hXRCC1, which is required for DNA single-strand break repair and genetic stability was produced as a histidine-tagged polypeptide in Escherichia coli, purified by affinity chromatography, and subjected to sedimentation and spectroscopic analyses. This study represents the first biophysical examination of full-length XRCC1. Sedimentation equilibrium measurements indicated that hXRCC1 exists as a monomer at lower protein concentrations but forms a dimer at higher protein concentrations with a K(d) of 5.7 x 10(-)(7) M. The size and shape of hXRCC1 in solution were determined by analytical ultracentrifugation studies. The protein exhibited an intrinsic sedimentation coefficient, s(0)(20,w), of 3.56 S and a Stokes radius, R(s), of 44.5 A, which together with the M(r) of 68000 suggested that hXRCC1 is a moderately asymmetric protein with an axial ratio of 7.2. Binding of model ligands, representing single-strand breaks with either a nick or a single nucleotide gap, quenched protein fluorescence, and binding affinities and stoichiometries were determined by carrying out fluorescence titrations as a function of ligand concentration. XRCC1 bound both nicked and 1 nucleotide-gapped DNA substrates tightly in a stoichiometric manner (1:1) with K(d) values of 65 and 34 nM, respectively. However, hXRCC1 exhibited lower affinities for a duplex with a 5 nucleotide gap, the intact duplex with no break, and a single-stranded oligonucleotide with K(d) values of 215, 230, and 260 nM, respectively. Our results suggest that hXRCC1 exhibits preferential binding to DNA with single-strand breaks with a gap size of <5 nucleotides.

Human polynucleotide kinase (hPNK) is required for processing and rejoining DNA strand break termini. The 5-DNA kinase and 3-phosphatase activities of hPNK can be stimulated by the "scaffold" protein XRCC1, but the mechanism remains to be fully elucidated. Using a variety of fluorescence techniques, we examined the interaction of hPNK with XRCC1 and substrates that model DNA single-strand breaks. hPNK binding to substrates with 5-OH termini was only ϳ5-fold tighter than that to identical DNA molecules with 5-phosphate termini, suggesting that hPNK remains bound to the product of its enzymatic activity. The presence of XRCC1 did not influence the binding of hPNK to substrates with 5-OH termini, but sharply reduced the interaction of hPNK with DNA bearing a 5-phosphate terminus. These data, together with kinetic data obtained at limiting enzyme concentration, indicate a dual function for the interaction of XRCC1 with hPNK. First, XRCC1 enhances the capacity of hPNK to discriminate between strand breaks with 5-OH termini and those with 5-phosphate termini; and second, XRCC1 stimulates hPNK activity by displacing hPNK from the phosphorylated DNA product.Scission of the DNA sugar-phosphate backbone is a common form of damage that can be induced not only by a broad range of genotoxic agents, but also as an intermediate product in several DNA repair pathways. The term "strand break" covers an array of diverse chemical structures. Aside from single-and doublestrand breaks, there are many chemically distinct end groups found at strand break termini. Repair of these strand interruptions is usually mediated by DNA polymerases and ligases. All DNA polymerases and ligases characterized to date are highly selective for the type of DNA ends that can be utilized. Both of these classes of enzymes require 3Ј-hydroxyl DNA termini, and the DNA ligases also require 5Ј-phosphate termini. However, the termini generated by several endonucleases, as well as those induced by ionizing radiation, frequently bear 5Ј-hydroxyl and/or 3Ј-phosphate groups (1-5) and must therefore be processed before they can be acted upon by DNA ligases or polymerases.Mammalian polynucleotide kinase (PNK) 3 /phosphatase is a bifunctional enzyme that can phosphorylate 5Ј-OH termini and dephosphorylate 3Ј-phosphate termini of DNA (6, 7). It is a DNA repair enzyme involved in the processing of strand break termini to a form suitable for other proteins to complete the replacement of missing nucleotides and strand rejoining (8 -11). PNK is implicated in the repair of both single-strand breaks (SSBs) and double-strand breaks. PNK stimulates SSB repair in both in vitro reconstitution experiments (5,10,12,13) and in vivo studies (14). Further evidence has indicated that, as a result of its required involvement in the repair of specific types of SSBs, PNK participates in the base excision repair pathway following the formation of strand breaks induced by DNA glycosylases such as NEIL1 and NEIL2 (5) and in the repair of topoisomerase-1 "dead-end" complexes (15,16). PNK i...

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.