The RIR motif in the scaffold protein XRCC1 mediates a low-affinity interaction with polynucleotide kinase/phosphatase (PNKP) during DNA single-strand break repair

The scaffold protein X-ray repair cross-complementing 1 (XRCC1)interacts with multiple enzymes involved in DNA base excision repair and single-strand break repair(SSBR) and is important for genetic integrity and normal neurological function. One of the most important interactions of XRCC1 is that with polynucleotide kinase/phosphatase(PNKP), a dual-function DNA kinase/phosphatase that processes damaged DNA termini and that, if mutated, results in ataxia with oculomotor apraxia 4 (AOA4) and microcephaly with early-onset seizures and developmental delay(MCSZ). XRCC1 and PNKP interact via a high-affinity phosphorylationdependent interaction site in XRCC1 and a fork-head associated domain in PNKP. Here, we identified using biochemical and biophysical approaches a second PNKP interaction site in XRCC1 that binds PNKP with lower affinity and independently of XRCC1 phosphorylation. However, this interaction nevertheless stimulated PNKP activity and promoted SSBR and cell survival. The low-affinity interaction site required the highly conserved REV1-interacting (RIR) motif in XRCC1 and included three critical and evolutionarily invariant phenylalanine residues. We propose a bipartite interaction model in which the previously identified highaffinity interaction acts as a molecular tether, holding XRCC1 and PNKP together and thereby promoting the low-affinity interaction identified here, which then stimulates PNKP directly

Phosphorylation of polynucleotide kinase/ phosphatase by DNA-dependent protein kinase and ataxia-telangiectasia mutated regulates its association with sites of DNA damage

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

Biophysical characterization of human XRCC1 and its binding to damaged and undamaged DNA

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