Biophysical Characterization of Human XRCC1 and Its Binding to Damaged and Undamaged DNA

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Abstract
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 Kd of 5.7 × 10-7 M. The size and shape of hXRCC1 in solution were determined by analytical ultracentrifugation studies. The protein exhibited an intrinsic sedimentation coefficient, s020,w, of 3.56 S and a Stokes radius, Rs, of 44.5 Å, which together with the Mr 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 Kd 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 Kd 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.