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.
Mani
Rajam S
RS
Department of Experimental Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta T6G 1Z2, Canada. rajam.mani@cancerboard.ab.ca
Karimi-Busheri
Feridoun
F
Fanta
Mesfin
M
Caldecott
Keith W
KW
Cass
Carol E
CE
Weinfeld
Michael
M
D002846:Chromatography, Affinity; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D013050:Spectrometry, Fluorescence; D000076105:X-ray Repair Cross Complementing Protein 1
D004268:DNA-Binding Proteins; D008024:Ligands; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D004247:DNA