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

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 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.

X-Ray Repair Cross-Complementing Group 1(XRCC1) Genetic Polymorphisms and Thyroid Carcinoma Risk: a Meta-Analysis

“…XRCC1 plays an important role in the DNA repair pathway because it could specifically interact with nicked and gapped DNA, rapidly and transiently responds to DNA damage in cells, thus may serve as a strand-break sensor (Mani et al, 2004). In addition, XRCC1 could interact with many proteins known to be involved in BER andSSBR, so it has been proposed that XRCC1 may function as a scaffold protein able to coordinate and facilitate the steps of various DNA repair pathways (Mani et al, 2007).…”

Which Gene is a Dominant Predictor of Response During FOLFOX Chemotherapy for the Treatment of Metastatic Colorectal Cancer, the MTHFR or XRCC1 Gene?

“…Secondly, XRCC1 protein repairs DNA replication errors specifically by the base excision repair (BER) mechanism which incorporates two enzymes, such as DNA ligase III and polymerase beta. [27][28][29] Unexpectedly, the large product of DNA adduct, which is the main action of oxaliplatin, cannot be repaired by BER but by nucleotide excision repair (NER). A newly found protein, XGP, is known to have dual actions for both BER and NER.…”

“…It is hypothesized that XGP is closely related with the XRCC1 protein and that it can work normally with normal XRCC1 protein. 8,[27][28][29] In combination chemotherapy, overall drug response is a net effect of individual drug responses. Although oxaliplatin is known to have more a potent cytotoxic effect than 5-FU in the treatment of CRC, it would appear to be important to determine which gene polymorphism is dominant when administering FOLFOX combination chemotherapy.…”

New Players in Recognition of Intact and Cleaved AP Sites: Implication in DNA Repair in Mammalian Cells

“…Addition of PNKP resulted in a 15-mer product with the 3′-OH termini (lane 3). XRCC1, a scaffold protein, stimulates the activity of BER proteins, such as DNA polymerase , PNKP, DNA ligase III (Mani et al, 2004;Caldecott, 2003;Whitehouse et al, 2001). Lastly, DNA polymerase replaces the missing DNA segment (lane 4) and DNA ligase reseals the DNA (lane 5).…”

“…By competition experiments using the THF-containing or regular DNA duplex it was demonstrated that XRCC1 binds DNA with an AP site, or its synthetic analogue, with considerably higher affinity than regular DNA duplex. XRCC1 is known to bind DNA with single-strand breaks with higher affinity that regular DNA duplex (Mani et al, 2004). We then investigated the relative affinities of XRCC1 to different DNA structures that could be BER intermediates in the repair of single or double DNA lesions.…”