Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase β

Abstract

Human X-ray cross-complementing group 1 (XRCC1) is a single-strand DNA break repair protein which forms a base excision repair (BER) complex with DNA polymerase β (β-Pol). Here we report a site-directed mutational analysis in which 16 mutated versions of the XRCC1 N-terminal domain (XRCC1-NTD) were constructed on the basis of previous NMR results that had implicated the proximity of various surface residues to β-Pol. Mutant proteins defective in XRCC1-NTD interaction with β-Pol and with a β-Pol-gapped DNA complex were determined by gel filtration chromatography and a gel mobility shift assay. The interaction surface determined from the mutated residues was found to encompass β-strand D and E of the five-stranded β-sheet (βABGDE) and the protruding α2 helix of the XRCC1-NTD. Mutations that included F67A (βD), E69K (βD), V86R (βE) on the five-stranded β-sheet and deletion of the α2 helix, but not mutations within α2, abolished binding of the XRCC1-NTD to β-Pol. A Y136A mutant abolished β-Pol binding, and a R109S mutant reduced β-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and δ-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the α2 helix and the βD and βE strands of XRCC1-NTD to the energetics of β-Pol binding. Establishing the direct contacts in the β-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.