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

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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.