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.
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 beta (beta-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 beta-Pol. Mutant proteins defective in XRCC1-NTD interaction with beta-Pol and with a beta-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 beta-strand D and E of the five-stranded beta-sheet (betaABGDE) and the protruding alpha2 helix of the XRCC1-NTD. Mutations that included F67A (betaD), E69K (betaD), V86R (betaE) on the five-stranded beta-sheet and deletion of the alpha2 helix, but not mutations within alpha2, abolished binding of the XRCC1-NTD to beta-Pol. A Y136A mutant abolished beta-Pol binding, and a R109S mutant reduced beta-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and delta-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the alpha2 helix and the betaD and betaE strands of XRCC1-NTD to the energetics of beta-Pol binding. Establishing the direct contacts in the beta-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.
Marintchev
Assen
A
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA.
Gryk
Michael R
MR
Mullen
Gregory P
GP
D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D019951:DNA Polymerase beta / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012995:Solubility; D013696:Temperature; D000076105:X-ray Repair Cross Complementing Protein 1
D004268:DNA-Binding Proteins; D000076105:X-ray Repair Cross Complementing Protein 1; C000619371:XRCC1 protein, human; D019951:DNA Polymerase beta