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
Copyright policy )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.
- University of Connecticut Health Center United States
Protein Folding, Binding Sites, Magnetic Resonance Spectroscopy, Protein Conformation, Temperature, DNA-Binding Proteins, X-ray Repair Cross Complementing Protein 1, Amino Acid Substitution, Solubility, Mutation, Mutagenesis, Site-Directed, Electrophoresis, Polyacrylamide Gel, Chromatography, High Pressure Liquid, DNA Polymerase beta, Protein Binding
Protein Folding, Binding Sites, Magnetic Resonance Spectroscopy, Protein Conformation, Temperature, DNA-Binding Proteins, X-ray Repair Cross Complementing Protein 1, Amino Acid Substitution, Solubility, Mutation, Mutagenesis, Site-Directed, Electrophoresis, Polyacrylamide Gel, Chromatography, High Pressure Liquid, DNA Polymerase beta, Protein Binding
citations This is an alternative to the "Influence" indicator, which also reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).37 popularity This indicator reflects the "current" impact/attention (the "hype") of an article in the research community at large, based on the underlying citation network.Top 10% influence This indicator reflects the overall/total impact of an article in the research community at large, based on the underlying citation network (diachronically).Top 10% impulse This indicator reflects the initial momentum of an article directly after its publication, based on the underlying citation network.Top 10%
Citations provided by BIP!Popularity provided by BIP!