Nucleic Acids Research, 2003, Vol. 31, No. 2 580-588
© 2003 Oxford University Press
Site-directed mutagenesis analysis of the structural interaction of the single-strand-break repair protein, X-ray cross-complementing group 1, with DNA polymerase ß
Department of Biochemistry, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06032, USA
*To whom correspondence should be addressed. Tel: +1 860 679 4785; Fax: +1 860 679 3408; Email: gryk{at}neuron.uchc.edu
Present address:
Assen Martintchev, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA
Gregory P. Mullen, deceased
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 ß (ß-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 ß-Pol. Mutant proteins defective in XRCC1-NTD interaction with ß-Pol and with a ß-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 ß-strand D and E of the five-stranded ß-sheet (ßABGDE) and the protruding 
2 helix of the XRCC1-NTD. Mutations that included F67A (ßD), E69K (ßD), V86R (ßE) on the five-stranded ß-sheet and deletion of the 2 helix, but not mutations within 2, abolished binding of the XRCC1-NTD to ß-Pol. A Y136A mutant abolished ß-Pol binding, and a R109S mutant reduced ß-Pol binding. E98K, E98A, N104A, Y136A, R109S, K129E, F142A, R31A/K32A/R34A and 
-helix-2 mutants displayed temperature dependent solubility. These findings confirm the importance of the 2 helix and the ßD and ßE strands of XRCC1-NTD to the energetics of ß-Pol binding. Establishing the direct contacts in the ß-Pol XRCC1 complex is a critical step in understanding how XRCC1 fulfills its numerous functions in DNA BER.
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