MUTATIONS IN HAMSTER SINGLE-STRAND BREAK REPAIR GENE XRCC1 CAUSING DEFECTIVE-DNA REPAIR

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing g roup 1 (XRCC1) is unknown and the exact role of the XRCC1 protein rema ins unclear, To help clarify the role of the XRCC1 gene we analyzed fo ur mutant cell lines of this complementation group and a revertant cel l line for XRCC1 protein content and for sequence alterations in the X RCC1 coding region, Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants co ntained a normal level of XRCC1, Although each of these cell lines exp ressed XRCC1 mRNA, we found in all cases a distinct point mutation res ulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids, Two of the mutations cause non-conservative amino ac id changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invar iant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains, A third debilitating mutation disrupts R NA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsect ion of the XRCC1 coding sequence. A fourth mutation results in a termi nation codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same cod on, Stop221-->Leu, permits translation of a full-length functional var iant protein. These mutational data indicate the importance of the put ative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.