RECOMBINATION ACTIVATING ACTIVITY OF XRCC1 ANALOGOUS GENES IN X-RAY SENSITIVE AND RESISTANT CHO CELL-LINES

The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CH O) mutant cell line EM9 but the mechanism of the correction is not elu cidated yet. XRCC1 shows substantial homology to the RAG2 gene (recomb ination activating gene) and we therefore tried to answer the question , whether structural similarities (sequence of a putative recombinatio n activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) woul d reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologou s to the structural part of human XRCC1 was present in cDNA of CHO. Di fferential display demonstrated two putative recombination activating domains in the parental CHO line AA8 and one in the radiosensitive mut ant EM9. Southern blot experiments showed the presence of several gene s with partial homology to human XRCC1. Recombination studies consiste d of expressing amplified target domains within chimeric proteins in r ecA(-) bacteria and subsequent detection of recombination events by se quencing the recombinant plasmids. Recombination experiments demonstra ted recombination activating activity of all putative recombination ac tivating domains amplified from AA8 and EM9 genomes as reflected by de letions within the inserts of the recombinant plasmids. The recombinat ion activating activity of XRCC1 analogues could explain a mechanism r esponsible for the correction of the DNA repair defect in EM9. (C) 199 7 Elsevier Science Ltd.