CLONING AND CHARACTERIZATION OF THE PROMOTER OF BABOON XRCC1, A GENE INVOLVED IN DNA STRAND-BREAK REPAIR

The DNA repair gene XRCC1 was the first cloned human DNA repair gene i nvolved in resistance to ionizing radiation. Previous studies have sho wn that rodent and baboon homologs of XRCC1 are expressed in all reste d tissues with significantly higher levels in testis. Furthermore, exp ression of murine XRCC1 is most abundant in pachytene spermatocytes an d round spermatids. To begin to study regulation of XRCC1 expression, the 5' region of baboon XRCC1 was cloned and characterized. 400 bp of 5'-flanking region showed the greatest promoter activity, while -194 t o -8 bp of the 5'-flanking region displayed core promoter activity in transient transfection assays. A comparison between baboon and human 5 '-flanking sequences in the core promoter region revealed a potential CAAT-box, an imperfect CREB-binding site and two putative Sp1-binding sites, Results from transient transfection assays in which each putati ve binding site was individually mutated indicated that the distal Sp1 -binding site has a functional role in transcription In comparison, bo th putative Sp1-binding sites bound protein(s)from HeLa cell nuclear e xtracts in vitro. In vitro binding was lost when mutated Sp1 sires wer e used in gel mobility shift assays. Finally anti-Sp1 antibodies produ ced mobility supershifts, thereby indicating Sp1 or an Sp1-like protei n bound to the DNA fragment in vitro.