Cloning and characterization of the promoter of baboon XRCC1, a gene involved in DNA strand-break repair.

ABSTRACT

The DNA repair gene XRCC1 was the first cloned human DNA repair gene involved in resistance to ionizing radiation. Previous studies have shown that rodent and baboon homologs of XRCC1 are expressed in all tested tissues with significantly higher levels in testis. Furthermore, expression of murine XRCC1 is most abundant in pachytene spermatocytes and 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 to -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 putative binding site was individually mutated, indicated that the distal Sp1-binding site has a functional role in transcription. In comparison, both putative Sp1-binding sites bound protein(s) from HeLa cell nuclear extracts in vitro. In vitro binding was lost when mutated Sp1 sites were used in gel mobility shift assays. Finally, anti-Sp1 antibodies produced mobility supershifts, thereby indicating Sp1 or an Sp1-like protein bound to the DNA fragment in vitro.