OPEN COMPLEX-FORMATION AROUND A LESION DURING NUCLEOTIDE EXCISION-REPAIR PROVIDES A STRUCTURE FOR CLEAVAGE BY HUMAN XPG PROTEIN

Human XPG nuclease makes the 3' incision during nucleotide excision re pair of DNA, The enzyme cleaves model DNA bubble structures specifical ly near the junction of unpaired DNA with a duplex region, It is not y et known, however, whether an unpaired structure is an intermediate du ring actual DNA repair, We find here that XPG requires opening of >5 b p for efficient cleavage, To seek direct evidence for formation of an open structure around a lesion in DNA during a nucleotide excision rep air reaction in vitro, KMnO4 footprinting experiments were performed o n a damaged DNA molecule bearing a uniquely placed cisplatin adduct, A n unwound open complex spanning similar to 25 nucleotides was observed that extended to the positions of 5' and 3' incision sites and was de pendent on XPA protein and on ATP. Opening during repair occurred prio r to strand incision by XPG.