CHARACTERIZATION OF THE APURINIC ENDONUCLEASE ACTIVITY OF DROSOPHILA RRP1

Drosophila Rrp1 (Recombination repair protein 1) belongs to a family o f DNA repair nucleases that includes Escherichia coli exonuclease III, Streptococcus pneumoniae exonuclease A, bovine BAP, mouse APEX endonu clease, and human APE. Within a 252 amino acid region, collinear homol ogy is shared between all members. Rrp1 is unique in that it includes a 427 amino acid N-terminal region not related to any known sequence. The protein copurifies with an apurinic endonuclease and a double-stra nded DNA 3'-exonuclease. In this study, a 5'-end-labeled 37 base pair oligonucleotide substrate containing a single apurinic site was used t o characterize the endonuclease activity of Rrp1. This substrate is ut ilized efficiently by Rrp1: the specific activity observed is 1 x 10(5 ) units/mg. The abasic double-stranded DNA oligonucleotide is cleaved only at the abasic site to create a single-strand break. Strand breaks are not detected in the complementary strand, in the single-stranded DNA oligonucleotide, or in the base-paired control substrate. After en donucleolytic cleavage at the abasic site, exonucleolytic processing a t the nick is slow and requires a molar excess of Rrp1, while exonucle ase III degrades the nicked substrate more efficiently. The Rrp1 cleav age product comigrates with a DNaseI cleavage product, and the newly f ormed terminus supports DNA synthesis by DNA polymerase. Therefore, Rr p1 cleaves the phosphodiester backbone at one position 5' to the apuri nic site and leaves a 3'-hydroxyl terminus. Rrp1 is a class II apurini c endonuclease and is likely to be important in DNA repair in Drosophi la.