Characterization of the apurinic endonuclease activity of Drosophila Rrp1.
Drosophila Rrp1 (Recombination repair protein 1) belongs to a family of DNA repair nucleases that includes Escherichia coli exonuclease III, Streptococcus pneumoniae exonuclease A, bovine BAP, mouse APEX endonuclease, and human APE. Within a 252 amino acid region, colinear homology 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-stranded DNA 3'-exonuclease. In this study, a 5'-end-labeled 37 base pair oligonucleotide substrate containing a single apurinic site was used to characterize the endonuclease activity of Rrp1. This substrate is utilized 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 endonucleolytic cleavage at the abasic site, exonucleolytic processing at the nick is slow and requires a molar excess of Rrp1, while exonuclease III degrades the nicked substrate more efficiently. The Rrp1 cleavage product comigrates with a DNaseI cleavage product, and the newly formed terminus supports DNA synthesis by DNA polymerase. Therefore, Rrp1 cleaves the phosphodiester backbone at one position 5' to the apurinic site and leaves a 3'-hydroxyl terminus. Rrp1 is a class II apurinic endonuclease and is likely to be important in DNA repair in Drosophila.
Nugent
M
M
Laboratory of Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Huang
S M
SM
Sander
M
M
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004330:Drosophila / Q000201:enzymology*; D029721:Drosophila Proteins; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D004277:DNA, Single-Stranded; D029721:Drosophila Proteins; D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D009713:Nucleotidyltransferases; C061900:Rrp1 protein, Drosophila; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D043223:Deoxyribonuclease IV (Phage T4-Induced); C468972:endonuclease IV, E coli; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase