Substrate specificity of the p53-associated 3'-5' exonuclease.
p53 exhibits 3'-5' exonuclease activity and the significance of this biochemical function is currently not defined. In order to gain information about the potential role(s) of this exonuclease activity, recombinant and wild-type human p53 was examined for excision of nucleotides from defined synthetic DNA substrates. p53 removes nucleotides threefold faster from single-strand DNA than from DNA duplexes, exhibits a 1.5-fold preference for 3'-terminals of DNA that contain a single nucleotide mispair (mismatch) as compared to correctly paired DNA and efficiently excises nucleotides from 3'-ends of blunt and cohesive (staggered) DNA double-strand breaks. The p53 exonuclease is predominantly non-processive on DNA which is 17 nucleotides long (or shorter) and processive on the longer 30-mers. The processivity of nucleotide excision is decreased in the presence of 50 mM potassium phosphate and eliminated when full-length p53 is replaced with the core domain, comprised of amino acids 82-292. Photoaffinity labeling indicates that (1) p53 monomers, rather than dimers, bind to single-strand forms of these oligomers; (2) complexes between p53 and 30-mers are more stable than those formed with 17-mers. The stability of these complexes determines processivity during nucleotide removal and modulates the 3'-5' exonuclease activity of p53. The relevance of substrate specificity of the p53 exonuclease to DNA repair is discussed.
Skalski
V
V
Department of Medical Biophysics, University of Toronto, Canada.
Lin
Z Y
ZY
Choi
B Y
BY
Brown
K R
KR
D000818:Animals; D020137:Base Pair Mismatch; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology; D013379:Substrate Specificity; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D004277:DNA, Single-Stranded; D009692:Nucleic Acid Heteroduplexes; D016159:Tumor Suppressor Protein p53; D004247:DNA; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V