Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair.
To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.
Lavrik
O I
OI
Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Prasad
R
R
Sobol
R W
RW
Horton
J K
JK
Ackerman
E J
EJ
Wilson
S H
SH
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002460:Cell Line; D004260:DNA Repair; D003845:Deoxycytosine Nucleotides / Q000378:metabolism; D005347:Fibroblasts / Q000201:enzymology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes / Q000378:metabolism*; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D019920:Photoaffinity Labels / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet
D003845:Deoxycytosine Nucleotides; D007527:Isoenzymes; D019920:Photoaffinity Labels; C024107:2'-deoxycytidine 5'-triphosphate; D008741:Methyl Methanesulfonate; D011065:Poly(ADP-ribose) Polymerases