Abstract
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.
Highlights
Introduction of Photoreactive FABdCMP Probe into DNA—The reaction conditions for evaluating DNA synthesis with photoreactive FAB-dCTP or dCTP were identical to those used for the cross-linking experiments
The extreme hypersensitivity imposed by the PARP inhibitors suggests that PARP is involved in both of the base excision repair (BER) sub-pathways, since our previous results indicated that each BER sub-pathway results in greater resistance to Methyl methanesulfonate (MMS) than that seen in the presence of 4-AN [25]
The poly(ADP-ribose) polymerase-1 (PARP-1) null cells were hypersensitive to MMS (Fig. 1B), and with PARP inhibitors the wild-type cells became even more sensitive than the PARP-1 null cells suggesting a role in BER for other proteins with PARP activity in addition to PARP-1
Summary
Vol 276, No 27, Issue of July 6, pp. 25541–25548, 2001 Printed in U.S.A. Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate. 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. 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 , and apurinic/apyrimidinic endonuclease. 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. We examined the question of whether proteins in MEF crude extracts can be selectively labeled by a photoaffinity DNA probe representing an early intermediate in long patch BER. The results described here reveal several important features of molecular interactions between BER enzymes and BER intermediates
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
