Suppression of base excision repair reactions by apoptotic 24kDa-fragment of poly(ADP-ribose) polymerase 1 in bovine testis nuclear extract.
In this study, we examined the interaction of PARP1 and its apoptotic 24kDa-fragment with DNA duplexes mimicking different stages/pathways of base excision repair (BER) using a photocross-linking technique combined to in vitro functional assay. We found that endogenous PARP1 was photocross-linked to the gapped, nicked and flap containing DNA structures and its apoptotic 24kDa-fragment (p24), like PARP1, can interact with the same BER DNA intermediates. Effects of exogenous p24 on the repair of DNA duplexes containing a one nucleotide gap with furan phosphate or phosphate group at the 5'-end of the downstream primer were studied in bovine testis nuclear extract. We showed that the interaction of p24 with DNA, as a whole, inhibited the BER reactions. However, gap filling and nick sealing catalyzed by the enzymes of the extract with DNA substrates characteristic for short patch (SP) BER pathway cannot be completely inhibited by p24. In contrast, binding of p24 to DNA duplex with a 5'-furan or a 5'-flap at the 5'-side of a nick inhibits strand-displacement DNA synthesis and activity of FEN1 in the repair of DNA via long patch (LP) BER pathway. Stimulation of the LP BER reactions induced by the addition of FEN1 or PCNA to the extract is suppressed by p24 thereby indicating that p24 can efficiently compete with these proteins of LP BER. Addition of pol beta to the extract can partially overcome the inhibitory effect of p24 and restore strand-displacement DNA synthesis. Thus, the apoptotic 24kDa-fragment of PARP1 may be considered as more efficient in inhibition of the LP than SP pathway and the effect may depend on the ratio of p24 to the repair enzymes catalyzing precise stages of BER.
Sukhanova
Maria
M
Institute of Chemical Biology and Fundamental Medicine, Prospect Lavrentieva 8, 630090 Novosibirsk, Russia.
Khodyreva
Svetlana
S
Lavrik
Olga
O
D000818:Animals; D017209:Apoptosis; D002417:Cattle; D002467:Cell Nucleus / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair; D045585:Flap Endonucleases / Q000378:metabolism; D008027:Light; D008297:Male; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding; D013737:Testis / Q000166:cytology / Q000201:enzymology*
D003432:Cross-Linking Reagents; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta; D045585:Flap Endonucleases