Poly(ADP-ribosyl)ation accelerates DNA repair in a pathway dependent on Cockayne syndrome B protein

Abstract

Activation of poly(ADP-ribose)polymerases 1 and 2 (PARP-1 and PARP-2) is one of the earliest responses of mammalian cells to DNA damage by numerous genotoxic agents. We have analysed the influence of PARP inhibition, either achieved by over-expression of the DNA binding domain of PARP-1 or by treatment with 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone, on the repair of single-strand breaks (SSB), pyrimidine dimers and oxidative base modifications sensitive to Fpg protein (mostly 8-hydroxyguanine) in mammalian cells at very low, non-cytotoxic levels of DNA damage. The data show that the repair rates of all three types of DNA damage are significantly lower in PARP-inhibited cells. Importantly, the retardation of the repair of base modifications is not associated with accumulation of intermediates such as SSB or abasic sites. Moreover, the influence of the PARP inhibition is not observed in cells deficient in Cockayne syndrome B protein (Csb). The results indicate that PARP activation and Csb are both involved in a novel mechanism that accelerates the global repair of various types of DNA modifications.

Figure 1

Repair kinetics of various oxidative DNA modifications in cells undergoing trans-dominant PARP inhibition (COM3 in the presence of Dex) and control cells (COM3 without Dex, COR3 in the presence or absence of Dex). (A) Repair of Fpg-sensitive oxidative modifications induced by Ro 19-8022 (0.05 µM) plus visible light. (B) Repair of cyclobutane pyrimidine dimers sensitive to T4 endonuclease V induced by UVB (2.4 J/m²). (C) Repair of SSB induced by tert-butylhydroperoxide (100 µM). Data represent means of three to six independent experiments (± SD).

Figure 2

Number of SSB and sites of base loss (sensitive to endonuclease IV) during the repair of Fpg-sensitive base modifications induced by Ro 19-8022 (0.05 µM) plus visible light in COM3 cells without (left panel) or with (right panel) trans-dominant inhibition of PARP.

Figure 3

Influence of PARP inhibition by DPQ (10 µM) on the repair of SSB, Fpg-sensitive DNA modifications and pyrimidine dimers at selected time points in COM3 cells. Data represent means of three to four independent experiments (± SD).

Figure 4

Cleavage of a 34mer duplex DNA containing a single 8-oxoG residue at position 16 by crude protein extracts of COR3 and COM3 cells, with or without PARP inhibition by pre-treatment of the cells with Dex (0.05 µM) or DPQ (10 µM). The 34mer oligodeoxyribonucleotide was 5′-³²P-labelled in the 8-oxoG-containing strand and incubated with 5 µg of the crude cell-free extracts for 30 min at 37°C. The cleavage products were analysed by 20% denaturing PAGE. Controls are [8-oxoG:C] duplex incubated with buffer alone (–) or with 10 ng purified human Ogg1 protein (+).

Figure 5

Repair of Fpg-sensitive oxidative base modifications induced by Ro 19-8022 plus light (A and B) and of cyclobutane pyrimidine dimers (C and D) in wild-type (F11.1; A and C) and Csb-deficient (B and D) MEFs with and without PARP inhibition by pre-treatment with DPQ (10 µM). Data represent the means of three to four independent experiments (± SD).