Differential effects of poly(ADP-ribose) polymerase inhibition on DNA break repair in human cells are revealed with Epstein-Barr virus

Chromosome Stability Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/2012; 109(17):6590-5. DOI: 10.1073/pnas.1118078109
Source: PubMed


Poly(ADP-ribose) polymerase (PARP) inhibitors can generate synthetic lethality in cancer cells defective in homologous recombination. However, the mechanism(s) by which they affect DNA repair has not been established. Here we directly determined the effects of PARP inhibition and PARP1 depletion on the repair of ionizing radiation-induced single- and double-strand breaks (SSBs and DSBs) in human lymphoid cell lines. To do this, we developed an in vivo repair assay based on large endogenous Epstein-Barr virus (EBV) circular episomes. The EBV break assay provides the opportunity to assess quantitatively and simultaneously the induction and repair of SSBs and DSBs in human cells. Repair was efficient in G1 and G2 cells and was not dependent on functional p53. shRNA-mediated knockdown of PARP1 demonstrated that the PARP1 protein was not essential for SSB repair. Among 10 widely used PARP inhibitors, none affected DSB repair, although an inhibitor of DNA-dependent protein kinase was highly effective at reducing DSB repair. Only Olaparib and Iniparib, which are in clinical cancer therapy trials, as well as 4-AN inhibited SSB repair. However, a decrease in PARP1 expression reversed the ability of Iniparib to reduce SSB repair. Because Iniparib disrupts PARP1-DNA binding, the mechanism of inhibition does not appear to involve trapping PARP at SSBs.

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    • "Frequently, at high doses of irradiation, two such nicks are present in complementary DNA strands within one helical turn leading to DSBs (Milligan et al. 1995). There are about 10 SSBs for each DSB created by IR (Ma et al. 2012). IR breakage frequently leaves " dirty ends, " consisting of phosphoglycolates and terminal nucleotides, that cannot be ligated to " clean " ends consisting of a 5 0 phosphate and 3 0 -OH group, such as those created by endonucleases (Weinfeld and Soderlind 1991). "
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    • "We used two conditions to ensure that strand breaks were quantitated accurately: for PFGE, DNA was deproteinised at room temperature because extra strand breaks are created at higher temperatures [68], and hybridisation was carried out in dried gels because the transfer of large DNA fragments onto membranes [9], [10] is not quantitative [69]. In another study [70] published while this manuscript was in preparation, a significant amount of minichromosome DNA remained in the sample well of PFGE gels and was interpreted as nicked circles, but here little or no DNA remained in the wells and nicked circular DNA migrated slowly into the gel, possibly reflecting methodological differences. A Poisson distribution of strand breaks was assumed in [70], but is not consistent with our finding that only one double strand break is formed in minichromosome DNA in irradiated cells (Figure 1 and [43]); this assumption is not supported strongly by experimental evidence and does not take into account the variable conformations and microenvironments of chromatin in the nucleus. "
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