Abstract
The efficient repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity. In mammalian cells, the nonhomologous end-joining process that represents the predominant repair pathway relies on the DNA-dependent protein kinase (DNA-PK) and the XRCC4-DNA ligase IV complex. Nonetheless, several in vitro and in vivo results indicate that mammalian cells use more than a single end-joining mechanism. While searching for a DNA-PK-independent end-joining activity, we found that the pretreatment of DNA-PK-proficient and -deficient rodent cells with an inhibitor of the poly(ADP-ribose) polymerase-1 enzyme (PARP-1) led to increased cytotoxicity of the highly efficient DNA double-strand breaking compound calicheamicin gamma1. In addition, the repair kinetics of the DSBs induced by calicheamicin gamma1 was delayed both in PARP-1-proficient cells pretreated with the PARP-1 inhibitor and in PARP-1-deficient cells. In order to get new insights into the mechanism of an alternative route for DSBs repair, we have established a new synapsis and end-joining two-step assay in vitro, operating on DSBs with either nuclear protein extracts or recombinant proteins. We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that actually required a novel synapsis activity of PARP-1 and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway. Taken together, these results strongly suggest that a PARP-1-dependent DSBs end-joining activity may exist in mammalian cells. We propose that this mechanism could act as an alternative route of DSBs repair that complements the DNA-PK/XRCC4/ligase IV-dependent nonhomologous end-joining.
Highlights
Introductiondouble-strand breaks (DSBs) are repaired through two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ) (for reviews see Refs. 3– 6)
In eukaryotic cells, double-strand breaks (DSBs) are repaired through two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ)
We found an end-joining activity independent of the DNA-PK/XRCC4-ligase IV complex but that required a novel synapsis activity of poly(ADP-ribose) polymerase-1 enzyme (PARP-1) and the ligation activity of the XRCC1-DNA ligase III complex, proteins otherwise involved in the base excision repair pathway
Summary
DSBs are repaired through two major pathways: homologous recombination (HR) and nonhomologous end-joining (NHEJ) (for reviews see Refs. 3– 6). The endjoining reaction has been studied in vitro by various assays, many of them using incubation with cell extracts of plasmid DNA linearized by enzymatic restriction as a model for DSBs containing substrates [17] Results of these in vitro repair experiments have brought substantial evidence for an alternative DNA-PK-independent end-joining pathway (18 –24). We developed a DNA pull-down assay using nuclear extracts (NE) from mammalian cells By using this approach, we have identified a new synapsis and end-joining activity independent of the classical NHEJ proteins. We have reconstituted this DSB end-joining activity with recombinant PARP-1, XRCC1, and DNA ligase III, proteins otherwise involved in BER [52] Taken together, these results suggest that a PARP-1-dependent DSB end-joining mechanism operates in cells as an alternative route that complements the DNA-PK/ XRCC4/ligase IV-dependent NHEJ
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