Poly ( ADP-RIBOSE ) polymerase-1 ( Parp-1 ) antagonizes topoisomerase I-dependent recombination stimulation by P 53

PARP-1 interacts with and poly(ADP-ribosyl)ates p53 and topoisomerase I, which both participate in DNA recombination. Previously, we showed that PARP-1 downregulates homology-directed doublestrand break (DSB) repair. We also discovered that, despite the well-established role of p53 as a global suppressor of error-prone recombination, p53 enhances homologous recombination (HR) at the RARa breakpoint cluster region (bcr) comprising topoisomerase I recognition sites. Using an SV40based assay and isogenic cell lines differing in the p53 and PARP-1 status we demonstrate that PARP-1 counteracts HR enhancement by p53, although DNA replication was largely unaffected. When the same DNA element was integrated in an episomal recombination plasmid, both p53 and PARP-1 exerted anti-recombinogenic rather than stimulatory activities. Strikingly, with DNA substrates integrated into cellular chromosomes, enhancement of HR by p53 and antagonistic PARP-1 action was seen, very similar to the HR of viral minichromosomes. siRNA-mediated knockdown revealed the essential role of topoisomerase I in this regulatory mechanism. However, after I-SceI-meganucleasemediated cleavage of the chromosomally integrated substrate, no topoisomerase I-dependent effects by p53 and PARP-1 were observed. Our data further indicate that PARP-1, probably through topoisomerase I interactions rather than poly(ADPribosyl)ation, prevents p53 from stimulating spontaneous HR on chromosomes via topoisomerase I activity. INTRODUCTION PARP-1 plays fundamental roles in the recruitment and modulation of enzymatic and regulatory factors involved in transcription, DNA replication, repair and recombination [reviewed in (1–3)]. Importantly, PARP-1 catalyses poly(ADP-ribosyl)ation of many of these proteins including itself and dissociates from DNA after auto-modification (4). PARP-1 is enzymatically activated by binding to single-strand breaks (SSBs) and participates in base excision repair (5,6). Given that PARP-1 also recognizes double-strand breaks (DSBs), interacts with Ku70/80, the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), and the RecQ helicase WRN, and regulates the biochemical activities of DNA-PKcs and WRN (7–10), several groups investigated potential activities of PARP-1 in DSB repair. Moreover, two recent studies showed that dysfunction of homologydirected DSB repair sensitizes cells to PARP inhibition suggesting that PARP enzymatic activity is required to avoid the accumulation of lesions which are repaired by homologous recombination (HR) (11,12). DSBs are caused spontaneously during physiological DNA processing in replication, immunoglobulin gene diversification and meiosis and can arise from exogenous DNAdamaging agents, including ionizing radiation or cancer chemotherapeutic agents. The two major pathways of DSB repair are nonhomologous end joining (NHEJ) and HR (13,14). In the NHEJ pathway, Ku70 and Ku80 bind the DSB, followed by recruitment and activation of DNA-PKcs, which mediates synapsis and recruits XRCC4 and DNA Ligase IV. In mammalian cells HR requires a protein complex comprising Mre11, Rad50 and Nbs1 for DSB recognition and end resection to yield 30-ssDNA tails. Subsequent strand exchange between the processed ssDNA and an intact homologous duplex are catalysed by Rad51. This reaction is facilitated by the DNA end-protecting protein Rad52, the DNAdependent ATPase and SNF2/SWI2 family member Rad54, as *To whom correspondence should be addressed. Tel: +49 731 50