Poly(ADP-ribose) polymerase (PARP-1) has a controlling role in homologous recombination

Abstract

Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-deficient cells fail to form Rad51 foci in response to DNA damage. Here, we show that PARP-1 mainly does not localise to Rad51 foci and that Rad51 foci form in PARP-1-/- cells, also in response to hydroxyurea. Furthermore, we show that homology directed repair following induction of a site-specific DSB is normal in PARP-1-inhibited cells. In contrast, inhibition or loss of PARP-1 increases spontaneous Rad51 foci formation, confirming a hyper recombination phenotype in these cells. Our data suggest that PARP-1 controls DNA damage recognised by HR and that it is not involved in executing HR as such.

Strand invasion involving short tract gene conversion is specifically suppressed in BRCA2-deficient hamster cells

“…The I-SceI restriction endonuclease recognizes an 18 bp long restriction site present in the S2neo gene and likely not elsewhere in the genome ( Figure 1a) (Johnson et al, 1999). An I-SceI-induced DSB induces HR in wild-type V79 Chinese hamster cells (Figure 2a) (Schultz et al, 2003). In contrast, the frequency of ISceI-induced neo R clones is lower in the BRCA2-deficient V-C8 cell line, which is also derived from the V79 Chinese hamster cell line (Kraakman-van der Zwet et al, 2002).…”

“…We stably transfected the SCneo vector by electroporation into the BRCA2-deficient V-C8 cell line (Kraakman-van der Zwet et al, 2002) and isolated independent hygromycin-resistant clones that were further expanded using methods previously described (Schultz et al, 2003). DNA was isolated from these clones to allow analysis of the integration of SCneo by Southern blotting.…”

“…We found that the recombination rate is 12 times lower in BRCA2-deficient hamster cells (Figure 2b), in agreement with Figure 1 The SCneo substrate for HR in V-C8 cells. (a) The SCneo recombination substrate, containing two unfunctional copies of the neo R gene, was electroporated into the V-C8 cell line and hyg R colonies were isolated as described previously (Schultz et al, 2003). (b) Following an I-SceI-induced DSB (by transient transfection of 1.5 Â 10 6 cells with the pCMV3xnlsI-SceI vector using lipofectamine 2000 (Invitrogen)), a functional neo R gene can be gained by HR through intrachromatid paring or sister chromatid pairing.…”

BRCA1, PARP, and 53BP1: conditional synthetic lethality and synthetic viability

“…These data support a model in which PARP1/2 activity is required for efficient recruitment of MRE11-dependent end resection activity to stalled replication forks. Of note, PARP1 2/2 cells and PARP inhibitor treated cells do not have drastic defects in HR-mediated repair of DNA DSBs induced by restriction enzymes and PARP1 2/2 ES cells do not have a dramatic defect in gene targeting (Schultz et al, 2003;Yang et al, 2004). These data together imply that PARP1/2 may have a specific role in HR-mediated replication fork restart, but not an essential role in HR-mediated DSBR in general.…”

Activation of PARP-1 in response to bleomycin depends on the Ku antigen and protein phosphatase 5

“…Genetic studies have established a role for PARP-1 in HR (Hochegger et al, 2006;Idogawa et al, 2007), whereas biochemical and genetic studies have linked PARP-1 to NHEJ (Schultz et al, 2003;Perrault et al, 2004;Audebert et al, 2006;Wang et al, 2006). PARP-1 has been shown to interact with the NHEJ proteins, Ku antigen and DNA-dependent protein kinase (DNA-PK) with reciprocal modifications and mutual alterations in activity suggested (Ruscetti et al, 1998;Ariumi et al, 1999;Galande and KohwiShigematsu, 1999).…”