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. 2003 Sep 1;31(17):4959-64.
doi: 10.1093/nar/gkg703.

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

Niklas Schultz  1 , Elena Lopez, Nasrollah Saleh-Gohari, Thomas Helleday
Affiliations

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

Niklas Schultz et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Localisation of PARP-1 and Rad51 in the human cell lines SKUT-1, DLD-1 and SW480. In merged images, DNA is displayed in blue, anti-Rad51 in red and anti-PARP-1 in green.
Figure 2
Figure 2
DNA (blue) and Rad51 foci (red) are visualised in A11 (PARP–/–) or A19 (PARP+/+) cells with or without a 24-h hydroxyurea treatment (0.2 mM).
Figure 3
Figure 3
Recombination substrate integrated in V79(SPD8) cells. (A) Structure of SCneo (29). (B) Predicted HR products resulting in G418 resistance. (C) Southern blot on DNA isolated from the SCneo transfected cell line S8SN.11 and probed with the S2neo fragment.
Figure 4
Figure 4
Recombination frequency in the S8SN.11 cell line with or without inhibition of PARP-1 using ISQ. A DSB was induced in the SCneo substrate by transient transfection with the pCMV3xnlsI-SceI vector.
Figure 5
Figure 5
Percentage cells containing more than 10 Rad51 foci in A11 (PARP–/–) or A19 (PARP+/+) cells following a 24-h treatment with 3-AB (2 mM), ISQ (0.6 mM) or NU1025 (0.1 mM). At least 300 nuclei were counted for each treatment and experiment. Error bars designate standard error from at least three experiments.
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