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J Biol Chem
2015 May 08;29019:12300-12. doi: 10.1074/jbc.M115.644005.
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14-3-3 proteins restrain the Exo1 nuclease to prevent overresection.
Chen X, Kim IK, Honaker Y, Paudyal SC, Koh WK, Sparks M, Li S, Piwnica-Worms H, Ellenberger T, You Z.
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The DNA end resection process dictates the cellular response to DNA double strand break damage and is essential for genome maintenance. Although insufficient DNA resection hinders homology-directed repair and ATR (ataxia telangiectasia and Rad3 related)-dependent checkpoint activation, overresection produces excessive single-stranded DNA that could lead to genomic instability. However, the mechanisms controlling DNA end resection are poorly understood. Here we show that the major resection nuclease Exo1 is regulated both positively and negatively by protein-protein interactions to ensure a proper level of DNA resection. We have shown previously that the sliding DNA clamp proliferating cell nuclear antigen (PCNA) associates with the C-terminal domain of Exo1 and promotes Exo1 damage association and DNA resection. In this report, we show that 14-3-3 proteins interact with a central region of Exo1 and negatively regulate Exo1 damage recruitment and subsequent resection. 14-3-3s limit Exo1 damage association, at least in part, by suppressing its association with PCNA. Disruption of the Exo1 interaction with 14-3-3 proteins results in elevated sensitivity of cells to DNA damage. Unlike Exo1, the Dna2 resection pathway is apparently not regulated by PCNA and 14-3-3s. Our results provide critical insights into the mechanism and regulation of the DNA end resection process and may have implications for cancer treatment.
FIGURE 5.
The central region of Exo1 limits DNA end resection activity in a Xenopus egg extract. A, immunodepletion (depl) of the Xenopus Dna2 protein (xDna2) or of both xDna2 and xExo1 from the Xenopus NPE. B, physical association of Exo1(WT)-His and Exo1(ΔCR)-His with a bead-immobilized, 2-kb DNA fragment in xExo1-depleted NPE. C, DNA end resection activity toward a 3′ 32P-labeled, 6-kb dsDNA fragment in the Exo1 and Dna2 codepleted NPE depicted in A that was supplemented with Exo1(WT)-His or Exo1(ΔCR)-His. Vertical lines were added for sample lane identification.
FIGURE 6.
14-3-3 proteins repress Exo1-mediated DNA end resection in a Xenopus egg extract. A, purified recombinant GST, GST-Difopein(WT), and GST-Difopein(MUT) were expressed in and purified from BL21(DE3) E. coli cells. MW, molecular weight. B, effects of GST, GST-Difopein(WT), or GST-Difopein(MUT) on the association of xExo1 with 14-3-3 proteins in the Xenopus NPE. The asterisk denotes a nonspecific band detected by the anti-14-3-3 antibody. IP, immunoprecipitation. C, effects of GST, GST-Difopein(WT) or GST-Difopein(MUT) on DNA end resection in the extract. Vertical lines were added for sample lane identification. D, effects of GST-Difopein(WT) or GST-Difopein(MUT) on DNA end resection in the mock-depleted (depl) and xExo1-depleted or xDna2-depleted extracts. Vertical lines were added for sample lane identification. E, effects of GST-Difopein(WT) or GST-Difopein(MUT) on DNA end resection mediated by added Exo1(WT)-His or Exo1(ΔCR)-His in the extract depleted of both xExo1 and xDna2. Vertical lines were added for sample lane identification.
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