Chen X et al. (2013), PCNA promotes processive DNA end resection by E...

XB-ART-47990

Nucleic Acids Res 2013 Nov 01;4120:9325-38. doi: 10.1093/nar/gkt672.

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PCNA promotes processive DNA end resection by Exo1.

Chen X , Paudyal SC , Chin RI , You Z .

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Exo1-mediated resection of DNA double-strand break ends generates 3' single-stranded DNA overhangs required for homology-based DNA repair and activation of the ATR-dependent checkpoint. Despite its critical importance in inducing the overall DNA damage response, the mechanisms and regulation of the Exo1 resection pathway remain incompletely understood. Here, we identify the ring-shaped DNA clamp PCNA as a new factor in the Exo1 resection pathway. Using mammalian cells, Xenopus nuclear extracts and purified proteins, we show that after DNA damage, PCNA loads onto double-strand breaks and promotes Exo1 damage association through direct interaction with Exo1. By tethering Exo1 to the DNA substrate, PCNA confers processivity to Exo1 in resection. This role of PCNA in DNA resection is analogous to its function in DNA replication where PCNA serves as a processivity co-factor for DNA polymerases.

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Species referenced: Xenopus
Genes referenced: atr blm cdkn1a dna2 exo1 fen1 mlh1 mmut nbn nsg1 pcna rad51 rbbp8 rpa1

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	Figure 1. A C-terminal region of Exo1 containing a PIP-Box facilitates Exo1 damage association. (A). Left panel: Diagram of Exo1 and its truncation mutants. Central panel: Representative images for the damage association of GFP-Exo1 and its mutants shown in the left panel. Red lines indicate the sites of laser irradiation in cells. Right panel: Quantified results for the damage association of GFP-Exo1 and its mutants shown in the left panel during the first 20 min after laser irradiation. Each data point is the average of independent measurements of five cells. Error bars represent standard deviation. (B). A conserved PIP-Box-like sequence in the C-terminus of Exo1 in vertebrates and Drosophila. In the PIP-Box consensus sequence, ‘h’ represents a hydrophobic residue and ‘a’ represents an aromatic residue. In all the ΔPIP mutants described in this study, the four key residues are mutated into alanine. (C). Association of xExo1 with xPCNA in the Xenopus nuclear extract. (D). Association of PCNA with FLAG-Exo1, but not FLAG-Exo1(ΔPIP) without a functional PIP-Box, expressed in HEK293T cells. Mutation of the PIP-Box in Exo1 did not affect its interaction with MLH1. (E). Purified, recombinant His-tagged PCNA, Exo1(WT), Exo1(ΔPIP) and Exo1(D173A) proteins expressed in Sf9 cells. (F). Direct interaction between recombinant PCNA with recombinant wild type Exo1, but not the Exo1(ΔPIP) mutant lacking a functional PIP-Box.
	Figure 2. PCNA promotes the damage association of Exo1. (A). Left panel: Representative images for the damage association of GFP-Exo1 and GFP-Exo1(ΔPIP) in human U2OS cells. Right panel: Quantified results for the damage association of GFP-Exo1 and GFP-Exo1(ΔPIP) during the first 20 min after laser irradiation. Each data point is the average of independent measurements of five cells. Error bars represent standard deviation. (B). Damage association of endogenous Exo1 in control- and PCNA-knowdown U2OS cells. γH2AX was used as a control to indicate the sites of DNA damage induced by laser irradiation. (C–E). Left panels: Representative images for the damage association of GFP-Exo1 in control-knockdown and PCNA-knockdown cells (C); of GFP-Exo1 in cells co-transfected with mCherry-NLS, mCherry-NLS-fused wild type or a mutant form of PIP-Box peptide derived from p21 (D); of GFP-Exo1, GFP-Exo1(PIPFen1-WT) and GFP-Exo1(PIPFen1-MUT) with the PIP-Box in Exo1 replaced with a wild-type or mutant PIP-Box in Fen1 (E). Red lines indicate the sites of laser irradiation in cells. Right panels: Quantified results for the damage association of the wild type or mutant GFP-Exo1 proteins depicted in the left panels during the first 20 min after laser irradiation. Each data point is the average of independent measurements of five cells. Error bars represent standard deviation. NLS, Nuclear Localization Signal.
	Figure 3. PCNA directly loads onto DSBs in an Exo1-independent manner. (A). Accumulation of endogenous PCNA at laser-induced DNA damage sites in control- or Exo1-knockdown U2OS cells. NBS1 was used as a control to indicate the sites of DSB damage in cells. (B). Left panel: Representative images for the damage association of GFP-PCNA in control- or Exo1-knockdown U2OS cells. Red lines indicate the sites of laser irradiation in cells. Right panel: Quantified results for the damage association of GFP-PCNA in control- or Exo1-knockdown U2OS cells during the first 20 min after laser irradiation. Each data point is the average of independent measurements of six cells. Error bars represent standard deviation. (C). Binding of xPCNA, xExo1, xNBS1, xRPA and xKu70 to a bead-immobilized 2 kb DNA fragment added to mock-depleted or xExo1-depleted Xenopus NPE. DNA-bound NBS1 was phosphorylated, resulting in its gel mobility shift. (D). Binding of purified His-PCNA to a bead-immobilized 2 kb DNA fragment in vitro, which was inhibited by Ethidium bromide (200 ng/µl).
	Figure 4. DNA end resection in the Xenopus nuclear extract by Exo1 requires its PIP-Box. (A) Immunodepletion of xExo1 from Xenopus NPE. (B) Depletion of xExo1 inhibited resection on a 3′ 32P-labeled 6 kb dsDNA fragment. Reactions were terminated at the indicated times, and resection products were resolved on a 0.8% agarose gel. (C) Recombinant human Exo1(WT) and Exo1(D173A) proteins, but not Exo1(ΔPIP), associated with a bead-immobilized DNA fragments in the Xenopus extract depleted of xExo1. (D) Exo1(WT) and Exo1(ΔPIP) exhibited similar levels of nuclease activity. Various amounts of purified recombinant Exo1(WT)-His or Exo1(ΔPIP)-His proteins were incubated with a 3′ 32P-labeled 6 kb dsDNA fragment. Reactions were terminated at the indicated times, and resection products were resolved on a 0.8% agarose gel. (E) Addition of recombinant human Exo1(WT), but not Exo1(ΔPIP) or Exo1(D173A), to xExo1-depleted extract rescued resection on a 3′ 32P-labeled 6 kb dsDNA fragment. Reactions were terminated at the indicated times, and resection products were resolved on a 0.8% agarose gel.
	Figure 5. Disruption of Exo1-PCNA interaction inhibited Exo1-mediated DNA resection in the Xenopus nuclear extract. (A). Addition of a wild-type, but not a mutant form, of PIP-Box peptide derived from p21 inhibited the xExo1-xPCNA interaction in the extract. (B). Addition of a wild-type, but not a mutant form, of p21 PIP-Box peptide inhibited DNA resection in the extract. (C). Addition of excessive recombinant PCNA protein to the extract neutralized the inhibitory effects of the wild-type p21 PIP-Box peptide on DNA resection. (D). xExo1-depletion abrogated the inhibitory effects of the wild type p21 PIP-Box peptide on DNA resection in the extract.
	Figure 6. PCNA enhances the processivity of Exo1 in resection. (A). Resection of a 3′ 32P-labeled 6 kb dsDNA fragment by recombinant, His-tagged Exo1(WT), Exo1(ΔPIP) or Exo1(D173A) in the absence or in the presence of PCNA. PCNA: 300 nM (monomer); All Exo1 proteins: 15 nM. (B). In the in vitro resection reactions containing purified Exo1 and PCNA or containing Exo1 alone, circular ssDNA derived from pBluescript SK(−), which binds and sequesters Exo1, was added at 0 or 2 min. PCNA exhibited stimulatory effects on DNA resection by Exo1(WT), but not Exo1(ΔPIP), when the sequestering ssDNA was added at 2 min, but not at 0 min, presumably because Exo1 (WT) was tethered to the DNA substrate by PCNA during the first 2 min incubation. PCNA: 300 nM (monomer); All Exo1 proteins: 15 nM; pBluescript SK(−) ssDNA: 3 nM.
	Figure 7. A model for DNA end resection. DNA end resection is initiated by MRN and CtIP through internal cleavage of 5′-strand DNA from the DNA ends. Subsequent resection extension is carried out by two parallel pathways mediated by Exo1 and Dna2, respectively. In the Exo1 pathway, PCNA loads onto the DSB from the ends and serves as a processivity factor for Exo1 in resection extension. MRN also enhances Exo1 processivity. The BLM helicase promotes Dna2-mediated resection extension. Binding of RPA to the 3′ ssDNA overhangs generated by end resection leads to recruitment and activation of ATR and subsequent checkpoint response. Replacement of RPA by Rad51 on the 3′ ssDNA overhangs leads to DNA repair by HR.

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