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PCNA promotes processive DNA end resection by Exo1.

Chen X, Paudyal SC, Chin RI, You Z - Nucleic Acids Res. (2013)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.

ABSTRACT
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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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).
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gkt672-F3: 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).

Mentions: To determine whether Exo1 is required for the loading of PCNA to DSBs, we examined the association of PCNA with DNA damage sites induced by laser irradiation in control- or Exo1-knockdown U2OS cells. Both endogenous PCNA and GFP-PCNA were efficiently recruited to DNA damage sites in Exo1-knockdown cells, and there was no significant difference in PCNA damage association in control- and Exo1-knockdown cells (Figure 3A and B and Supplementary Figure S3B). Moreover, depletion of Exo1 from the Xenopus extract also did not cause obvious effects on PCNA damage association (Figure 3C). Furthermore, purified recombinant PCNA protein alone could bind to a dsDNA fragment in vitro (Figure 3D). Taken together, these data indicate that PCNA loads onto DSBs after DNA damage and that this loading does not require Exo1.Figure 3.


PCNA promotes processive DNA end resection by Exo1.

Chen X, Paudyal SC, Chin RI, You Z - Nucleic Acids Res. (2013)

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).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3814391&req=5

gkt672-F3: 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).
Mentions: To determine whether Exo1 is required for the loading of PCNA to DSBs, we examined the association of PCNA with DNA damage sites induced by laser irradiation in control- or Exo1-knockdown U2OS cells. Both endogenous PCNA and GFP-PCNA were efficiently recruited to DNA damage sites in Exo1-knockdown cells, and there was no significant difference in PCNA damage association in control- and Exo1-knockdown cells (Figure 3A and B and Supplementary Figure S3B). Moreover, depletion of Exo1 from the Xenopus extract also did not cause obvious effects on PCNA damage association (Figure 3C). Furthermore, purified recombinant PCNA protein alone could bind to a dsDNA fragment in vitro (Figure 3D). Taken together, these data indicate that PCNA loads onto DSBs after DNA damage and that this loading does not require Exo1.Figure 3.

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO 63110, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus