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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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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.
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gkt672-F4: 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.

Mentions: The role of PCNA in facilitating the damage association of Exo1 suggests that PCNA promotes DNA end resection. Directly testing this in cells could be difficult, as PCNA is also involved in DNA replication and is essential for cell viability, which could confound the functional analysis of PCNA specifically in DSB resection (49,50). To circumvent this issue, we chose to use the Xenopus nuclear extract, which maintains the proper DSB resection process but alone lacks DNA replication (42,51). To assay DNA end resection in the extract, we used a 3′-end 32P-labeled 6 kb dsDNA fragment and examined the length changes of the DNA substrate resulted from resection on agarose gels. Consistent with previous studies in Xenopus egg cytosol (52), we found that immunodepletion of xExo1 from the Xenopus nuclear extract partially inhibited DNA end resection (Figure 4A and B). The partial effects of Exo1-depletion on resection are consistent with the notion that Exo1 and Dna2 function in parallel in resection (23–25,51–55).Figure 4.


PCNA promotes processive DNA end resection by Exo1.

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

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.
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gkt672-F4: 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.
Mentions: The role of PCNA in facilitating the damage association of Exo1 suggests that PCNA promotes DNA end resection. Directly testing this in cells could be difficult, as PCNA is also involved in DNA replication and is essential for cell viability, which could confound the functional analysis of PCNA specifically in DSB resection (49,50). To circumvent this issue, we chose to use the Xenopus nuclear extract, which maintains the proper DSB resection process but alone lacks DNA replication (42,51). To assay DNA end resection in the extract, we used a 3′-end 32P-labeled 6 kb dsDNA fragment and examined the length changes of the DNA substrate resulted from resection on agarose gels. Consistent with previous studies in Xenopus egg cytosol (52), we found that immunodepletion of xExo1 from the Xenopus nuclear extract partially inhibited DNA end resection (Figure 4A and B). The partial effects of Exo1-depletion on resection are consistent with the notion that Exo1 and Dna2 function in parallel in resection (23–25,51–55).Figure 4.

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