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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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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.
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gkt672-F1: 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.

Mentions: To elucidate the Exo1-mediated resection pathway, we first examined the regulation of the damage recruitment of Exo1 in human cells. Both endogenous Exo1 and GFP-tagged Exo1 were efficiently recruited to DNA damage sites induced by laser irradiation (Supplementary Figure S1 and Figure 1A) (38,43,44). Interestingly, deletion of the C-terminal region (aa 751–846) of Exo1 drastically reduced the damage association of GFP-Exo1 (see result for GFP-Exo1(1–750) in Figure 1A). This C-terminal region fused to GFP was also efficiently recruited to DNA damage sites [see result for GFP-Exo1(751–846) in Figure 1A]. These data indicate that the C-terminal region of Exo1 facilitates its damage association.Figure 1.


PCNA promotes processive DNA end resection by Exo1.

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

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

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gkt672-F1: 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.
Mentions: To elucidate the Exo1-mediated resection pathway, we first examined the regulation of the damage recruitment of Exo1 in human cells. Both endogenous Exo1 and GFP-tagged Exo1 were efficiently recruited to DNA damage sites induced by laser irradiation (Supplementary Figure S1 and Figure 1A) (38,43,44). Interestingly, deletion of the C-terminal region (aa 751–846) of Exo1 drastically reduced the damage association of GFP-Exo1 (see result for GFP-Exo1(1–750) in Figure 1A). This C-terminal region fused to GFP was also efficiently recruited to DNA damage sites [see result for GFP-Exo1(751–846) in Figure 1A]. These data indicate that the C-terminal region of Exo1 facilitates its damage association.Figure 1.

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