Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Atypon full text link Atypon Free PMC article
Full text links

Actions

Share

.2003 Aug;23(16):5919-27.
doi: 10.1128/MCB.23.16.5919-5927.2003.

Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair

Affiliations

Physical and functional interaction between DNA ligase IIIalpha and poly(ADP-Ribose) polymerase 1 in DNA single-strand break repair

John B Leppard et al. Mol Cell Biol.2003 Aug.

Abstract

The repair of DNA single-strand breaks in mammalian cells is mediated by poly(ADP-ribose) polymerase 1 (PARP-1), DNA ligase IIIalpha, and XRCC1. Since these proteins are not found in lower eukaryotes, this DNA repair pathway plays a unique role in maintaining genome stability in more complex organisms. XRCC1 not only forms a stable complex with DNA ligase IIIalpha but also interacts with several other DNA repair factors. Here we have used affinity chromatography to identify proteins that associate with DNA ligase III. PARP-1 binds directly to an N-terminal region of DNA ligase III immediately adjacent to its zinc finger. In further studies, we have shown that DNA ligase III also binds directly to poly(ADP-ribose) and preferentially associates with poly(ADP-ribosyl)ated PARP-1 in vitro and in vivo. Our biochemical studies have revealed that the zinc finger of DNA ligase III increases DNA joining in the presence of either poly(ADP-ribosyl)ated PARP-1 or poly(ADP-ribose). This provides a mechanism for the recruitment of the DNA ligase IIIalpha-XRCC1 complex to in vivo DNA single-strand breaks and suggests that the zinc finger of DNA ligase III enables this complex and associated repair factors to locate the strand break in the presence of the negatively charged poly(ADP-ribose) polymer.

PubMed Disclaimer

Figures

FIG. 1.
FIG. 1.
Identification of DNA ligase III-associated proteins by affinity chromatography. A HeLa nuclear extract was fractionated by using a DNA ligase III affinity chromatography column as described in Materials and Methods. (A) Proteins in comparable fractions eluted with 0.3 M NaCl from the beads liganded by GST and by GST-DNA ligase III (Lig III) were detected by silver staining after separation by SDS-PAGE. The polypeptides indicated were identified by MALDI-TOF mass spectrometry. The identities of PARP-1, Ku70, and Ku80 were verified by immunoblotting with PARP-1 (B) and Ku70 and Ku80 (C) monoclonal antibodies.
FIG. 2.
FIG. 2.
(A) The yeast two-hybrid genetic screen was employed to determine whether DNA ligase III interacts directly with PARP-1. Yeast strains were constructed as described in Materials and Methods. Colonies were tested for growth on triple dropout medium (-Leu,-Trp,-His) and subjected to a β-galactosidase (β-Gal) filter assay to detect protein-protein interactions as indicated. P/LIII, yeast strain expressing PARP-1 fused to the GAL4 activation domain (P) and DNA ligase III fused to the GAL4 DNA binding domain (LIII); P/V, yeast strain expressing the GAL4 binding domain (V) and PARP-1 fused to the GAL4 activation domain (P). (B) Mapping of the region of DNA ligase III that interacts with PARP-1. Pull-down assays with glutathione Sepharose and nickel beads were performed as described in Materials and Methods with purified PARP-1 (10 nM) and tagged versions of DNA ligase III. Ni, nickel beads only; Ni-Lig III, nickel beads liganded by His-tagged full-length DNA ligase III; Ni-ΔZf-LIII, nickel beads liganded by His-tagged DNA ligase III lacking the N-terminal 55 amino acids; GST, glutathione Sepharose beads liganded by GST; GST-692, glutathione Sepharose beads liganded by a GST fusion protein containing the C-terminal 692 residues of DNA ligase III; GST-152, glutathione Sepharose beads liganded by a GST fusion protein containing the N-terminal 152 residues of DNA ligase III; Zf, zinc finger; cat. domain, catalytic domain. The binding of PARP-1 to the beads was detected by immunoblotting (IB) with PARP-1 monoclonal antibody.
FIG. 3.
FIG. 3.
Binding of DNA ligase III and PARP-1 to DNA single-strand interruptions. Effects of NAD on DNA binding by PARP-1 are shown. (A) Analysis of the binding of DNA ligase III to a DNA single-strand break by surface plasmon resonance. (Top panel) Schematic representation of the nicked hairpin oligonucleotide immobilized on the chip surface. (Bottom panel) Representative sensorgram showing the binding and release of untagged full-length DNA ligase III (LigIII), injected at the concentrations indicated, from the nicked hairpin oligonucleotide immobilized on the chip surface. (B) Visualization of DNA ligase III bound to a DNA single-strand break by DNase I footprinting. Intact DNA ligase III (LigIII; 20 nM) was preincubated with the labeled, nicked DNA substrate (16 nM) indicated and then incubated with DNase I as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled oligonucleotides were detected by autoradiography. −, no enzyme. The vertical line indicates the region protected from DNase I activity. (C) Effect of DNA strand break binding by DNA ligase III and PARP-1 on T4 polynucleotide kinase activity. Intact DNA ligase III (150 nM) or PARP-1 (150 nM) was preincubated with the indicated DNA substrate containing a single-nucleotide (1 nt) gap in the presence (+) or absence (−) of NAD as indicated prior to incubation with polynucleotide kinase and [γ-32P]ATP as described in Materials and Methods. After separation by denaturing gel electrophoresis, labeled 29-mer oligonucleotide was detected and quantitated by phosphorimager analysis. Lane C, no PARP-1 or DNA ligase III.
FIG. 4.
FIG. 4.
Effect of DNA binding by PARP-1 on DNA joining. (A) Purified PARP-1 (200 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 2.5 nM), a truncated version lacking the zinc finger (ΔZf-Lig III; 2.5 nM), or DNA ligase I (Lig I; 2.5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, intact DNA ligase III; grey bars, truncated version of DNA ligase III lacking the zinc finger; black bars, DNA ligase I. (C) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (100 nM) in the presence (+) or absence (−) of NAD. After separation by SDS-PAGE, poly(ADP-ribosyl)ated PARP-1 was detected by immunoblotting (IB) with a monoclonal antibody against PAR. The positions of unmodified and poly(ADP-ribosyl)ated PARP-1 are indicated.
FIG. 5.
FIG. 5.
Interaction of DNA ligase III with PAR. Effects of PAR on DNA joining by DNA ligase III are shown. (A) Intact DNA ligase III (Lig III; 10 nM) or a truncated version lacking the zinc finger (ΔZf-LIII; 10 nM) was incubated with PAR antibody in the presence (+) or absence (−) of PAR. After separation of immunoprecipitated (IP) proteins by SDS-PAGE, DNA ligase III was detected by immunoblotting (IB). (B) DNA joining reactions with intact DNA ligase III (5 nM) or a truncated version lacking the zinc finger (5 nM) were carried out as described in Materials and Methods in the presence or absence of 0.5 nM PAR as indicated. The results of four independent DNA joining assays are shown graphically. White bars, intact DNA ligase III; grey bars, truncated version of DNA ligase III lacking the zinc finger.
FIG. 6.
FIG. 6.
Effect of poly(ADP-ribosyl)ated PARP-1 on DNA joining by DNA ligase III. (A) Purified PARP-1 (100 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (50 nM) in the presence (+) or absence (−) of NAD. Subsequently, intact DNA ligase III (Lig III; 5 nM) or a truncated version lacking the zinc finger (ΔZf-Lig III; 5 nM) was added to the reaction mixture as indicated. After incubation for 10 min at 25°C, labeled oligonucleotides were separated by denaturing gel electrophoresis. The labeled substrate (30-mer) and ligated product (50-mer) were detected and quantitated by phosphorimager analysis. (B) The results of three independent DNA joining assays are shown graphically. White bars, full-length DNA ligase IIIβ; grey bars, truncated version of DNA ligase IIIβ lacking the zinc finger.
FIG. 7.
FIG. 7.
DNA ligase III preferentially binds to poly(ADP-ribosyl)ated PARP-1 in vitro. Effects of DNA damage on the association between PARP-1 and DNA ligase III-XRCC1 in vivo are shown. (A) Purified PARP-1 (500 nM) was preincubated with a labeled DNA duplex containing a single ligatable nick (300 nM) in the presence (+) or absence (−) of NAD. After treatment with DNase I, intact DNA ligase III (10 nM) or a truncated version lacking the zinc finger (10 nM) was added to the reaction mixture as indicated. Proteins immunoprecipitated (IP) by PARP-1 antibody were separated by SDS-PAGE and then detected by immunblotting (IB) with the indicated antibody. Upper panel, intact DNA ligase III (Lig III); middle panel, truncated version of DNA ligase III lacking the zinc finger (ΔZf-Lig III); lower panel, unmodified PARP-1 y(ADP-ribosyl)ated PARP-1 [P-(ADPR)n]. (B) Effect of H2O2 treatment on the association of PARP-1, DNA ligase IIIα, and XRCC1. Whole cell extracts (WCE) were prepared from undamaged (−) or damaged (+) HeLa cells as described in Materials and Methods. Equivalent aliquots of the cells to be damaged were pretreated with 1,5-isoquinolinediol (DiQ; 100 μM) as indicated for 1 h prior to and during H2O2 treatment. Proteins immunoprecipitated by PARP-1 antibody were separated by SDS-PAGE and then detected by immunoblotting with the indicated antibody. DNA ligase IIIα and XRCC1 in the extracts from undamaged cells were detected by direct immunoblotting.
FIG. 8.
FIG. 8.
Model of the repair of DNA single-strand breaks by PARP-1 and DNA ligase III-XRCC1. The binding of PARP-1 to a DNA single-strand break activates PARP-1's polymerase activity that in turn results in automodification and dissociation of poly(ADP-ribosyl)ated PARP-1 from the strand break. DNA ligase IIIα (LigIII)-XRCC1 (XR1) associates with poly(ADP-ribosyl)ated PARP-1 in the vicinity of the DNA strand break. The zinc finger of DNA ligase IIIα enables this ternary complex to specifically recognize and bind to the DNA strand break despite the presence of negatively charged PARs. Additional repair factors such as polynucleotide kinase (PNK), Pol β, and AP endonuclease (APE) that process damaged termini are recruited via interactions with XRCC1. After the generation of ligatable termini, repair is completed by DNA ligase IIIα.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ausubel, F. M., R. Brent, R. Kingston, D. Morre, J. Seidman, A. Smith, and K. Struhl. 1994. Current protocols in molecular biology. John Wiley and Sons, Inc., New York, N.Y.
    1. Caldecott, K. W., S. Aoufouchi, P. Johnson, and S. Shall. 1996. XRCC1 polypeptide interacts with DNA polymerase β and possibly poly (ADP-ribose) polymerase, and DNA ligase III is a novel molecular nick-sensor in vitro. Nucleic Acids Res. 24:4387-4394. - PMC - PubMed
    1. Caldecott, K. W., C. K. McKeown, J. D. Tucker, S. Ljunquist, and L. H. Thompson. 1994. An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III. Mol. Cell. Biol. 14:68-76. - PMC - PubMed
    1. Caldecott, K. W., C. K. McKeown, J. D. Tucker, L. Stanker, and L. H. Thompson. 1996. Characterization of the Xrcc1-DNA ligase III complex in vitro and its absence from mutant hamster cells. Nucleic Acids Res. 23:4836-4843. - PMC - PubMed
    1. Callebaut, I., and J. P. Mornon. 1997. From BRCA1 to RAP1: a widespread BRCT module closely associated with DNA repair. FEBS Lett. 400:25-30. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Atypon full text link Atypon Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp