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Cockayne syndrome B protein stimulates apurinic endonuclease 1 activity and protects against agents that introduce base excision repair intermediates.

Wong HK, Muftuoglu M, Beck G, Imam SZ, Bohr VA, Wilson DM - Nucleic Acids Res. (2007)

Bottom Line: This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment.CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity.Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.

ABSTRACT
The Cockayne syndrome B (CSB) protein--defective in a majority of patients suffering from the rare autosomal disorder CS--is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP-DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

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CSB associates with APE1. Interaction between CSB and APE1 in indirect ELISA. Mouse anti-APE1 (panel A) or anti-CSB (panel B) was coated to the well. Purified APE1 (30 fmol, 1 ng) or CSB (6 fmol, 1 ng) was then bound to the appropriate antibody. These complexes were next incubated with different concentrations of CSB (panel A) or APE1 (panel B) as indicated. Ethidium bromide (EtBr) or DNase I was added where indicated. Values are means ± standard errors (n = 3). (C) CSB and Ape1 are in a common complex in human cell extracts. Lysates from CS1AN cells transfected with the ECFP-CSB plasmid were immunoprecipitated (IP) with either ECFP (left) or APE1 (right) antibodies. The IPs were then analyzed by immunoblotting (IB) with either anti-CSB or anti-APE1 antibodies. Lysates of 10% of the amount used for IP were loaded as input comparisons. As controls, lysates IPed with rabbit IgG only were loaded (IgG Control), and IPs with anti-ECFP were performed on extracts from ECFP-only expressing cells (Vector Control).
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Figure 1: CSB associates with APE1. Interaction between CSB and APE1 in indirect ELISA. Mouse anti-APE1 (panel A) or anti-CSB (panel B) was coated to the well. Purified APE1 (30 fmol, 1 ng) or CSB (6 fmol, 1 ng) was then bound to the appropriate antibody. These complexes were next incubated with different concentrations of CSB (panel A) or APE1 (panel B) as indicated. Ethidium bromide (EtBr) or DNase I was added where indicated. Values are means ± standard errors (n = 3). (C) CSB and Ape1 are in a common complex in human cell extracts. Lysates from CS1AN cells transfected with the ECFP-CSB plasmid were immunoprecipitated (IP) with either ECFP (left) or APE1 (right) antibodies. The IPs were then analyzed by immunoblotting (IB) with either anti-CSB or anti-APE1 antibodies. Lysates of 10% of the amount used for IP were loaded as input comparisons. As controls, lysates IPed with rabbit IgG only were loaded (IgG Control), and IPs with anti-ECFP were performed on extracts from ECFP-only expressing cells (Vector Control).

Mentions: Evidence suggests a role for CSB in facilitating global genome, and possibly transcription-coupled BER (1). As a means of elucidating the molecular involvement of CSB in BER, we examined for physical interactions of CSB with proteins operating in this and related DNA repair pathways. Specifically, initial studies employed a dot blot technique, where select proteins were (i) spotted and fixed to a capture membrane, (ii) incubated with purified CSB in solution and (iii) probed for binding of CSB using CSB-specific antibodies. Potential interactors identified using this technique were the strand break sensor protein PARP-1 (data not shown), the structure-specific endonuclease FEN1, the tyrosine kinase c-Abl and the major human abasic endonuclease APE1 (Supplementary Figure 1A: See online supplementary material for a color version of this figure). No significant interaction was seen with the tumor suppressor p53, the replication processivity factor PCNA, the end-joining binding complex Ku70/80, the Werner helicase, the protein defective in Nijmegen Breakage Syndrome NBS1, the telomere repeat binding protein TRF1, the recombination protein Rad51 and the single-stranded DNA binding protein RPA (Supplemental Figure 1A and data not shown). Detailed studies describing the interaction of CSB with PARP-1 have been reported elsewhere (35), and experiments with other putative binding partners (e.g. c-Abl and FEN1) are ongoing.Figure 1.


Cockayne syndrome B protein stimulates apurinic endonuclease 1 activity and protects against agents that introduce base excision repair intermediates.

Wong HK, Muftuoglu M, Beck G, Imam SZ, Bohr VA, Wilson DM - Nucleic Acids Res. (2007)

CSB associates with APE1. Interaction between CSB and APE1 in indirect ELISA. Mouse anti-APE1 (panel A) or anti-CSB (panel B) was coated to the well. Purified APE1 (30 fmol, 1 ng) or CSB (6 fmol, 1 ng) was then bound to the appropriate antibody. These complexes were next incubated with different concentrations of CSB (panel A) or APE1 (panel B) as indicated. Ethidium bromide (EtBr) or DNase I was added where indicated. Values are means ± standard errors (n = 3). (C) CSB and Ape1 are in a common complex in human cell extracts. Lysates from CS1AN cells transfected with the ECFP-CSB plasmid were immunoprecipitated (IP) with either ECFP (left) or APE1 (right) antibodies. The IPs were then analyzed by immunoblotting (IB) with either anti-CSB or anti-APE1 antibodies. Lysates of 10% of the amount used for IP were loaded as input comparisons. As controls, lysates IPed with rabbit IgG only were loaded (IgG Control), and IPs with anti-ECFP were performed on extracts from ECFP-only expressing cells (Vector Control).
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Related In: Results  -  Collection

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Figure 1: CSB associates with APE1. Interaction between CSB and APE1 in indirect ELISA. Mouse anti-APE1 (panel A) or anti-CSB (panel B) was coated to the well. Purified APE1 (30 fmol, 1 ng) or CSB (6 fmol, 1 ng) was then bound to the appropriate antibody. These complexes were next incubated with different concentrations of CSB (panel A) or APE1 (panel B) as indicated. Ethidium bromide (EtBr) or DNase I was added where indicated. Values are means ± standard errors (n = 3). (C) CSB and Ape1 are in a common complex in human cell extracts. Lysates from CS1AN cells transfected with the ECFP-CSB plasmid were immunoprecipitated (IP) with either ECFP (left) or APE1 (right) antibodies. The IPs were then analyzed by immunoblotting (IB) with either anti-CSB or anti-APE1 antibodies. Lysates of 10% of the amount used for IP were loaded as input comparisons. As controls, lysates IPed with rabbit IgG only were loaded (IgG Control), and IPs with anti-ECFP were performed on extracts from ECFP-only expressing cells (Vector Control).
Mentions: Evidence suggests a role for CSB in facilitating global genome, and possibly transcription-coupled BER (1). As a means of elucidating the molecular involvement of CSB in BER, we examined for physical interactions of CSB with proteins operating in this and related DNA repair pathways. Specifically, initial studies employed a dot blot technique, where select proteins were (i) spotted and fixed to a capture membrane, (ii) incubated with purified CSB in solution and (iii) probed for binding of CSB using CSB-specific antibodies. Potential interactors identified using this technique were the strand break sensor protein PARP-1 (data not shown), the structure-specific endonuclease FEN1, the tyrosine kinase c-Abl and the major human abasic endonuclease APE1 (Supplementary Figure 1A: See online supplementary material for a color version of this figure). No significant interaction was seen with the tumor suppressor p53, the replication processivity factor PCNA, the end-joining binding complex Ku70/80, the Werner helicase, the protein defective in Nijmegen Breakage Syndrome NBS1, the telomere repeat binding protein TRF1, the recombination protein Rad51 and the single-stranded DNA binding protein RPA (Supplemental Figure 1A and data not shown). Detailed studies describing the interaction of CSB with PARP-1 have been reported elsewhere (35), and experiments with other putative binding partners (e.g. c-Abl and FEN1) are ongoing.Figure 1.

Bottom Line: This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment.CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity.Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.

ABSTRACT
The Cockayne syndrome B (CSB) protein--defective in a majority of patients suffering from the rare autosomal disorder CS--is a member of the SWI2/SNF2 family with roles in DNA repair and transcription. We demonstrate herein that purified recombinant CSB and the major human apurinic/apyrimidinic (AP) endonuclease, APE1, physically and functionally interact. CSB stimulates the AP site incision activity of APE1 on normal (i.e. fully paired) and bubble AP-DNA substrates, with the latter being more pronounced (up to 6-fold). This activation is ATP-independent, and specific for the human CSB and full-length APE1 protein, as no CSB-dependent stimulation was observed with Escherichia coli endonuclease IV or an N-terminal truncated APE1 fragment. CSB and APE1 were also found in a common protein complex in human cell extracts, and recombinant CSB, when added back to CSB-deficient whole cell extracts, resulted in increased total AP site incision capacity. Moreover, human fibroblasts defective in CSB were found to be hypersensitive to both methyl methanesulfonate (MMS) and 5-hydroxymethyl-2'-deoxyuridine, agents that introduce base excision repair (BER) DNA substrates/intermediates.

Show MeSH
Related in: MedlinePlus