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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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Global AP site repair in CSB-V and CSB-WT cells. (A) Steady-state AP site levels in total chromosomal DNA from CSB-V and CSB-WT cells. AP sites measured per 106 nucleotides (nt) are shown. Values represent the average and standard deviation of three independent measurements. (B) Total AP endonuclease activity of CSB-V and CSB-WT whole cell extracts. Shown is a representative gel of reactions performed at the indicated concentration of CSB-V or CSB-WT whole cell extract (μg/10 μl) with 34F duplex substrates (44). NE = no enzyme control. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane. (C) AP site incision kinetics.Three microgram/10 μl of the indicated extract was incubated for the time specified, and the percentage of S converted to P was determined. Plotted is the average and standard deviation of at least three data points. (D) Recombinant CSB activates AP site incision in whole cell extracts. Extracts (0.2 μg) were prepared from CSB-V cells and assayed for AP site incision efficiency, without or with supplemented recombinant CSB protein (10, 30, 100, 300 fmol).
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Figure 4: Global AP site repair in CSB-V and CSB-WT cells. (A) Steady-state AP site levels in total chromosomal DNA from CSB-V and CSB-WT cells. AP sites measured per 106 nucleotides (nt) are shown. Values represent the average and standard deviation of three independent measurements. (B) Total AP endonuclease activity of CSB-V and CSB-WT whole cell extracts. Shown is a representative gel of reactions performed at the indicated concentration of CSB-V or CSB-WT whole cell extract (μg/10 μl) with 34F duplex substrates (44). NE = no enzyme control. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane. (C) AP site incision kinetics.Three microgram/10 μl of the indicated extract was incubated for the time specified, and the percentage of S converted to P was determined. Plotted is the average and standard deviation of at least three data points. (D) Recombinant CSB activates AP site incision in whole cell extracts. Extracts (0.2 μg) were prepared from CSB-V cells and assayed for AP site incision efficiency, without or with supplemented recombinant CSB protein (10, 30, 100, 300 fmol).

Mentions: As a means of evaluating the function of CSB in in vivo AP site repair, we measured the steady-state level of abasic lesions in total chromosomal DNA isolated from both CSB-V and CSB-WT fibroblasts using an established aldehyde reactive probe method (54). These studies found that both human cell lines maintain similar AP site levels (Figure 4A). Consistent with this, both CSB-WT and CSB-V whole cell extracts exhibited comparable total AP site incision capacities on fully paired 34-mer duplex substrates (Figure 4B and C). In addition, as with the whole cell extracts, we did not detect any difference in incision efficiency of normal duplex or bubble substrate AP site-containing DNAs using nuclear extracts prepared from CSB-WT or CSB-V cells (data not shown). These results suggest that CSB does not obviously modulate global genome repair of abasic lesions. However, the data does not exclude a more specialized role for the CSB–APE1 interaction in the repair of a subset of APE1 substrates, perhaps in regions of complex DNA structure. Significantly, addition of recombinant CSB protein to whole cell extracts prepared from CSB-V cells resulted in a concentration-dependent stimulation of AP site incision efficiency (Figure 4D), supportive of the idea that situations of high-localized concentrations of APE1 and CSB may indeed foster AP site repair. We note that semi-quantitative western blot analysis using CSB-WT whole cell extracts indicated an overall ratio of APE1:CSB of ≥100:1 (data not shown).Figure 4.


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)

Global AP site repair in CSB-V and CSB-WT cells. (A) Steady-state AP site levels in total chromosomal DNA from CSB-V and CSB-WT cells. AP sites measured per 106 nucleotides (nt) are shown. Values represent the average and standard deviation of three independent measurements. (B) Total AP endonuclease activity of CSB-V and CSB-WT whole cell extracts. Shown is a representative gel of reactions performed at the indicated concentration of CSB-V or CSB-WT whole cell extract (μg/10 μl) with 34F duplex substrates (44). NE = no enzyme control. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane. (C) AP site incision kinetics.Three microgram/10 μl of the indicated extract was incubated for the time specified, and the percentage of S converted to P was determined. Plotted is the average and standard deviation of at least three data points. (D) Recombinant CSB activates AP site incision in whole cell extracts. Extracts (0.2 μg) were prepared from CSB-V cells and assayed for AP site incision efficiency, without or with supplemented recombinant CSB protein (10, 30, 100, 300 fmol).
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Figure 4: Global AP site repair in CSB-V and CSB-WT cells. (A) Steady-state AP site levels in total chromosomal DNA from CSB-V and CSB-WT cells. AP sites measured per 106 nucleotides (nt) are shown. Values represent the average and standard deviation of three independent measurements. (B) Total AP endonuclease activity of CSB-V and CSB-WT whole cell extracts. Shown is a representative gel of reactions performed at the indicated concentration of CSB-V or CSB-WT whole cell extract (μg/10 μl) with 34F duplex substrates (44). NE = no enzyme control. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane. (C) AP site incision kinetics.Three microgram/10 μl of the indicated extract was incubated for the time specified, and the percentage of S converted to P was determined. Plotted is the average and standard deviation of at least three data points. (D) Recombinant CSB activates AP site incision in whole cell extracts. Extracts (0.2 μg) were prepared from CSB-V cells and assayed for AP site incision efficiency, without or with supplemented recombinant CSB protein (10, 30, 100, 300 fmol).
Mentions: As a means of evaluating the function of CSB in in vivo AP site repair, we measured the steady-state level of abasic lesions in total chromosomal DNA isolated from both CSB-V and CSB-WT fibroblasts using an established aldehyde reactive probe method (54). These studies found that both human cell lines maintain similar AP site levels (Figure 4A). Consistent with this, both CSB-WT and CSB-V whole cell extracts exhibited comparable total AP site incision capacities on fully paired 34-mer duplex substrates (Figure 4B and C). In addition, as with the whole cell extracts, we did not detect any difference in incision efficiency of normal duplex or bubble substrate AP site-containing DNAs using nuclear extracts prepared from CSB-WT or CSB-V cells (data not shown). These results suggest that CSB does not obviously modulate global genome repair of abasic lesions. However, the data does not exclude a more specialized role for the CSB–APE1 interaction in the repair of a subset of APE1 substrates, perhaps in regions of complex DNA structure. Significantly, addition of recombinant CSB protein to whole cell extracts prepared from CSB-V cells resulted in a concentration-dependent stimulation of AP site incision efficiency (Figure 4D), supportive of the idea that situations of high-localized concentrations of APE1 and CSB may indeed foster AP site repair. We note that semi-quantitative western blot analysis using CSB-WT whole cell extracts indicated an overall ratio of APE1:CSB of ≥100:1 (data not shown).Figure 4.

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