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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 deficient cells exhibit hypersensitivity to MMS and HmdU. (A) MMS sensitivity. CSB-V, CSB-E646Q, and CSB-WT cells were exposed to MMS at different concentrations (indicated) for 1 h, and subsequently incubated for 10 days in drug-free medium. The surviving fraction of cells (% survival) was calculated by dividing the number of colonies in treated dishes by those counted in the untreated control. (B) HmdU sensitivity. The indicated cell lines were exposed to HmdU at different concentrations (denoted) for 24 h and plated in complete media. The colony formation ability was determined after 10 days. Values shown in both panels represent the mean and standard deviation of six independent data points.
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Figure 5: CSB deficient cells exhibit hypersensitivity to MMS and HmdU. (A) MMS sensitivity. CSB-V, CSB-E646Q, and CSB-WT cells were exposed to MMS at different concentrations (indicated) for 1 h, and subsequently incubated for 10 days in drug-free medium. The surviving fraction of cells (% survival) was calculated by dividing the number of colonies in treated dishes by those counted in the untreated control. (B) HmdU sensitivity. The indicated cell lines were exposed to HmdU at different concentrations (denoted) for 24 h and plated in complete media. The colony formation ability was determined after 10 days. Values shown in both panels represent the mean and standard deviation of six independent data points.

Mentions: As an additional means of evaluating the cellular contribution of CSB to the repair of BER substrates/intermediates, we determined the sensitivity of the SV40-transformed CSB mutant fibroblast cell line CS1AN.S3.G2 complemented with either a control vector (CSB-V) or a recombinant plasmid expressing wild-type human CSB (CSB-WT) (48) to the monofunctional alkylating agent MMS and the thymidine analog HmdU. For MMS, the most critical biological lesion is presumed to be the N-methylation base products, which frequently give rise to AP sites via enhanced hydrolysis of the N-glycosylic bond or DNA glycosylase-mediated base release (55). HmdU is an oxidative base product formed by attack of intracellular ROS as well as by way of deamination of HmdC, another oxidative base lesion. Repair of HmdU in chromosomal DNA proceeds through a SMUG1-initiated BER response (56). Prior studies have established that exposure to exogenous HmdU in the culture medium induces cell killing in a manner that is dependent on the formation of BER intermediates in DNA, and not the base itself (57). As shown in Figure 5, CSB-V cells exhibit an ∼3-fold increase in sensitivity to both MMS (panel A, 0.48 mM) and HmdU (panel B, 5.4 μM), as determined by the LD37 doses relative to the CSB-WT control (MMS, 1.3 mM; HmdU, 17.1 μM). These findings provide novel evidence for a direct role of CSB in the repair of BER substrates/intermediates, possibly AP sites or single-strand breaks (SSBs), which are common to the two agents. As seen previously (48), we did not detect an increased sensitivity of the CSB-V cells (studied here) to hydrogen peroxide (data not shown).Figure 5.


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 deficient cells exhibit hypersensitivity to MMS and HmdU. (A) MMS sensitivity. CSB-V, CSB-E646Q, and CSB-WT cells were exposed to MMS at different concentrations (indicated) for 1 h, and subsequently incubated for 10 days in drug-free medium. The surviving fraction of cells (% survival) was calculated by dividing the number of colonies in treated dishes by those counted in the untreated control. (B) HmdU sensitivity. The indicated cell lines were exposed to HmdU at different concentrations (denoted) for 24 h and plated in complete media. The colony formation ability was determined after 10 days. Values shown in both panels represent the mean and standard deviation of six independent data points.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919475&req=5

Figure 5: CSB deficient cells exhibit hypersensitivity to MMS and HmdU. (A) MMS sensitivity. CSB-V, CSB-E646Q, and CSB-WT cells were exposed to MMS at different concentrations (indicated) for 1 h, and subsequently incubated for 10 days in drug-free medium. The surviving fraction of cells (% survival) was calculated by dividing the number of colonies in treated dishes by those counted in the untreated control. (B) HmdU sensitivity. The indicated cell lines were exposed to HmdU at different concentrations (denoted) for 24 h and plated in complete media. The colony formation ability was determined after 10 days. Values shown in both panels represent the mean and standard deviation of six independent data points.
Mentions: As an additional means of evaluating the cellular contribution of CSB to the repair of BER substrates/intermediates, we determined the sensitivity of the SV40-transformed CSB mutant fibroblast cell line CS1AN.S3.G2 complemented with either a control vector (CSB-V) or a recombinant plasmid expressing wild-type human CSB (CSB-WT) (48) to the monofunctional alkylating agent MMS and the thymidine analog HmdU. For MMS, the most critical biological lesion is presumed to be the N-methylation base products, which frequently give rise to AP sites via enhanced hydrolysis of the N-glycosylic bond or DNA glycosylase-mediated base release (55). HmdU is an oxidative base product formed by attack of intracellular ROS as well as by way of deamination of HmdC, another oxidative base lesion. Repair of HmdU in chromosomal DNA proceeds through a SMUG1-initiated BER response (56). Prior studies have established that exposure to exogenous HmdU in the culture medium induces cell killing in a manner that is dependent on the formation of BER intermediates in DNA, and not the base itself (57). As shown in Figure 5, CSB-V cells exhibit an ∼3-fold increase in sensitivity to both MMS (panel A, 0.48 mM) and HmdU (panel B, 5.4 μM), as determined by the LD37 doses relative to the CSB-WT control (MMS, 1.3 mM; HmdU, 17.1 μM). These findings provide novel evidence for a direct role of CSB in the repair of BER substrates/intermediates, possibly AP sites or single-strand breaks (SSBs), which are common to the two agents. As seen previously (48), we did not detect an increased sensitivity of the CSB-V cells (studied here) to hydrogen peroxide (data not shown).Figure 5.

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