Limits...
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.

Show MeSH

Related in: MedlinePlus

CSB specifically activates full-length human APE1. (A) APE1 stimulation is dependent on CSB. Reactions were carried out as in Figure 2. Immunodepleted supernatant (ID) or immunoprecipitated (IP) CSB fractions were prepared as described in Materials and Methods section. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane, and compared with the effects of purified, recombinant CSB protein. (B) CSB stimulation is specific for human APE1. Reactions were performed with E. coli AP endonuclease IV (0.1 fmol; ∼3 pg) and increasing amounts of CSB (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 fmol). (C) Incision activity of truncated APE1 protein is not activated by CSB. Truncated APE1 lacking the first 29 amino acids (5 fmol) was incubated with increasing concentrations of CSB (1, 5, 20, 50 and 100 fmol) as in Figure 2. Representative gel images are shown in panels A, B and C. In panels B and C, a full-length APE1 (30 fmol) and CSB (300 fmol) control reaction was simultaneously performed. Percentage of S converted to P is denoted.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1919475&req=5

Figure 3: CSB specifically activates full-length human APE1. (A) APE1 stimulation is dependent on CSB. Reactions were carried out as in Figure 2. Immunodepleted supernatant (ID) or immunoprecipitated (IP) CSB fractions were prepared as described in Materials and Methods section. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane, and compared with the effects of purified, recombinant CSB protein. (B) CSB stimulation is specific for human APE1. Reactions were performed with E. coli AP endonuclease IV (0.1 fmol; ∼3 pg) and increasing amounts of CSB (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 fmol). (C) Incision activity of truncated APE1 protein is not activated by CSB. Truncated APE1 lacking the first 29 amino acids (5 fmol) was incubated with increasing concentrations of CSB (1, 5, 20, 50 and 100 fmol) as in Figure 2. Representative gel images are shown in panels A, B and C. In panels B and C, a full-length APE1 (30 fmol) and CSB (300 fmol) control reaction was simultaneously performed. Percentage of S converted to P is denoted.

Mentions: To determine whether the activation of APE1 was indeed dependent on the CSB protein, we performed incision assays using immunodepleted and immunoprecipitated CSB protein fractions (see Materials and Methods section). As revealed by silver staining, immunodepletion of CSB with anti-HA antibodies reduced CSB protein levels in the recovered supernatant by ∼90% (data not shown). Thus, as expected, and supportive of the stimulation being CSB-dependent, the immunodepleted supernatant (ID CSB) had a ∼10-fold reduced ability to stimulate APE1 activity; that is, roughly equal stimulation was observed at a 10-fold higher amount of the immunodepleted CSB extract, relative to the purified CSB protein or the immunoprecipitated (i.e. immunopurified, IP CSB) CSB protein (Figure 3A).Figure 3.


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 specifically activates full-length human APE1. (A) APE1 stimulation is dependent on CSB. Reactions were carried out as in Figure 2. Immunodepleted supernatant (ID) or immunoprecipitated (IP) CSB fractions were prepared as described in Materials and Methods section. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane, and compared with the effects of purified, recombinant CSB protein. (B) CSB stimulation is specific for human APE1. Reactions were performed with E. coli AP endonuclease IV (0.1 fmol; ∼3 pg) and increasing amounts of CSB (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 fmol). (C) Incision activity of truncated APE1 protein is not activated by CSB. Truncated APE1 lacking the first 29 amino acids (5 fmol) was incubated with increasing concentrations of CSB (1, 5, 20, 50 and 100 fmol) as in Figure 2. Representative gel images are shown in panels A, B and C. In panels B and C, a full-length APE1 (30 fmol) and CSB (300 fmol) control reaction was simultaneously performed. Percentage of S converted to P is denoted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: CSB specifically activates full-length human APE1. (A) APE1 stimulation is dependent on CSB. Reactions were carried out as in Figure 2. Immunodepleted supernatant (ID) or immunoprecipitated (IP) CSB fractions were prepared as described in Materials and Methods section. Percentage of intact substrate (S) converted to incised product (P) is denoted under each lane, and compared with the effects of purified, recombinant CSB protein. (B) CSB stimulation is specific for human APE1. Reactions were performed with E. coli AP endonuclease IV (0.1 fmol; ∼3 pg) and increasing amounts of CSB (0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 fmol). (C) Incision activity of truncated APE1 protein is not activated by CSB. Truncated APE1 lacking the first 29 amino acids (5 fmol) was incubated with increasing concentrations of CSB (1, 5, 20, 50 and 100 fmol) as in Figure 2. Representative gel images are shown in panels A, B and C. In panels B and C, a full-length APE1 (30 fmol) and CSB (300 fmol) control reaction was simultaneously performed. Percentage of S converted to P is denoted.
Mentions: To determine whether the activation of APE1 was indeed dependent on the CSB protein, we performed incision assays using immunodepleted and immunoprecipitated CSB protein fractions (see Materials and Methods section). As revealed by silver staining, immunodepletion of CSB with anti-HA antibodies reduced CSB protein levels in the recovered supernatant by ∼90% (data not shown). Thus, as expected, and supportive of the stimulation being CSB-dependent, the immunodepleted supernatant (ID CSB) had a ∼10-fold reduced ability to stimulate APE1 activity; that is, roughly equal stimulation was observed at a 10-fold higher amount of the immunodepleted CSB extract, relative to the purified CSB protein or the immunoprecipitated (i.e. immunopurified, IP CSB) CSB protein (Figure 3A).Figure 3.

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