Purification and characterization of a uracil-DNA glycosylase from the yeast, saccharomyces cerevisiae

Bill Crosby; Louise Prakash; Howard Davis; David C. Hinkle

doi:10.1093/nar/9.21.5797

Purification and characterization of a uracil-DNA glycosylase from the yeast, saccharomyces cerevisiae

Bill Crosby, Louise Prakash, Howard Davis, David C. Hinkle

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Abstract

An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharornyces cerevisiae. The enzyme is active on both native and denatured PBSl UNA and is active in the absence of divalent cation, and in the presence of 1 mM EDTA. The enzyme has a native molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gelfiltration. Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel. This analysis suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons. Normal levels of uracil-DNA glycosylase activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.

Original language	English (US)
Pages (from-to)	5797-5810
Number of pages	14
Journal	Nucleic acids research
Volume	9
Issue number	21
DOIs	https://doi.org/10.1093/nar/9.21.5797
State	Published - Nov 11 1981
Externally published	Yes

ASJC Scopus subject areas

Genetics

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title = "Purification and characterization of a uracil-DNA glycosylase from the yeast, saccharomyces cerevisiae",

abstract = "An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharornyces cerevisiae. The enzyme is active on both native and denatured PBSl UNA and is active in the absence of divalent cation, and in the presence of 1 mM EDTA. The enzyme has a native molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gelfiltration. Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel. This analysis suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons. Normal levels of uracil-DNA glycosylase activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.",

author = "Bill Crosby and Louise Prakash and Howard Davis and Hinkle, {David C.}",

note = "Funding Information: We are grateful to Dr. David Swinton for performing the HPLC analysis of PBS1 [3H]-DNA and to Drs. Dayle Hager and Richard Burgess for providing us with their procedure for recovering enzymatic activity from SDS-polyacrylamide gels prior to publication. This paper Is based on work performed partially under NIH grants GM21782 and GM19261, grant IN-18from theAmerican Cancer Society andunder Contract Number DE-AC02-76EVO349O with the U.S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-2000.",

year = "1981",

month = nov,

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doi = "10.1093/nar/9.21.5797",

language = "English (US)",

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T1 - Purification and characterization of a uracil-DNA glycosylase from the yeast, saccharomyces cerevisiae

AU - Crosby, Bill

AU - Prakash, Louise

AU - Davis, Howard

AU - Hinkle, David C.

N1 - Funding Information: We are grateful to Dr. David Swinton for performing the HPLC analysis of PBS1 [3H]-DNA and to Drs. Dayle Hager and Richard Burgess for providing us with their procedure for recovering enzymatic activity from SDS-polyacrylamide gels prior to publication. This paper Is based on work performed partially under NIH grants GM21782 and GM19261, grant IN-18from theAmerican Cancer Society andunder Contract Number DE-AC02-76EVO349O with the U.S. Department of Energy at the University of Rochester Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-2000.

PY - 1981/11/11

Y1 - 1981/11/11

N2 - An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharornyces cerevisiae. The enzyme is active on both native and denatured PBSl UNA and is active in the absence of divalent cation, and in the presence of 1 mM EDTA. The enzyme has a native molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gelfiltration. Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel. This analysis suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons. Normal levels of uracil-DNA glycosylase activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.

AB - An activity which releases free uracil from bacteriophage PBS1 DNA has been purified over 10,000 fold from extracts of Saccharornyces cerevisiae. The enzyme is active on both native and denatured PBSl UNA and is active in the absence of divalent cation, and in the presence of 1 mM EDTA. The enzyme has a native molecular weight of 27,800 as estimated by glycerol gradient centrifugation and gelfiltration. Enzyme activity has been recovered after denaturation in SDS and electrophoresis in an SDS polyacrylamide gel. This analysis suggests that the enzyme consists of a single polypeptide chain of about 27,000 daltons. Normal levels of uracil-DNA glycosylase activity were found in partially purified extracts of the nitrous-acid sensitive rad18-2 mutant of yeast.

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JO - Nucleic acids research

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Purification and characterization of a uracil-DNA glycosylase from the yeast, saccharomyces cerevisiae

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