Cloning and expression in Escherichia coli of a gene for an alkylbase DNA glycosylase from Saccharomyces cerevisiae; a homologue to the bacterial alkA gene.
An alkylation repair deficient mutant of Escherichia coli (tag ada), lacking DNA glycosylase activity for removal of alkylated bases, was transformed by a genomic yeast DNA library and clones selected which survived plating on medium containing the alkylating agent methylmethane sulphonate. Three distinct yeast clones were identified which were able to suppress the alkylation sensitive phenotype of the bacterial mutant. Restriction enzyme analysis revealed common DNA fragments present in all three clones spanning 2 kb of yeast DNA. DNA from this region was sequenced and analysed for possible translation of polypeptides with any homology to either the Tag or the AlkA DNA glycosylases of E. coli. One open reading frame of 296 amino acids was identified encoding a putative protein with significant homology to AlkA. DNA containing the open reading frame was subcloned in E. coli expression vectors and cell extracts assayed for alkylbase DNA glycosylase activity. It appeared that such activity was expressed at levels sufficiently high for enzyme purification. The molecular weight of the purified protein was determined by SDS-PAGE to be 35,000 daltons, in good agreement with the 34,340 value calculated from the sequence. The yeast enzyme was able to excise 7-methylguanine as well as 3-methyladenine from dimethyl sulphate treated DNA, confirming the related nature of this enzyme to the AlkA DNA glycosylase from E. coli.
Berdal
K G
KG
Norwegian Defence Research Establishment, Division for Environmental Toxicology, Kjeller, Norway.
Bjørås
M
M
Bjelland
S
S
Seeberg
E
E
D000225:Adenine / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D045647:DNA Glycosylases; D004260:DNA Repair; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D005800:Genes, Fungal; D006147:Guanine / Q000031:analogs & derivatives / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D016366:Open Reading Frames; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
C025946:3-methyladenine; D006147:Guanine; C008450:7-methylguanine; D008741:Methyl Methanesulfonate; C021565:3-methyladenine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C076131:DNA-3-methyladenine glycosidase II; D000225:Adenine