Mutagenesis, genotoxicity, and repair of 1-methyladenine, 3-alkylcytosines, 1-methylguanine, and 3-methylthymine in alkB Escherichia coli.
AlkB repairs 1-alkyladenine and 3-methylcytosine lesions in DNA by directly reversing the base damage. Although repair studies with randomly alkylated substrates have been performed, the miscoding nature of these and related individually alkylated bases and the suppression of mutagenesis by AlkB within cells have not yet been explored. Here, we address the miscoding potential of 1-methyldeoxyadenosine (m1A), 3-methyldeoxycytidine (m3C), 3-ethyldeoxycytidine (e3C), 1-methyldeoxyguanosine (m1G), and 3-methyldeoxythymidine (m3T) by synthesizing single-stranded vectors containing each alkylated base, followed by vector passage through Escherichia coli. In SOS(-), AlkB-deficient cells, m1A was only 1% mutagenic; however, m3C and e3C were 30% mutagenic, rising to 70% in SOS(+) cells. In contrast, the mutagenicity of m1G and m3T in AlkB(-) cells dropped slightly when SOS polymerases were expressed (m1G from 80% to 66% and m3T from 60% to 53%). Mutagenicity was abrogated for m1A, m3C, and e3C in wild-type (AlkB(+)) cells, whereas m3T mutagenicity was only partially reduced. Remarkably, m1G mutagenicity was also eliminated in AlkB(+) cells, establishing it as a natural AlkB substrate. All lesions were blocks to replication in AlkB-deficient cells. The m1A, m3C, and e3C blockades were completely removed in wild-type cells; the m1G blockade was partially removed and that for m3T was unaffected by the presence of AlkB. All lesions demonstrated enhanced bypass when SOS polymerases were induced. This work provides direct evidence that AlkB suppresses both genotoxicity and mutagenesis by physiologically realistic low doses of 1-alkylpurine and 3-alkylpyrimidine DNA damage in vivo.
Delaney
James C
JC
Department of Chemistry and Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Essigmann
John M
JM
D000225:Adenine / Q000031:analogs & derivatives* / Q000502:physiology; D001483:Base Sequence; D003596:Cytosine / Q000031:analogs & derivatives / Q000502:physiology; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D019175:DNA Methylation; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology; D004269:DNA, Bacterial / Q000502:physiology; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000502:physiology*; D005821:Genetic Techniques; D006147:Guanine / Q000031:analogs & derivatives* / Q000502:physiology; D016296:Mutagenesis / Q000502:physiology*; D009711:Nucleotides / Q000502:physiology*; D013014:SOS Response, Genetics / Q000502:physiology; D013941:Thymine / Q000031:analogs & derivatives* / Q000502:physiology
D018736:DNA Adducts; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009711:Nucleotides; C017515:1-methylguanine; C030630:1-methylthymine; C008407:1-methyladenine; D006147:Guanine; D003596:Cytosine; D000225:Adenine; D013941:Thymine