Mutagenic consequences of the incorporation of 6-thioguanine into DNA.
6-Thioguanine (S6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508-514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M13lacISaXb in reactions that omitted dGTP (-G) or dATP (-A). LacI mutation frequency was determined by beta-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducer). When a high concentration of SdGTP (24 microM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the -G and -A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the -G + S6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the -A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S6G with thymine leads to mutagenic effects in this assay.
Uribe-Luna
S
S
Department of Biochemistry, Baylor College of Medicine, Houston, TX 77030, U.S.A.
Quintana-Hau
J D
JD
Maldonado-Rodriguez
R
R
Espinosa-Lara
M
M
Beattie
K L
KL
Farquhar
D
D
Nelson
J A
JA
D001426:Bacterial Proteins / Q000235:genetics; D004247:DNA / Q000096:biosynthesis / Q000235:genetics*; D004261:DNA Replication; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D056968:Lac Repressors; D009153:Mutagens; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics; D013866:Thioguanine / Q000378:metabolism*; D013873:Thionucleotides / Q000378:metabolism*
D001426:Bacterial Proteins; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D009153:Mutagens; D012097:Repressor Proteins; D013873:Thionucleotides; C021173:2'-deoxy-6-thioguanosine 5'-triphosphate; D004247:DNA; D013866:Thioguanine