The mutagenicity of thymidine glycol in Escherichia coli is increased when it is part of a tandem lesion.
Tandem lesions are comprised of two contiguously damaged nucleotides. Tandem lesions make up the major family of reaction products generated from a pyrimidine nucleobase radical, which are formed in large amounts by ionizing radiation. One of these tandem lesions contains a thymidine glycol lesion flanked on its 5'-side by 2-deoxyribonolactone (LTg). The replication of this tandem lesion was investigated in Escherichia coli using single-stranded genomes. LTg is a much more potent replication block than thymidine glycol and is bypassed only under SOS-induced conditions. The adjacent thymidine glycol does not significantly affect nucleotide incorporation opposite 2-deoxyribonolactone in wild-type cells. In contrast, the misinsertion frequency opposite thymidine glycol, which is negligible in the absence of 2-deoxyribonolactone, increases to 10% in wild-type cells when LTg is flanked by a 3'-dG. Experiments in which the flanking nucleotides are varied and in cells lacking one of the SOS-induced bypass polymerases indicate that the mutations are due to a mechanism in which the primer misaligns prior to bypassing the lesion, which allows for an additional nucleotide to be incorporated across from the 3'-flanking nucleotide. Subsequent realignment and extension results in the observed mutations. DNA polymerases II and IV are responsible for misalignment induced mutations and compete with DNA polymerase V which reads through the tandem lesion. These experiments reveal that incorporation of the thymidine glycol into a tandem lesion indirectly induces increases in mutations by blocking replication, which enables the misalignment-realignment mechanism to compete with direct bypass by DNA polymerase V.
Huang
Haidong
H
Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Imoto
Shuhei
S
Greenberg
Marc M
MM
D020121:5' Untranslated Regions / Q000187:drug effects / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics; D004249:DNA Damage; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics; D004269:DNA, Bacterial / Q000138:chemical synthesis / Q000187:drug effects*; D018900:Dinucleotide Repeats / Q000187:drug effects* / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D016680:Genome, Bacterial / Q000187:drug effects; D017665:Hydroxyl Radical / Q000633:toxicity; D016296:Mutagenesis; D013014:SOS Response, Genetics / Q000187:drug effects; D017384:Sequence Deletion / Q000187:drug effects; D013400:Sugar Acids / Q000633:toxicity; D013936:Thymidine / Q000031:analogs & derivatives* / Q000633:toxicity
D020121:5' Untranslated Regions; D004269:DNA, Bacterial; D013400:Sugar Acids; C029390:thymidine glycol; D017665:Hydroxyl Radical; C056405:2,4,5-trihydroxypentanoic acid gamma-lactone; D013936:Thymidine