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The mutagenicity of thymidine glycol in Escherichia coli is increased when it is part of a tandem lesion.

ABSTRACT: 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.

SUBMITTER: Huang H

PROVIDER: S-EPMC2728776 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The mutagenicity of thymidine glycol in Escherichia coli is increased when it is part of a tandem lesion.

Huang Haidong H Imoto Shuhei S Greenberg Marc M MM

Biochemistry 20090801 33

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 blo ...[more]

PMID: 19618962

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Project description:BackgroundA considerable challenge in the development of bioprocesses for producing chemicals and fuels has been the high cost of feedstocks relative to oil prices, making it difficult for these processes to compete with their conventional petrochemical counterparts. Hence, in the absence of high oil prices in the near future, there has been a shift in the industry to produce higher value compounds such as fragrances for cosmetics. Yet, there is still a need to address climate change and develop biotechnological approaches for producing large market, lower value chemicals and fuels.ResultsIn this work, we study ethylene glycol (EG), a novel feedstock that we believe has promise to address this challenge. We engineer Escherichia coli (E. coli) to consume EG and examine glycolate production as a case study for chemical production. Using a combination of modeling and experimental studies, we identify oxygen concentration as an important metabolic valve in the assimilation and use of EG as a substrate. Two oxygen-based strategies are thus developed and tested in fed-batch bioreactors. Ultimately, the best glycolate production strategy employed a target respiratory quotient leading to the highest observed fermentation performance. With this strategy, a glycolate titer of 10.4 g/L was reached after 112 h of production time in a fed-batch bioreactor. Correspondingly, a yield of 0.8 g/g from EG and productivity of 0.1 g/L h were measured during the production stage. Our modeling and experimental results clearly suggest that oxygen concentration is an important factor in the assimilation and use of EG as a substrate. Finally, our use of metabolic modeling also sheds light on the intracellular distribution through central metabolism, implicating flux to 2-phosphoglycerate as the primary route for EG assimilation.ConclusionOverall, our work suggests that EG could provide a renewable starting material for commercial biosynthesis of fuels and chemicals that may achieve economic parity with petrochemical feedstocks while sequestering carbon dioxide.

Dataset Information

The mutagenicity of thymidine glycol in Escherichia coli is increased when it is part of a tandem lesion.

Publications

The mutagenicity of thymidine glycol in Escherichia coli is increased when it is part of a tandem lesion.

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