Dataset Information

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh.

ABSTRACT: Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-prone motifs in Sulfolobus chromosomes and used them to measure the efficiency and accuracy of bypass in normal and dbh(-) strains of Sulfolobus acidocaldarius. Oligonucleotide-mediated transformation allowed low levels of abasic-site bypass to be observed in S. acidocaldarius and demonstrated that the local sequence context affected bypass specificity; in addition, most erroneous TLS did not require Dbh function. Applying the technique to another common lesion, 7,8-dihydro-8-oxo-deoxyguanosine (8-oxo-dG), revealed an antimutagenic role of Dbh. The efficiency and accuracy of replication past 8-oxo-dG was higher in the presence of Dbh, and up to 90% of the Dbh-dependent events inserted dC. A third set of assays, based on phenotypic reversion, showed no effect of Dbh function on spontaneous -1 frameshifts in mononucleotide tracts in vivo, despite the extremely frequent slippage at these motifs documented in vitro. Taken together, the results indicate that a primary genetic role of Dbh is to avoid mutations at 8-oxo-dG that occur when other Sulfolobus enzymes replicate past this lesion. The genetic evidence that Dbh is recruited to 8-oxo-dG raises questions regarding the mechanism of recruitment, since Sulfolobus spp. have eukaryotic-like replisomes but no ubiquitin.

SUBMITTER: Sakofsky CJ

PROVIDER: S-EPMC4596666 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh.

Sakofsky Cynthia J CJ Grogan Dennis W DW

Genetics 20150729 2

Hyperthermophilic archaea offer certain advantages as models of genome replication, and Sulfolobus Y-family polymerases Dpo4 (S. solfataricus) and Dbh (S. acidocaldarius) have been studied intensively in vitro as biochemical and structural models of trans-lesion DNA synthesis (TLS). However, the genetic functions of these enzymes have not been determined in the native context of living cells. We developed the first quantitative genetic assays of replication past defined DNA lesions and error-pro ...[more]

PMID: 26224736

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Project description:Glycogen is a polysaccharide that comprises α-1,4-linked glucose backbone and α-1,6-linked glucose polymers at the branching points. It is widely found in organisms ranging from bacteria to eukaryotes. The physiological role of glycogen is not confined to being an energy reservoir and carbon source but varies depending on organisms. Sulfolobus acidocaldarius, a thermoacidophilic archaeon, was observed to accumulate granular glycogen in the cell. However, the role of glycogen and genes that are responsible for glycogen metabolism in S. acidocaldarius has not been identified clearly. The objective of this study is to identify the gene cluster, which is composed of enzymes that are predicted to be involved in the glycogen metabolism, and confirm the role of each of these genes by constructing deletion mutants. This study also compares the glycogen content of mutant and wild type and elucidates the role of glycogen in this archaeon. The glycogen content of S. acidocaldarius MR31, which is used as a parent strain for constructing the deletion mutant in this study, was increased in the early and middle exponential growth phases and decreased during the late exponential and stationary growth phases. The pattern of the accumulated glycogen was independent to the type of supplemented sugar. In the comparison of the glycogen content between the gene deletion mutant and MR31, glycogen synthase (GlgA) and α-amylase (AmyA) were shown to be responsible for the synthesis of glycogen, whereas glycogen debranching enzyme (GlgX) and glucoamylase (Gaa) appeared to affect the degradation of glycogen. The expressions of glgC-gaa-glgX and amyA-glgA were detected by the promoter assay. This result suggests that the gradual decrease of glycogen content in the late exponential and stationary phases occurs due to the increase in the gene expression of glgC-gaa-glgX. When the death rate in nutrient limited condition was compared among the wild type strain, the glycogen deficient strain and the strain with increased glycogen content, the death rate of the glycogen deficient strain was found to be higher than any other strain, thereby suggesting that the glycogen in S. acidocaldarius supports cell maintenance in harsh conditions.

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Dataset Information

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh.

Publications

Lesion-Induced Mutation in the Hyperthermophilic Archaeon Sulfolobus acidocaldarius and Its Avoidance by the Y-Family DNA Polymerase Dbh.

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