Mutation spectra in supF: approaches to elucidating sequence context effects

Canella, Karen A; Seidman, Michael M.

doi:10.1016/s0027-5107(00)00016-6

Cited by 55 publications

(54 citation statements)

References 120 publications

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“…Given that other regions of the TK enzyme may have a much higher degree of plasticity of substituted residues, the overall ratio of undetectable tk mutants may be higher than 56%. On the other hand, about 96% of the 255 (3 ϫ 85) possible changes within the 85 bases of the supF gene have been isolated (3). This indicates that the supF mutagenesis system is more sensitive in detecting mutations than the tk system.…”

Section: Discussionmentioning

confidence: 99%

Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

Hwang

Liu

Hwang

2002

J Virol

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Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene. A mutation frequency of approximately 10 ؊4 was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene. However, recombinants derived from the PAA r 5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three-to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA r 5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.DNA replication in mammalian cells is a complicated process (reviewed in references 2 and 25). It requires intricate DNA replication machinery that both duplicates chromosomal DNA and maintains genomic integrity. As the pivotal enzymes, DNA polymerases (Pols) are critical for ensuring the accuracy of DNA synthesis via their roles in the selection of nucleotides to be incorporated during DNA replication (polymerization) and in the removal of misinserted nucleotides from the 3Ј-OH end of the primer strand (proofreading) (reviewed in references 20 and 23

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Section: Discussionmentioning

confidence: 99%

Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

Hwang

Liu

Hwang

2002

J Virol

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“…However, from the detection of TCHQ-DNA adducts, we hypothesize that TCHQ may react with plasmid DNA to form TCHQ-DNA adducts, and G:C base pairs are the preferential source for generating these TCHQ-DNA adducts. TCHQ-DNA adducts may pair with T, G or protonated adenine, in addition to C [25,26,31]. However, the levels of AP sites and 8-OH-dG were estimated to be approximately two orders of magnitude greater than the previously reported levels from direct DNA adducts [37].…”

Section: Discussionmentioning

confidence: 74%

“…Gene mutations result from an accumulation of errors during DNA replication and repair [30]. The pSP189 shuttle vector plasmid provides an excellent platform to detect DNA damage caused by many physical or chemical mutagens in mammalian cells [31][32][33]. The supF assay has been previously [21,[34][35][36].…”

Section: Discussionmentioning

confidence: 99%

Characterization of TCHQ-induced genotoxicity and mutagenesis using the pSP189 shuttle vector in mammalian cells

Wang

Jiao

et al. 2012

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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a b s t r a c tTetrachlorohydroquinone (TCHQ) is a major toxic metabolite of the widely used wood preservative, pentachlorophenol (PCP), and it has also been implicated in PCP genotoxicity. However, the underlying mechanisms of genotoxicity and mutagenesis induced by TCHQ remain unclear. In this study, we examined the genotoxicity of TCHQ by using comet assays to detect DNA breakage and formation of TCHQ-DNA adducts. Then, we further verified the levels of mutagenesis by using the pSP189 shuttle vector in A549 human lung carcinoma cells. We demonstrated that TCHQ causes significant genotoxicity by inducing DNA breakage and forming DNA adducts. Additionally, DNA sequence analysis of the TCHQ-induced mutations revealed that 85.36% were single base substitutions, 9.76% were single base insertions, and 4.88% were large fragment deletions. More than 80% of the base substitutions occurred at G:C base pairs, and the mutations were G:C to C:G, G:C to T:A or G:C to A:T transversions and transitions. The most common types of mutations in A549 cells were G:C to A:T (37.14%) and A:T to C:G transitions (14.29%) and G:C to C:G (34.29%) and G:C to T:A (11.43%) transversions. We identified hotspots at nucleotides 129, 141, and 155 in the supF gene of plasmid pSP189. These mutation hotspots accounted for 63% of all single base substitutions. We conclude that TCHQ induces sequence-specific DNA mutations at high frequencies. Therefore, the safety of using this product would be carefully examined.

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“…The cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum in a 5% CO2 humidified incubator. The pSP189 plasmid (including a randomly generated 8-base pair signature sequence at the 3'-end of the supF gene) was kindly provided by Michael Seidman (26). The bacterial strain, E. coli MBM7070, was cultured on agar containing ampicillin and isopropyl β-D-thiogalactoside, a β-galactosidase inducer, as well as X-gal as a color indicator.…”

Section: Cell Lines and Plasmidsmentioning

confidence: 99%

“…The plasmids were isolated from the white, mutant colonies, and the supF gene was sequenced. The sibling plasmids that originated from the same mutational event were identified by the repeated appearance of a signature sequence adjacent to the supF gene (26). The siblings were excluded from the total number of white, mutant colonies.…”

Section: Introductionmentioning

confidence: 99%

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

Kim

Kim²,

Cha³

et al. 2008

BMB Reports

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Mutation spectra in supF: approaches to elucidating sequence context effects

Cited by 55 publications

References 120 publications

Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

Replication Fidelity of the supF Gene Integrated in the Thymidine Kinase Locus of Herpes Simplex Virus Type 1

Characterization of TCHQ-induced genotoxicity and mutagenesis using the pSP189 shuttle vector in mammalian cells

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

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