Replication of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion by Escherichia coil cell extracts

Wang, G; Pm, Dunman; Mz, Humayun

doi:10.1038/cr.1997.1

Cited by 5 publications

(1 citation statement)

References 24 publications

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“…(iii) Primer removal and gap filling are carried out by DNA polymerase I, the nick is sealed by DNA ligase, and the DNA is negatively supercoiled by gyrase. We have shown recently (Wang et al ., 1997a) that an in vitro replication system based on crude cell‐free extracts appears to model the in vivo replication processes by the following criteria: (i) DNA replication is sensitive to rifampicin, which is expected to block initiation by RNA polymerase at Ori − ; (ii) DNA replication does not require host DNA polymerases I or II; (iii) replication of ssDNA bearing site‐specific lesions shows the same pattern of lesion‐specific inhibition as that obtained in vivo , e.g. DNA O 6 ‐methylguanine does not block replication, whereas abasic sites act as strong blocks to replication.…”

Section: Resultsmentioning

confidence: 80%

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA‐dependent error‐prone DNA replication activity

Mamun

Rahman

Humayun

1999

Molecular Microbiology

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SummaryA base substitution mutation (mutA) in the Escherichia coli glyV tRNA gene potentiates asp ! gly mistranslation and confers a strong mutator phenotype that is SOS independent, but requires recA, recB and recC genes. Here, we demonstrate that mutA cells express an error-prone DNA polymerase by using an in vitro experimental system based on the conversion of phage M13 single-stranded viral DNA bearing a model mutagenic lesion to the double-stranded replicative form. Ampli®cation of the newly synthesized strand followed by multiplex DNA sequence analysis revealed that mutation ®xation at 3,N 4 -ethenocytosine («C) was <3% when the DNA was replicated by normal cell extracts, <48% when replicated by mutA cell extracts and <3% when replicated by mutA recA double mutant cell extracts, in complete agreement with previous in vivo results. Mutagenesis at undamaged DNA sites was signi®cantly elevated by mutA cell-free extracts in the M13 lacZ(a) forward mutagenesis system. Neither polA (DNA polymerase I) nor polB (DNA polymerase II) genes are required for the mutA phenotype, suggesting that the phenotype is mediated through a modi®cation of DNA polymerase III or the activation of a previously unidenti®ed DNA polymerase. These ®ndings de®ne the major features of a novel mutagenic pathway and imply the existence of previously unrecognized links between translation, recombination and replication.

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Section: Resultsmentioning

confidence: 80%

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA‐dependent error‐prone DNA replication activity

Mamun

Rahman

Humayun

1999

Molecular Microbiology

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Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo

et al. 2004

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DNA base flipping analytical pipeline

Zhang

Hastert

Ludwig

et al. 2017

Biology Methods and Protocols

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DNA base modifications and mutations are observed in all genomes throughout the kingdoms of life. Proteins involved in their establishment and removal were shown to use a base flipping mechanism to access their substrates. To better understand how proteins flip DNA bases to modify or remove them, we optimized and developed a pipeline of methods to step-by-step detect the process starting with protein–DNA interaction, base flipping itself and the ensuing DNA base modification or excision. As methylcytosine is the best-studied DNA modification, here we focus on the process of writing, modifying and reading this DNA base. Using multicolor electrophoretic mobility shift assays, we show that the methylcytosine modifier Tet1 exhibits little DNA sequence specificity with only a slight preference for methylated CpG containing DNA. A combination of chloroacetaldehyde treatment and high-resolution melting temperature analysis allowed us to detect base flipping induced by the methylcytosine modifier Tet1 as well as the methylcytosine writer M.HpaII. Finally, we show that high-resolution melting temperature analysis can be used to detect the activity of glycosylases, methyltransferases and dioxigenases on DNA substrates. Taken together, this DNA base flipping analytical pipeline (BaFAP) provide a complete toolbox for the fast and sensitive analysis of proteins that bind, flip and modify or excise DNA bases.

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Replication of M13 single-stranded DNA bearing a site-specific ethenocytosine lesion by Escherichia coil cell extracts

Cited by 5 publications

References 24 publications

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA‐dependent error‐prone DNA replication activity

Escherichia coli cells bearing mutA, a mutant glyV tRNA gene, express a recA‐dependent error‐prone DNA replication activity

Role of mismatch-specific uracil-DNA glycosylase in repair of 3,N4-ethenocytosine in vivo

DNA base flipping analytical pipeline

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