Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus

Sedliaková, Milena; Brozmanová, J; Mašek, František; Kleibl, K.

doi:10.1016/s0006-3495(81)84742-x

Cited by 21 publications

(2 citation statements)

References 44 publications

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“…This would indicate that there is some alteration in the structure of the duplex opposite the replicated pyrimidine dimer. A related observation was reported by two groups (31,32), who found that after replication of UV-irradiated DNA in vivo, in E. coli (31) 10 mM Tris HCl at pH 8, 10 mM EDTA, 0.1 M NaCl, bovine serum albumin at 0.1 mg/ml, and 2 x iOs units of T4 endonuclease V. Mixtures were incubated at 37°C for 60 min, after which they were analyzed by alkaline agarose gel electrophoresis as described in Materials and Methods and in the legend to Fig. 2A.…”

Section: Discussionsupporting

confidence: 75%

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers.

Livneh¹

1986

Proc. Natl. Acad. Sci. U.S.A.

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Replication of UV-irradiated circular singlestranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase HI holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage 4X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase m holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins.Pyrimidine photodimers, the major UV-induced lesions in DNA, are believed to constitute an absolute block to DNA replication, which then resumes at a site distal to the dimer, generating a single-stranded gap within the DNA duplex (refs. 1-3; but see ref. 4). Current models suggest that recA protein binds to the single-stranded region, resulting in its activation as a specific protease, which can then lead to the cleavage of lexA repressor. As a consequence, at least 16 cellular genes are induced, generating a diversity of phenomena including inhibition of cell division, enhanced postreplication repair, and mutagenesis. The process of mutagenesis, which requires the recA (1), umuC, and umuD (5, 6) gene products, is of particular interest. It has been suggested that mutagenesis occurs by error-prone replication through the pyrimidine dimers, possibly by an altered, error-prone, DNA polymerase (1). In vitro studies have suggested that DNA polymerase III (EC 2.7.7.7) is involved in UV mutagenesis (7,8).'Lackey et al. (9) have isolated an altered DNA polymerase I from SOS-induced cells that shows a high frequency of miscoding in vitro. However, the significance of this activity in vivo is unclear in view of the normal UV-mutability observed with polA mutants (1).In undertaking a biochemical analysis of UV-induced mutagenesis, we began by examining the replication of UV-irradiated circular single-stranded DNAs from phages M13, G4, and 4X174 with purified enzymes isolated from wild-type cells ["SS -* RF reaction" (10)]. We have found that, contrary to expectations, pyrimidine photodimers do not constitute an absolute block to DNA replication and can be bypassed to a significant extent, possibly with formation of mutations, even in the absence of SOS-induced proteins. MATERIALS AND METHODSDNA Preparations. Ml3mp8 single-stranded (ss) DNA (11) and OX174 ss DNA were prepared as described (12, 13).M13Goril ss DNA was a gift from D. Soltis and G4 ss DNA was a gift from M. Stayton.Enzymes and Proteins. Escherichia coli DNA polymerase III holoenzyme and ss DNA-...

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Section: Discussionsupporting

confidence: 75%

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers.

Livneh¹

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…This was also true when we tried to measure the amount of pyrimidine dimers in the unreplicated DNA (HH peak). This insensitivity of UV lesions to dimerspecific enzyme has already been reported for dimer-containing DNA in E. coli B/r DNA after replication (36). When these bacteria receive a sublethal UV dose before UV irradiation they do not efficiently excise the lesion.…”

Section: Methodssupporting

confidence: 64%

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA

et al. 1989

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Agents that interfere with DNA replication in Escherichia coli induce physiological adaptations that increase the probability of survival after DNA damage and the frequency of mutants among the survivors (the SOS response). Such agents also increase the survival rate and mutation frequency of irradiated bacteriophage after infection of treated bacteria, a phenomenon known as Weigle reactivation. In UV-irradiated single-stranded DNA phage, Weigle reactivation is thought to occur via induced, error-prone replication through template lesions (translesion synthesis [P. Caillet-Fauquet, M. Defais, and M. Radman, J. Mol. Biol. 117:95-112, 1977]). Weigle reactivation occurs with higher efficiency in double-stranded DNA phages such as X, and we therefore asked if another process, recombination between partially replicated daughter molecules, plays a major role in this case. To distinguish between translesion synthesis and recombinational repair, we studied the early replication of UV-irradiated bacteriophage X in SOS-induced and uninduced bacteria. To avoid complications arising from excision of UV lesions, we used bacterial uvrA mutants, in which such excision does not occur. Our evidence suggests that translesion synthesis is the primary component of Weigle reactivation of X phage in the absence of excision repair. The greater efficiency in Weigle reactivation of double-stranded DNA phage could thus be attributed to some inducible excision repair unable to occur on single-stranded DNA. In addition, after irradiation, X phage replication seems to switch prematurely from the theta mode to the rolling circle mode.Exposure of Escherichia coli to agents that interfere with DNA replication leads to RecA-mediated cleavage of the LexA repressor, which induces a number of diverse physiological functions called the SOS response (32,45). Among the multiple functions expressed during this response is a transient error-prone DNA processing event that seems to be responsible for most of the radiation-or chemical-induced mutagenesis (42). This phenomenon requires RecA-mediated cleavage of the UmuD protein and formation of a complex between the COOH fragment and the UmuC protein (3, 31, 37). The UmuDC complex may act by facilitating replication with a high level of misincorporation past noninstructive lesions in the DNA (2). Mutagenesis is observed not only on the bacterial chromosome but also on extrachromosomal DNA, and it can easily be studied using viruses as probes (14). Induction of the host SOS response results in an increased survival of UV-irradiated bacteriophage (Weigle reactivation), which is accompanied by an increased mutation frequency in the progeny (13, 43). Genetic and biochemical evidence indicates that in singlestranded DNA phage, Weigle reactivation occurs via induced replication through the UV lesions (translesion synthesis) (6). Moreover in vitro studies of the extent of DNA synthesis on UV-irradiated XX174 DNA have shown a large increase in the turnover of nucleoside triphosphates to free monophosphates throu...

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The presence of alkali‐labile sites in DNA daughter chains of UV‐irradiated Escherichia coli

Slezáriková

Sedliaková

1982

FEBS Letters

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DNA newly synthesized in UV irradiated Escherichia coli B/r Her+ was 2 min pulse-labeled at various periods, then denatured and analysed by sucrose gradient centrifugation either in neutral or in alkaline conditions. Data indicate that in DNA of damaged cells alkali-labile sites are produced. In cells saturated with inducible proteins production of alkali-labile sites disappears in -1 h. In the absence of inducible proteins production of alkali-labile sites continues. Escherichia coliUltraviolet light DNA repair Alkali-labile sites Stable DNA replication Chloramphenicol

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Evidence that dimers remaining in preinduced Escherichia coli B/r Hcr+ become insensitive after DNA replication to the extract from Micrococcus luteus

Cited by 21 publications

References 44 publications

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers.

Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers.

Translesion synthesis is the main component of SOS repair in bacteriophage lambda DNA

The presence of alkali‐labile sites in DNA daughter chains of UV‐irradiated Escherichia coli

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