UV mutagenesis in E. coli with excision repair initiated by uvrABC or denV gene products

Bockrath, Richard; Hodes, Marquis Z.; Mosbaugh, P.; Valerie, Kristoffer; Riel, J K de

doi:10.1016/0167-8817(88)90039-9

Cited by 7 publications

(4 citation statements)

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“…These suppressor mutations have been shown to arise from C-to-T transitions at DNA sites affecting the anticodon, i.e., -ATCAAAA-to -ATTAAAA-(the transcribed strand encoding the anticodon loop) (5). Either T=C or T(6-4)C photoproducts could form at this mutation site, and targeting by a variable proportion of T=C and T(6-4)C has been proposed (1,2). Back mutations at the ochre defect (-TTA-in the transcribed strand) apparently are not targeted by photoreversible dimers (1,14).…”

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“…Plasmid pKY33 was constructed from the vector pBR322 with insertion of a 2.8-kilobase AccI fragment from pBG6 that contained the phr gene (23). Previous studies with other E. coli strains showed no effects by pBR322 on the types of mutation frequency response described here (1 When we compared UV mutagenesis in control and transformed strains of JC3890 by measuring the induction of Arg+ prototrophic mutants, substantially fewer mutants resulted with the transformed strain. Individual mutant colonies were isolated and tested with bacteriophage T4 mutants to distinguish de novo glutamine tRNA ochre suppressor mutations at the glnU gene and back mutations at the argE3(0c) defect (2; Ruiz-Rubio and Bockrath, in press), which together accounted for essentially all of the Arg+ mutants.…”

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“…Either T=C or T(6-4)C photoproducts could form at this mutation site, and targeting by a variable proportion of T=C and T(6-4)C has been proposed (1,2). Back mutations at the ochre defect (-TTA-in the transcribed strand) apparently are not targeted by photoreversible dimers (1,14). Thus, in strain JC3890 (pKY33) carrying a large amount of photolyase, there is a specific reduction of mutations associated with targeting by T=C.…”

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An in vivo complex with DNA photolyase blocks UV mutagenesis targeted at a thymine-cytosine dimer in Escherichia coli

1988

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UV mutation frequency responses for two types of Escherichia coli prototrophic mutant were measured. Only the response associated with a mutation targeted by a thymine-cytosine pyrimidine dimer was reduced in the dark in cells with amplified DNA photolyase. This specific reduction is attributed to the interruption of mutational DNA synthesis by a photolyase complex at the targeting dimer.The thymine-cytosine cyclobutane dimer (T=C) has been implicated as the UV photoproduct at the site of mutagenesis causing (targeting) C-to-T transitions in certain DNA sequences in Escherichia coli (2, lOa,12). DNA photolyase is a cellular protein that specifically binds to cyclobutanepyrimidine dimers (17), the major photoproduct produced in DNA after exposure to germicidal UV light (13). We find that with cells containing amplified levels of photolyase, mutations otherwise targeted at T=C are eliminated in the dark.Possibly the specialized DNA synthesis required for misincorporation during translesion replication at T=C in template DNA (2, 4) is blocked by photolyase bound to T=C. The presence of photolyase-T=C complexes in cells with amplified DNA photolyase also is indicated by a reduced rate of T=C-to-T=U deamination and by a reduced magnitude of delayed photoreversal (PR) mutagenesis.Normally E. coli contains only a few DNA photolyase molecules per cell. These molecules cycle from dimer to dimer, under a flux of PR light, to monomerize dimers (8, 16). A photolyase-dimer complex, which can form in the dark, dissociates upon addition of light and monomerization of the dimer (17). Strains transformed with appropriate plasmids have amplified levels of the enzyme (16, 23). We found the transformed E. coli K-12 strain JC3890(pKY33) to have at least 600 enzyme molecules per cell, sufficient to form complexes with all of the dimers in a cell after biologically significant UV irradiation (a fluence of 1.0 J/m2 would produce about 60 dimers per genome [13]). Strain JC3890 without the plasmid could form only on the order of 10 complexes..These estimates were drawn from the effects of a single flash of PR light on the viability of the two different strains exposed to UV light (9) (Fig. 1) excision repair defective (AuvrB301) and the immediate precursor to TK501 (for details of strains and procedures, see M. Ruiz-Rubio and R. Bockrath, Mutat. Res,, in press).When we compared UV mutagenesis in control and transformed strains of JC3890 by measuring the induction of Arg+ prototrophic mutants, substantially fewer mutants resulted with the transformed strain. Individual mutant colonies were isolated and tested with bacteriophage T4 mutants to distinguish de novo glutamine tRNA ochre suppressor mutations at the glnU gene and back mutations at the argE3(0c) defect (2; Ruiz-Rubio and Bockrath, in press), which together accounted for essentially all of the Arg+ mutants. The diminished mutation frequency response in cells with amplified photolyase was found to be due exclusively to reduction of the suppressor mutations (Fig. 2).Since the produc...

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An in vivo complex with DNA photolyase blocks UV mutagenesis targeted at a thymine-cytosine dimer in Escherichia coli

1988

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“…Metabolic labelling and immunoblotting experiments showed that low, but detectable, levels of endonuclease V are produced in uninduced cells (data not shown). Our previous work demonstrated that the uninduced ptac-denV/ AB2480(F"IacIQI) strain produces -5-fold more endonuclease V than the pdenV-7/AB2480 strain (13,35). After induction with IPTG a pronounced increase of the 16 kDa endonuclease V protein was detected within 15 min which accounted for 30% of total protein made (data not shown).…”

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Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activityin vitroandin vivo

Valerie

1995

Nucl Acids Res

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Ultraviolet photoproducts at the ochre suppressor mutation site in the gln U gene of Escherichia coli: Relevance to “mutation frequency decline”

1989

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Ochre suppressor mutations induced by UV in the Escherichia coli glnU tRNA gene are CG to TA transitions at the first letter of the anticodon-encoding triplet, CAA. Premutational UV photoproducts at this site have long been known to exhibit an excision repair anomaly ("mutation frequency decline" or MFD), whereby postirradiation inhibition of protein synthesis enhances their excision and reduces suppressor mutation yields ten-fold. We sought to clarify the basis of this unique repair response by determining the spectrum of UV photoproducts on both strands of a 36 bp region of glnU which includes the anticodon-encoding triplet. We found that four different photolesions are produced within the 3 bp sequence corresponding to the tRNA anticodon: (i) on the transcribed strand, TC (6-4) photoproducts and TC cyclobutane dimers are formed in equal numbers at the site of the C to T transition, indicating that this site is a hotspot for the usually less frequent (6-4) photoproduct; (ii) on the nontranscribed strand, TT dimers are found opposite the second and third letters of the anticodon-encoding triplet, adjacent to the mutation site; and (iii) on the nontranscribed strand, an alkali-sensitive lesion other than a (6-4) photoproduct is formed, apparently at the G in the mutation site. We suggest that mutation frequency decline may reflect excision repair activity at closely spaced UV lesions on opposite strands, resulting in double-strand breaks and the death of potential mutants.

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UV mutagenesis in E. coli with excision repair initiated by uvrABC or denV gene products

Cited by 7 publications

References 35 publications

An in vivo complex with DNA photolyase blocks UV mutagenesis targeted at a thymine-cytosine dimer in Escherichia coli

An in vivo complex with DNA photolyase blocks UV mutagenesis targeted at a thymine-cytosine dimer in Escherichia coli

Replacing tryptophan-128 of T4 endonuclease V with a serine residue results in decreased enzymatic activityin vitroandin vivo

Ultraviolet photoproducts at the ochre suppressor mutation site in the gln U gene of Escherichia coli: Relevance to “mutation frequency decline”

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