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

Abstract

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...

Anti-mutagenic Activity of DNA Damage-binding Proteins Mediated by Direct Inhibition of Translesion Replication

“…The strains used were constructed as described under "Experimental Procedures. Consistent with our suggestion is the observation that overproduction of DNA photolyase reduced in vivo UV mutagenesis (39). A related observation was the report that mutations due to a site-specific O 6 -butylguanine in phage x174 increased 8-fold in a uvrA6 mutant.…”

Mechanism of SOS mutagenesis of UV-irradiated DNA: mostly error-free processing of deaminated cytosine.

“…There would then seem to be some discrepancy between the time measured in vitro for deamination to occur (=30 min in single-stranded DNA) and the bypass delay time (-100 min). This could be explained if the time needed for deamination were lengthened by the intracellular environment of the lesion, reminiscent of the inhibition of deamination in S13 DNA by the viral capsid (8) and in E. coli DNA by DNA photolyase (11). Fig.…”

“…More detailed experiments with infectious S13 DNA show that the transition from negligible to nearly complete mutagenesis occurs between 25 and 30 min of DNA storage at 37°C (unpublished data). Earlier studies of mutagenesis by delayed photoreactivation had already indicated that dimerized cytosines in the double-stranded DNA of E. coli are deaminated in vivo within 90 min at 37°C (11). We predicted that if a sufficiently long delay were an intrinsic aspect of the SOS repair process, deamination of the cytosines in UV-induced dimers could have time to occur and would then be a source of the C -+ T mutations that are so commonly induced by UV irradiation.…”

Characterization of Spontaneous Mutation in the oxyR Strain of Escherichia coli

“…The spontaneous mutation frequencies in oxyR::Km and ⌬oxyR strains were determined by using four different mutation assays; Rif R may occur by base substitution in the rpoB gene (22), resistance to valine may occur by forward mutations leading to a decreased ability to transport valine into the cell or a mutation leading to an altered acetohydroxy acid synthetase I or III (ilvB or ilvHI genes, respectively) (23,24), Arg ϩ His ϩ reversions of AB1157 cells may occur by G:C3 A:T base substitution in suppresser tRNA genes (25) and ColB R may arise by forward mutations of deletions, insertions, frameshifts, or any of the six possible base-substitution mutations in the tonB gene (12).…”