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In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl-or O6-n-butyl-guanine in a T:G base pair

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Summary

We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MeG) or an O6-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage ΦX174 form I'DNA (Chambers et al. 1985). We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contranst to this, the transition frequency from T:MeG is high (5±2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process. Using the BuG data reported here and in our previous paper, we calculate that BuG pairs with a thymine residue 0.5%–0.62% of the time during replication in vivo, and that BuG markedly inhibits replication of the strand that contains it. Because of the complication introduced by alkyl transfer repair, similar calculations for MeG cannot be made from the current data.

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Abbreviations

MeG and BuG:

O6-methyl-or O6-n-butyl-guanine moiety in ΦX DNA (in each case, the plus strand nucleotide is specified first)

form I'DNA:

relaxed, covalently closed, circular, double-stranded DNA

Wt:

wild-type phenotype

Am:

“amber” phenotype

pfu:

plaque forming units

MNNG:

N-methyl-N'-nitro-N-nitrosoguanidine

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Author notes

  1. Ewa Sledziewska-Gojska

    Present address: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, ul Rakowiecka 36, 02-532, Warsaw, Poland

  2. Samim Hirani-Hojatti

    Present address: Department of Medical Biophysics, Ontario Cancer Institute, 500 Sherbourne Street, M4X 1K9, Toronto, Ontario, Canada

Authors and Affiliations

  1. Department of Biochemistry, Dalhousie University, Sir Charles Tupper Building, B3H 4H7, Halifax, Nova Scotia, Canada

    Robert W. Chambers, Ewa Sledziewska-Gojska & Samim Hirani-Hojatti

Authors
  1. Robert W. Chambers
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  2. Ewa Sledziewska-Gojska
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  3. Samim Hirani-Hojatti
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Additional information

Communicated by B.J. Kilbey

ΦX mutants are named by designating the gene, the type of mutation (e.g. ms=missense), the codon number, the mutant codon and the new amino acid (where pertinent) in that order (e.g. ΦXGam3) carries an amber in the third codon of gene G, and should not be confused with the classical am3 mutant used in the older literature to designate what is now known to be ΦXEam7

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Chambers, R.W., Sledziewska-Gojska, E. & Hirani-Hojatti, S. In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl-or O6-n-butyl-guanine in a T:G base pair. Mol Gen Genet 213, 325–331 (1988). https://doi.org/10.1007/BF00339598

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  • DOI: https://doi.org/10.1007/BF00339598

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