Molecular and General Genetics MGG

, Volume 213, Issue 2–3, pp 325–331 | Cite as

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

  • Robert W. Chambers
  • Ewa Sledziewska-Gojska
  • Samim Hirani-Hojatti
Article
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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.

Key words

Site-specific mutagenesis O6-alkyl-guanine Transitions from T:MeG and T:BuG DNA repair Bacteriophage ΦX174 

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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Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Robert W. Chambers
    • 1
  • Ewa Sledziewska-Gojska
    • 1
  • Samim Hirani-Hojatti
    • 1
  1. 1.Department of BiochemistryDalhousie UniversityHalifaxCanada

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