Robert W. Chambers, Ewa Sledziewska-Gojska*, and Samim Hirani-Hojatti**. Department of Biochemistry, Sir Charles Tupper Building, Dalhousie University, ...
Mol Gen Genet (1988) 213:325-331 © Springer-Verlag 1988
In vivo effect of D N A 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*, and Samim Hirani-Hojatti** Department of Biochemistry, Sir Charles Tupper Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
Summary. We have previously reported some effects of D N A repair on the transition frequencies produced by an O6-methyl-guanine (MEG) or an Or-n-butyl-guanine (BUG) paired with C at the first position of the third codon in gene G of bacteriophage ~X174 form I' D N A (Chambers et al. 1985). We now report experiments in which the transition is produced from T : M e G or T:BuG, instead of C :MeG or C :BUG, located at this site. The site-modified DNAs were transfected into cells with normal D N A 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 eoli ABlI57) 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 contrast to this, the transition frequency from T : M e G 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
* Present address: Polish Academy of Sciences, Institute of Biochemistry and Biophysics, ul Rakowiecka 36, 02-532, Warsaw, Poland ** Present address. Department of Medical Biophysics, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9 Offprint requests to: R.W. Chambers Abbreviations: MeG and BuG, O6-methyl- or O6-n-butyl-guanine moiety in q~X 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; ~X 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. o~XGam3 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 qSXEam7); pfu, plaque forming units; MNNG, N-methyl-N'-nitr o-N-nitrosoguanidine
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 - D N A repair - Bacteriophage q~X174
Introduction In a previous paper we described the use of site-specific mutagenesis to study the effect of excision repair, alkyl transfer repair and recombination on the transition frequency produced by C : M e G (O6-methyl,guanine) or C : B u G (O6-n-butyl-guanine) pairs located at position 2401 in gene G of ~X174 D N A (Chambers et al. 1985). We have now carried out a similar study involving T : M e G or T: BuG pairs located at the same position in the DNA. The experiments were designed to investigate two questions. The first concerns the effect of the initial base pair on the transition frequency produced from MeG or BuG. The second deals with the effect that D N A repair has on these transition frequencies.
Materials and methods The mutant, ~ X G a m 3 , used to isolate template D N A has been described (Bhanot et al. 1979). The following ~ X resistant strains, obtained from Dr. Barbara Bachmann at the Yale Escherichia coli Stock Center, were used to prepare spheroplasts: A B l I 5 7 F - thr-1 leuB6 thi-1 argE3 hisG4 A(gpt-proA)62 lacY1 galK2 2- mtl-1 xyl-5 ara-14 rpsL31 tsx-33 supE44 (Bachmann 1972); AB1886 as ABl157 but uvrA6, AB1885 as ABl157 but uvrB5, AB1884 as ABl157 but uvrC34 (Bachmann 1972; Howard-Flanders etal. 1966), AB2463 as AB1157 but recA13 (Bachmann 1972). The uvrB5 mutation is a 2 bp deletion; a truncated protein is formed (Backendorf et al. 1986). AB2480 was made by a cross between AB2437 Hfr J2 uvrA6 and 3033 F - recA13 A (gpt proA)62 lacY1 tsx-33? supE44? gaIK2 2recA13 rpsL8 (or rpsL31) xyl-5 mtL1 thi-i ; it is not isogenic to the other strains but carries the same uvrA and recA mutations (Howard-Flanders et al. 1969). The ~X-sensitive
326 indicator strains C and HF4714 Su2 + were used to screen the spheroplast lysates after transfection with D N A (Bhanot et al. 1979). The suppressor inserts glutamine (Bhanot e t a l . 1979; R. Chambers, unpublished experiments). F o r m I' D N A was synthesized by the primer extension m e t h o d using 2.4 p m o l o f ~ X G a m 3 D N A as the template and 30 p m o l o f the a p p r o p r i a t e primer (see Fig. 1). The D N A was purified by electrophoresis on an agarose gel in the presence o f ethidium bromide. The form I' b a n d was identified by comparison with a k n o w n s t a n d a r d of wild-type form I D N A , which runs slightly faster than form I' D N A and much faster than form II or III. The D N A was isolated and spheroplasts were p r e p a r e d as described previously (Chambers et al. 1985). Each spheroplast preparation was checked for competency by transfection with wild-type form I D N A . These spheroplasts were stored at 0 ° C and used immediately after their Competency h a d been determined. Transfection was carried out with 5 x 10 s molecules o f form I' D N A and 5 x l0 s cells. After a b s o r p t i o n for 15 min at 3 2 ° C in 300 ~tl o f buffer the volume was adjusted to 1 ml with p r e w a r m e d P A M medium (Guthrie and Sinsheimer 1963). Incubation was continued for 2 h at 32 ° C. The incubation mixture was diluted ten fold with b o r a t e - E D T A buffer and shaken vigorously to lyse the cells. Titers were determined by mixing duplicate aliquots o f the spheroplast lysate with 10 s a p p r o p r i a t e tester cells (see Fig. I) in soft agar and pouring onto nutrient agar plates. The plates were incubated at 38 ° C and read after 5-6 h.
Results Site-specific transitions f r o m T: MeG and T: BuG
The experimental design is outlined in Fig. 1. Detection o f the site-specific transition ( T A G ~ C A G ) p r o d u c e d from
ii ~
I
i
s-
(-) TACAAA TGAAAA (+)---ATGTTT ~TAGIACTTTT-5" amber 3" DNA polymerase
[large fragment) DNA ligase
1
Transition
dNTP's
ATP wt
HF4714 JTotal phage
1. Purify form I' DNA ~, 2. Transfect s p h e r o p l a s t s , 3 2 ° C ~ 3. Plate lysate
C wt phage
Fig. 1. Experimental design. Form I' DNA was synthesized as described in Materials and methods. The synthetic heteroduplex carried TAG as the third codon of gene G in the ( + ) strand and either (O6-n-butyl-guanine) BuG or (O6-methyl-guanine) MeG paired with T in the complementary strand as shown by the boxed area in the figure. The purified DNA was transfected into spheroplasts prepared from cells with normal DNA repair as well as from those with defects in excision repair, in recombination, and in both (see Materials and methods). In each case the spheroplasts carried a suppressor that inserts the wild-type amino acid, Gln, at the amber codon. The screen for total phage (Wt+Am) utilized a ~X-sensitive suppressor strain (Eseheriehia eoli HF4714). Transition "mutants" (Wt phenotype) were measured on E. eoli C Su-
GTC
~ TAG am GTC wt CAG~._GTC ATc~/TAG am TAG
~ transferalk~yl
/4 1
GTC CAG wt
~
a ~ - ~ T
ATC ~ TAG am
Fig. 2. The effect of replication and alkyl transfer repair on the transition frequency produced from a T:G* base pair. G*, 06methyl- or O6-n-butyl-guanine Table 1. The effect that inducing methyl transfer repair has on the transition frequency produced from T:MeG (O6-methyl-guanine) and C:MeG located in the third codon of ~ X gene G Spheroplasts a
DNA
Transitions (% of total phage)
Uninduced Induced
T: MeG b T: MeG
3c 14 c
Uninduced Induced
C: MeG d C: MeG
12 ° 4°
" Spheroplasts were Escherichia coli ABl157 supE/pO)XG. The plasmid, p ~ X G , was not required for the experiments with T:MeG since we were measuring A m ~ W t , and Su2 + (supE) inserts the correct amino acid (Gin). The plasmid, was required for the experiments utilizing C: MeG where we were measuring Lethal --*Ts. All of the experiments were done with a single spheroplast preparation: cells were grown to 2.5 x 10 s and split. Half was induced with 1 gg/ml MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) and washed free of the inducing agent. Both halves were converted to spheroplasts and transfected as described previously [footnote to Table I in Chambers et al. 1985] b The T residue is part of the TAG codon in the ( + ) strand. The MeG or BuG (O%n-butyl-guanine) residue is part of the complementary codon in the ( - ) strand. Progeny from both strands are packaged as ( + ) strands in the infectious virus particle. A transition from in this DNA is measured by the phenotypic change Am ~ Wt c Calculated from the mean pfu on E. eoli C (Wt) divided by pfu on E. coli HF4714 Su2 + ( A m + W t = t o t a l phage). Incubation was at 38° C. Titers were measured in duplicate at dilutions that gave about t00 pfu/plate. Since the plating efficiencies of E. eoli C and E. eoli HF4714 differ by only 10% corrections were not made a The preparation of this DNA has been described (Chambers et al. 1985) e A transition from this DNA is measured by the phenotypic change Lethal--* Ts as described previously (Chambers et al. 1985)
either M e G or B u G located in the first position o f the third c o d o n ~bX gene G was straightforward since the phenotype o f the phage progeny changes from a m b e r (Am) to wild type (Wt). As shown in Fig. 2, the frequency with which replication o f T: G* produces a T ~ C transition in vivo is dependent u p o n the frequency with which the O6-alkyl-guanine pairs with C. R e p a i r o f G* by alkyl transfer, which generates a T: G pair, should increase the transition frequency as shown in Fig. 2. To test this we induced the methyl transfer repair system by splitting a culture o f A B l 1 5 7 and pretreating h a l f with a low dose o f N - m e t h y l - N ' - n i t r o - N - n i t r o soguanidine ( M N N G ) . The transition frequency produced by D N A carrying T: M e G after transfection of spheroplasts
327 Table 2. A site-specific T ~ C transition produced by T:MeG (0 6-
Table 3. The effect of uvrA, uvrB and uvrC mutations on the transi-
methyl-guanine) and T:BuG (O6-n-butyl-guanine) located at position 2401 in double-stranded ~ X174 DNA
tion frequency produced from (O6-methyl-guanine) T:MeG and (O6-n-butyl-guanine) T:BuG located in the third codon of ~ X gene G
Spheroplasts" phenotype
DNA base pair
Spheroplasts a
DNA base pair b
UvrA ÷ RecA +
T:A ~ T: MeG T:BuG
0.03 6(7) d 0.3
Transition mutants c %
UvrA-/p qsXG
T :A T : MeG T:BuG
< 0.06 16(14) d 2
T:A T:MeG T: BuG
4 17 7
13 3
UvrB /p q~XG
T:A T:MeG T: BuG
0.02 11 0.4
T:A T:MeG T:BuG
3 19 8
16 4
UvrC-/p q~XG
T:A T:MeG T: BuG
3 16 9
13 6
UvrA - RecA *
UvrA + RecA-
UvrA RecA-
T:A T: MeG T:BuG
Transition mutants b % of total phage
