Molec. gen. Genet. 168, 319-321 (1979) © by Springer-Verlag 1979

Recombination in Phage T4 Gene-43 (DNA Polymerase) Mutants A m i r a m R o n e n a n d C a l o m i r a Halevy Department of Genetics, The Hebrew University of Jerusalem, Israel

Summary. The effect of phage T4 gene 43 ( D N A polymerase) m u t a t i o n s o n r e c o m b i n a t i o n between adj a c e n t base pairs was m e a s u r e d in r H a m b e r a n d opal mutants. The m u t a t o r allele tsL56 did n o t p r o m o t e rec o m b i n a t i o n frequencies at the two sites in which its effect was studied. The a n t i m u t a t o r allele tsCB87 caused slight or n o r e d u c t i o n in r e c o m b i n a t i o n frequencies at five sites,

Introduction M u t a t o r a n d a n t i m u t a t o r properties Of phage T 4 D N A p o l y m e r a s e have been described (Speyer e t a l . , 1966; D r a k e a n d Allen, 1968; D r a k e e t a l . , 1969), a n d a t t r i b u t e d to altered specificity of the enzyme d u r i n g the process of D N A replication ( G o u l i a n et al., 1968; Brutlag a n d K o r n b e r g , 1972). As some D N A replication is involved in the process of rec o m b i n a t i o n ( T o m i z a w a , 1967), several studies have been u n d e r t a k e n to test whether m u t a n t D N A polymerase affects the frequency of r e c o m b i n a t i o n in phage T4. However, the m a r k e r s used were separated by genetic distances exceeding 0.5 u n i t s a n d little or n o effect was f o u n d (Bernstein, 1967; Speyer a n d R o s e n b e r g , 1968; D r a k e a n d Allen, 1968; Berger et al., 1969; Davis a n d S y m o n d s , 1974). In this report we are c o n c e r n e d with the effects of m u t a t o r a n d a n t i m u t a t o r phage T4 D N A polymerases o n recombin a t i o n between very close markers, i.e. the second a n d third nucleotides of allelic a m b e r a n d opal triplets ~ ( R o n e n a n d Salts, 1971). 1 Unless otherwise indicated, mRNA language is used to describe DNA sequences Abbreviations." A, T, G and C are adenine, thymine, guanine and 5-hydroxymethylcytosine,respectively

Materials and Methods Bacteria. E. coli B was used in crosses and for non-selectiveplating. r + recombinants were selected on the K12(2) strain KB. Phage. The rH mutants have been described before (Ronen and Salts 1971; Salts and Ronen 1971; Ronen and Rahat 1976). They are all derived from T4B. The gene-43 mutants tsCB87 and tsL56 were given to us by J.W. Drake. Since they are derived from T4D, we transferred the temperature-sensitive mutations into T4 background by repeated backcrossing, before 43ts rlI double mutants were constructed (Ronen, Halevy and Kass, in press). Media. Bacto tryptone broth and agar were used. Ultrahigh-sensitivity Crosses (Tessman, 1965) were performed essentiallyas described by Ronen and Salts (1971). The multiplicity of infection was eight of each parental phage. Plates were incubated at 37°. Burst Size. To measure burst size in any particular cross, E. coli B cells were infected at 37° with a mixture of amber and opal phages, in the presence of 3 x t0-3M KCN. The multiplicity of infection was eight of each phage. Adsorption was terminated by 1:2 dilution into anti-T4 serum (k=3 rain ~). After 5 rain at 37°, the infective centers were diluted 105 fold in broth, and incubated at 30° for 90 min. Efficiency of Detection was measured by plating 100-200 progeny of each of three isolated, wild-type recombinants from each cross, on two plates: one plate was seeded with E. coli B; the other piate contained E. coli KB and, in addition, 107 E. coli B preinfected with 1.6 x 108 r1272 phage particles (r1272 is a mutant which carries a deletion encompassing the whole rlI region). The efficiency of detection is the ratio of the number of plaques in the KB plates to that in the B plates. Recombination Index is the average number of r + plaques per plate, divided by the burst size and the efficiency of detection.

Results The results of the crosses are presented in T a b l e 1. A t sites YH119, Y H I 2 2 a n d YH320, the e s t i m a t i o n of r e c o m b i n a t i o n frequency is difficult in presence of the gene-43 m u t a t o r allele tsL56 because of reversion at high frequency of either the amber, the opal or both alleles ( R o n e n , Halevy a n d Kass, in press). As a conse-

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320

A. R o n e n and C. Halevy: Recombination in T4 Gene-43 M u t a n t s

Table 1. Effect of the gene-43 mutations tsL56 and t s C B 8 7 on recombination between allelic amber and opal mutations Cross

No. of plaques per plate

Efficiency of detection

Relative burst size

Recombination index

ts + r Y H l l 5 a m x ts + r Y H l l 5 o p tsCB87 rYHll5am x tsCB87 rYHll5op

663 + 85 538 ,+ 130 560 ,+ 166

0.43 0.87 0.78

1.0 1.7 1.1

1542 +_ 198 588 ,+ 88 653 ,+ 193

46 ± 24 62 ± 24

0.68 0.70

0.78 1.3

87 _+45 68 _+26

0.78

47 ,+ 16 3 8 + 10

0.57 0.71

0.54 1.3

153 +_52 41 _+ 11

0.27*

451 ,+ 197 1016 ,+ 376 1341 +__691

1.0 0.79 0.64

0.71 1.5 0.77

629+_277 857 _+317 2721 _+ 1402

1.4 4.3

66 ,+ 23 120,+97

0.70 0.62

0.63 1.3

150 +_52 148 _+ 120

0.99

tsL56 r Y H l I 5 a m

× tsL56 r Y H l l 5 o p

ts + r Y H l l9arn x ts + r Y H l l 9 o p tsCB87 r YHl19arn x tsCB87 r YH119op ts + r Y H 1 2 2 a m x ts + r Y H 1 2 2 o p tsCB87 r YH122am x tsCB87 r YHi22op ts + r Y H 1 3 2 a r n x ts + r Y H 1 3 2 o p tsCB87 rYH132am x tsCB87 rYH132op tsL56 r YH132am x tsL56 r YH132op ts + r Y H 3 2 0 a m x ts + r Y H 3 2 0 o p tsCB87 r YH320am x tsCB87 r YH320op

Ratio"

0.38** 0.42**

Burst size, efficiency of detection and recombination index are explained under Materials and Methods. 6-9 crosses were performed for each site Recombination index of ts . A n asterisk denotes that the difference between the indices is significant at the 0.05 level. Two asterisks Recombination index of ts + denote a significance at the 0.01 level a

quence, the effect of tsL56 on allelic amber x opal recombination could be measured only at sites Y H l l 5 and YH132. Only in 3 cases do we see a significant effect of the gene-43 mutation on recombination frequencies. At site YH122, the presence of tsCB87 reduces the frequency of wild-type recombinants to 27 per cent. At site YHI15 the reduction in recombination frequency caused by the antimutator allele is even smaller (38 per cent). At site Y H l l 5 , the mutator allele tsL56 reduces the frequency of wild-type recombinants to 42 per cent. These effects, though statistically significant, do not correlate in size or in generality (and in the case of tsL56, also in direction) with the mutator or antimutator phenotypes of the gene 43 mutants.

Discussion T4 D N A polymerase, coded by gene 43, has been implicated (Miller, 1975) in repairing the single-strand gaps of the joint molecules which are formed in the process of recombination (Tomizawa, 1967; Anraku and Lehman, 1969). When alMic U A G and U G A codons recombine to produce a wild type (UGG) codon, such gap filling must (a) start, or (b) end with a mismatched nucleotide (Fig. 1). The first possibility is depicted in Fig. 1 a, where gap filling must begin by using the mismatched G as primer. A D N A polymerase with a more exacting editing function (an-

~

AT C

~_

~

~>

T A G

AT C

~ = [] c>

~

X

ACT :~ T G A

>

/

OR :~ T A G

.~

[~ a Fig. 1. Mismatching at the site of allelic U A G x U G A recombination. Heavy lines: parental D N A strands (polarity indicated by arrows). Thin lines: repair D N A (gap filling)

timutator phenotype) may have higher tendency to correct the mismatch before inserting the new G. As suggested by Ronen and Salts (1971), this will result in the conversion of the heteroduplex to a homoduplex containing an amber (UAG) codon. Conversely, an enzyme which has a mutator phenotype, because it is defective in the editing step, will have a higher tendency to preserve the mismatch which is required for the production of a U G G codon. The gene-43 mutants employed in this study are defective in the " e d i t i n g " function (Muzyczka et al., 1972; Bessman et al., 1974), but fail to affect the production of wild-type codons in allelic U A G x U G A

A. Ronen and C. Halevy: Recombination in T4 Gene-43 Mutants r e c o m b i n a t i o n . The three cases where r e c o m b i n a t i o n frequencies in presence o f the gene-43 m u t a t i o n differ significantly f r o m the n o r m a l frequencies d o n o t seem to b e a r out the e x p e c t a t i o n s m e n t i o n e d above. Several e x p l a n a t i o n s m a y be considered. First, the m u t a t i o n s in tsL56 a n d tsCB87 m a y have no special editing effect on the p a r t i c u l a r mism a t c h e s s h o w n in Fig. l. I n d e e d , tsCB87 does n o t reduce s p o n t a n e o u s r e v e r s i o n o f the a m b e r m u t a t i o n s e m p l o y e d here ( R o n e n , H a l e v y a n d Kass, in press). H o w e v e r , tsL56 increases s p o n t a n e o u s reversion o f r Y H 1 1 5 a m a n d r Y H 1 3 2 a m 50- a n d 22- fold, respectively ( R o n e n , H a l e v y a n d Kass, in press), I f this increase involves the U A G ~ U G G reversion p a t h w a y , which is superficially similar to the U A G x U G A - ~ U G G r e c o m b i n a t i o n p a t h w a y , one m i g h t expect tsL56 to p r o m o t e r e c o m b i n a t i o n at sites Y H l l 5 a n d YHl32. Second, l i g a t i o n o f m i s m a t c h e d n u c l e o t i d e s at the 5' end n e a r the g a p m a y be efficient e n o u g h to a l l o w r e c o m b i n a t i o n to p r o c e e d a l o n g the p a t h w a y d e p i c t e d in Fig. l b . In this case, o n l y p a r t o f the p o t e n t i a ! r e c o m b i n a t i o n events s t u d i e d in this w o r k will be a m e n a b l e to the effect o f m u t a t i o n s in gene 43. Third, D N A p o l y m e r a s e c o d e d b y gene 43 m a y be d i s p e n s a b l e in the p r o c e s s o f g a p filling. P a r t i c i p a t i o n o f h o s t f u n c t i o n s in g a p filling m a y m a s k p o s s i b l e effects o f m u t a t i o n s in T4 gene 43 on U A G × U G A r e c o m b i n a t i o n . O t h e r viral a n d b a c t e r i a l genes i n v o l v e d in resolving the h e t e r o d u p l e x r e g i o n in the j o i n t m o l e c u l e ( M o s i g et al., 1972) m a y also influence the fate o f the m i s m a t c h e d nucleotides. W e also d o n o t k n o w t h a t either one o f the h e t e r o d u p l e x structures shown in Fig. 1 is i n d e e d an i n t e r m e d i a t e in the f o r m a t i o n o f w i l d - t y p e r e c o m b i n a n t s in U A G x U G A crosses. W e may, however, dismiss the p o s s i b i l i t y t h a t U G G c o d o n s are f o r m e d by " e d i t i o n " in two j u x t a p o s e d , intact U A G a n d U G A sites. This is s u p p o r t e d by the finding ( R o n e n a n d A t i d i a , u n p u b l i s h e d results), t h a t the p r o d u c t i o n o f w i l d - t y p e p h a g e in allelic a m b e r x o p a l crosses is a s s o c i a t e d with free r e c o m b i n a t i o n between o u t s i d e markers.

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321 tion due to amber mutations in T4D bacteriophage. J. Virol. 3, 171-175 (1969) Bernstein, H. : The effect on recombination of mutational defects in the DNA-polymerase and deoxycytidylate hydroxymethylase of phage T4D. Genetics 56, 755 769 (1967) Bessman, M.J., Muzyczka, N., Goodman, M.F., Schnaar, R.L.: Studies on the biochemical basis of spontaneous mutation. II. The incorporation of a base and its analogue into DNA by wild-type, mutator and antimutator DNA polymerases. J. Mol. Biol. 88, 409 421 (1974) Brutlag, D., Kornberg, A.: Enzymatic synthesis of deoxyribonucleic acid. XXXVI. A proofreading function for the 3'45' exonuclease activity in deoxyribonucleic acid polymerases. J. Biol. Chem. 247, 241-248 (1972) Davis, K.J., Symonds, N. : The pathway of recombination in phage T4. A genetic study. Mol. Gen. Genet. 132, 173-180 (1974) Drake, J.W., Allen, E.F. : Antimutagenic DNA polymerase of bacteriophage T4. Cold Spring Harbor Syrup. Quant. Biol. 33, 339-344 (1968) Drake, J.W., Allen, E.F., Forsberg, S.A., Preparata, R.M., Greening, E.O. : Genetic control of mutation rates in bacteriophage T4. Nature 221, 1128-1132 (1969) Goulian, M., Lucas, Z.J., Kornberg, A.: Enzymatic synthesis of deoxyribonucleic acid. XXV. Purification and properties of deoxyribonucleic acid polymerase induced by infection with phage T4. J. Biol. Chem. 243, 627-638 (1968) Miller, R.C.: T4 DNA polymerase (gene 43) is required in vivo for repair of gaps in recombinants. J. Virol. 15, 316-321 (1975) Mosig, G., Bowden, D.W., Bock, S.: E. coli DNA polymerase I and other host functions participate in T4 DNA replication and recombination. Nature New Biol. 240, 12-15 (1972) Muzyczka, N., Poland, R.L., Bessman, M.J.: Studies on the biochemical basis of spontaneous mutation. I. A comparison of the deoxyribonucleic acid polymerases of mutator, antimutator, and wild type strains of bacteriophage T4. J. Biol. Chem. 247, 7116 7122 (1972) Ronen, A., Halevy, C., Kass, N.: Site specificity and variability in the mutator and antimutator effects of phage T4 gene 43 mutants. Genetics (in press) Ronen, A., Rahat, A. : Mutagen specificity and position effects on mutation in T4rH nonsense sites. Mutat. Res. 34, 21-34 (1976) Ronen, A., Salts, Y.: Genetic distances separating adjacent base pairs in bacteriophage T4. Virology 45, 496 502 (1971) Salts, Y., Ronen, A.: Neighbor effects in the mutation of ochre triplets in the T4rH gene. Mutat. Res. 13, 109-113 (197i) Speyer, J.F., Karam, J.D., Lenny, A.B.: On the role of DNA polymerase in base selection. Cold Spring Harbor Syrup. Quant. Biol. 31, 693-697 (1966) Speyer, J.F., Rosenberg, D. : The function of T4 DNA polymerase. Cold Spring Harbor Syrup. Quant. Biol. 33, 345 350 (1968) Tessman, I.: Genetic ultrafine structure in the T4rH region. Genetics 51, 63-75 (1965) Tomizawa, J.: Molecular mechanisms of genetic recombination in bacteriophage: Joint molecules and their conversion to recombinant molecules. J. Cell Physiol. 70, Sup. 1, 201-213 (1967) C o m m u n i c a t e d by B.A. Bridges Received July 24, 1978