marker-dependent recombination in t4 bacteriophage. 111. … · 2002. 7. 8. · he122 x 375 70...

Copyright 0 1991 by the Genetics Society of America

Marker-Dependent Recombination in T4 Bacteriophage. 111. Structural Prerequisites for Marker Discrimination

V. P. Shcherbakov and L. A. Plugina

Institute of Chemical Physics, U.S.S.R. Academy of Sciences, Chernogolouka 142432, Mascow Region, U.S.S.R. Manuscript received June 29, 1990

Accepted April 22, 199 1

ABSTRACT Distance- as well as marker-dependence of genetic recombination of bacteriophage T 4 was studied

in crosses between rIIB mutants with known base sequences. The notion of a “basic recombination,” which is the recombination within distances shorter than hybrid DNA length in the absence of mismatch repair and any marker effects, was substantiated. The basic recombination frequency per base pair can serve as an objective parameter (natural constant) of general recombination reflecting its intensity. Comparative studies of the recombination properties of rIIB mutants with various sequence changes in the mutated sites showed that the main factor determining the probability of mismatch repair in recombination heteroduplexes is the length of a continuous heterologous region. A run of A:T pairs immediately adjoining the mismatch appears to stimulate its repair. In the case of mismatches with DNA strands of unequal length, formed by frameshift mutations, the repair is asymmetric, the longer strand (bulge) being preferentially removed. A pathway for mismatch repair including sequential action of endonuclease VI1 (gp49) -+ 3‘- 5’ exonuclease (gp43) -+ DNA polymerase (gp43) + DNA ligase (gp30) was proposed. A possible identity of the recombinational mismatch repair mechanism to that operating to produce mutations via sequence conversion is discussed.

I N previous papers (SHCHERBAKOV, SIZOVA and PLU- GINA 1979; SHCHERBAKOV et al. 1982a,b) we de-

scribe an “extra” recombination mechanism in T4 phage, which contributed to general recombination only when particular mutations were used as genetic markers (high recombination or HR markers), whereas it was practically inactive towards other rZZB mutations (low recombination or LR markers). This marker-dependent recombination demonstrates a number of properties which identify it as a repair of mismatches in recombination heteroduplexes: (1) Being marker-dependent, this extra recombination is also highly sensitive to a DNA base sequence change at the same site on the chromosome of the other parent. This suggests the DNA structure composed of strands originating from different parents (heteroduplex) as a substrate for the recombination. (2) The marker-dependent recombination mechanism generates exclusively double exchanges separated by about 20 base pairs (bp). Mismatch repair most easily ex- plains such ultra closely linked double exchanges, these in turn accounting partly for high negative interference in T4 crosses over short distances (CHASE and DOERMANN 1958). (3) The frequencies of reciprocal recombinants in mass lysates from crosses of the rZZB mutants susceptible to the marker-dependent recombination were found to be unequal (SHCHER- BAKOV, SIZOVA and PLUGINA 1979). This asymmetry of recombination may reflect the unequal rates of

( k w t i c b 128: 673-685 (August. 1991)

repair of the corresponding mismatches to the rZZ+ or rZ1- strands. (4) The quantitative comparison of four recombination phenomena: allele-specificity of recombination frequencies, genetic map contraction, high negative interference (SHCHERBAKOV et al. 1982b) and asymmetry of recombination (SHCHERBAKOV, SIZOVA and PLUGINA 1979) suggested that these were different manifestations of one and the same physical process, mismatch repair.

In the present paper, we attempted to elucidate the structural features of genetic markers and related mismatches which make them susceptible to the repair. Determination of the base sequences of rZZ genes and of sequence changes in many rZZ mutants (PRIB- NOW et al. 1981; SUGINO and DRAKE 1984; SHINE- DLING et al. 1987) has made the present work possible and eventually successful.

MATERIALS AND METHODS

Bacteriophages: The physical locations of T 4 rIIB mutations used in this study and the nucleotide sequences in corresponding mutated sites are presented in Figure 1. The origin of some of the mutant strains was described earlier (SHCHERBAKOV et al. 1982a). Strains FC55 and FC47 are from S. P. CHAMPE’S collection; mutants P53, FC302, ac19, 0 ~ x 5 0 4 , o p X 5 0 4 , F C 3 0 1 , X 6 5 5 and N 2 4 were generously supplied by B. S. SINGER. All the T 4 strains used were made isogenic to T4D as described (SHCHERBAKOV et al. 1982a).

Bacteria: Escherichia coli BB (BARNETT et al. 1967) was used as a host in all the phage crosses, for preparing phage stocks, for phage titration and for measuring the total phage

674 V. P. Shcherbakov and L. A. Plugina

a c l 9

F C 3 0 2 F C 3 0 1 A C

P 5 3 T A A B B B

F C l A C

B bl lb 5 ' A T G T A :,+,A T A T TAA A T , g o C C TGA C C A A,+,A A C G A A-

I G HE122 uv375 $ I 1 5 O C X 5 0 4

G A O p X 5 0 4

T G am360 a m ~ ~ 3 5 7 A G A FC40 ? ' ? ' C A A o F o C T G A A A T T G,ToT A A A C T G T A,'J;,T C A A G T G G T$,A T T- 4.J. 4. T G op360 T X 5 1 1 + + uv357 FC21 T A 360 o p U V 3 5 7 G

. ? + v " A

C F C 5 5 B T N24

A C A C C ~ S + o A C A G G A A T T S ~ o G C T G A T T G ~ 5 ~ 0 A A G G T G T A T - ?'

T 375 X655 A

C G F C 4 7

,TOG G T T G A C A C 5 e o A T C C G T C G T 5 S o T T T T G A 3" T o

yields in the crosses; E. coli strains 594(X) (KRYLOV 1973) and CA244(X) (BRENNER and BECKWITH 1965), which are nonpermissive for all dl mutants, were used to titrate wild- type ( r l P ) recombinants. Opal suppressor E. coli strain K223(X)su9 (SAMBROOK, FAN and BRENNER 1967), ocher suppressor strain CA167(X)suc and amber suppressor E. cola CA265(h)su3 (BRENNER and BECKWITH 1965) were used for titration of ocher- and amber-type recombinants, respectively. All E. coli strains used are permissive for T4 dl+.

Media, management of bacterial cultures and the pro- cedure for standard crosses: These were essentially as described earlier (SHCHERBAKOV et al. 1982a). The frequencies of recombinants were determined as the lysate titer on the relevant X-lysogen divided by that on E. coli BB. About 500 plaques were counted for each titer determination. The apparent recombinant frequencies were corrected for rela- tive plating efficiency of a given recombinant on the X- lysogenic indicator strain and on E. coli BB, as described earlier (SHCHERBAKOV et al. 1982a).

FIGURE 1.-Mutations and base sequences in the promotor proximal rIIB gene segment of T4 phage in accordance with SHINEDLINC et al. 1987. The names of the mutations are given in italic type. t or means base substitution; V and A designate insertion or deletion, respectively, of one or more base pairs, new (mutant) nucleotides being bold-printed: b , and ,b are termination codons out of phase (underlined). The nucleotides are numbered according to PRIBNOW et al. (198 1).

RESULTS

Physical scale of genetic recombination: Embark- ing on the study of the relationship between the recombination frequencies and the physical distances, one may foresee a priori that it might not be linear. There are at least three factors that will influence this ideal relationship. First, the contribution of mismatch repair to recombination generally is not distance-dependent (SHCHERBAKOV et al. 1982a,b). Second, within distances of the order of several base pairs, the recombination is strongly inhibited (TESSMAN 1965; KATZ 1968; KATZ and BRENNER 1969; RONEN and SALTS 197 1). This inhibition results from interference of the markers themselves with the recombination process (FINCHAM and HOLLIDAY 1970). Third, recombination contribution of the hybrid regions with parental flanks (patches) becomes distance-independent after marker separation longer than the length of

FIGURE 2,"Basic frequency of recombinants as a function of the physical distance between base-substitution markers within the proximal rlIB gene segment. R,, designates the frequency of wild- type recombinants in a two-factor cross ij X Ij, S, being the length of the i-j interval in base pairs. Closed circles correspond to the crosses at ultrashort distances. The straight line was drawn through the open circles by the least-square method. Data are from Table 1.

the patch (STAHL 1979; TOOMPUU and SHCHERBAKOV 1980).

The above distorting effects are expected to be greatly reduced in crosses between LR-markers separated by distances which, on one hand, are long enough to avoid marker interference, but on the other hand, are significantly shorter than the length of the hybrid DNA (hDNA).

Crosses which meet the above "ideal" conditions are presented in Figure 2 (crosses bs X bs in Table 1). We chose for these crosses only mutants with a single base substitution (transition or transversion) since all such

Mismatch Repair in Phage T4 675

TABLE 1

Frequencies of wild-type recombinants (R,,) in two-factor crosses iJ X Zj between rZZB mutants of T4 phage

Cross (i X j ) s, (bP)

Crosses bs X bs (Figure 2) tiV375 X 360 3 360 X X51 I 6 tiV375 X X51 1 9 X51 I X opW357 19 X511 X tiV357 19 HE122 X W 3 7 5 25 0 ~ x 5 0 4 X W 3 7 5 25 360 X opW357 25 360 X W 3 5 7 25 HE122 X 360 28 tiV375 X opW357 28 w 3 7 5 x W 3 5 7 28 HE122 X X51 1 34 0 ~ x 5 0 4 X X51 I 34 W 3 5 7 X X655 37 W 3 5 7 X N24 38 HE122 X opW357 53 HE122 X W 3 5 7 53 ocX504 X W 3 5 7 53 HE122 X 375 70 0 ~ x 5 0 4 X 375 70 ocX504 X X655 90 0 ~ x 5 0 4 X N24 91

Crosses bs X)+ (0 in Figure 3) HE122 X FC3Ol 3 opUV357 X FC40 4 optiV357 X FC55 18 W 3 5 7 X FC55 18 UV375 X FC301 24 tiV375 X FC302 28 360 X FC302 31 X51 I X FC55 37 360 X FC55 43 UV375 X FC55 46 tiV357 X FC301 52 opUV357 X FC302 56 tiV357 X FC302 56 HE122 X FC40 57 HE122 X FC55 71

Crosses bs X FC47 (A in Figure 3) N24 X FC47 31 X655 X FC47 32 375 X FC47 52 tiV357 x FC47 69

UV375 x FC47 97 X51 I X FC47 88

HE122 X FC47 122

w 3 7 5 X P53 29 CI osses bs X fs- (0 in Figure 3 )

(RIJ f SD) X 10'

'0.0030

'0.24 f 0.01 121 .02 f 0.02 '0.91 f 0.07

51.17 f 0.09 161.34 f 0.04 61.22 f 0.07 '1.33 f 0.07 "1.37 & 0.04 '1.21 f 0.15 51.40 f 0.02 51 .47 f 0.05 51.68 f 0.20 51.59 f 0.05

"2.26 f 0.06 '2.24 f 0.05 '2.38 f 0.07 '2.89 f 0.06 52.96 f 0.10 '3.58 f 0.40 '3.84 f 0.35

40.22 f 0.02

51.21 -+ 0.05

60.036 60.20 f 0.02

'0.87 f 0.03 52.13 f 0.12 51.15 f 0.04 '1 .48 f 0.06 '1.71 f 0.04 62.30 f 0.04 62. 17 f 0.09 53.26 f 0.22

"2.65 f 0.03 52.35 f 0.20 '2.26 f 0.1 1 63. 11 f 0.25

'1.51 ? 0.05 '1 .44 f 0.04 62.12 f 0.11

'3.78 f 0.21 '3.74 f 0.20 54.57 f 0.22

5 1 . 1 7 f 0.07

51.01 f 1.20

73.02 f 0.06

~

Cross (i X j ) S,, (bp) (RJJ k SD) X 1 0 "

ti11375 X FC21 30 '1.43 -+ 0.13 HE122 X FC21 55 52.22 f 0.24 tiv357 x P53 57 52.44 f 0.25

Crosses bs X 2bs; f i X 26s (0 in Figure 3 ) 360 X amUV37-5 UV375 X op360 UV375 X am360 X51 1 X am360 X51 I X op36O X51 1 X amW375 FC55 X amW357 X51 I X amUV357 opUV357 X am360 W 3 5 7 X am360 opUV357 X op360 UV357 X op360 HE122 X amW375 UV375 X opX504 FC21 X am360 opW357 X amW375 FC21 X op360 FC40 X am360 HE122 X op360 HE122 X am360 FC21 X amtiV375 FC40 X 0 ~ 3 6 0

FC302 X 0 ~ 3 6 0 FC302 X am360 X51 I X opX504 FC55 X am360 FC55 X op36O HE122 X amW357 tiV357 X opX504 FC302 X amUV357 375 X obX504

FC40 X amUV375

1 3 3 4 5 7

17 19 23 23 24 24 25 25 25 26 26 27 28 28 28 28 30 31 31 34 41 42 53 53 56 70

50.00 16 '0.0041 '0.00 13

'0.28 f 0.03 40.60 f 0.08 '0.27 f 0.04 '3.76 f 0.10 '3.07 f 0.10 61.87 f 0.18 51.69 f 0.12 '2.06 f 0.07 '2.07 f 0.17 71.37 f 0.06 51.44 f 0.12 '1.67 f 0.16 '1.69 f 0.24 '1.89 f 0.18 '1.93 f 0.23 71.95 f 0.13 61.71 f 0.05 51.31 f 0.15

'1.40 f 0.08 '2.26 f 0.18 '1.92 f 0.12 51.85 f 0.12 62.80 f 0.23 53.15 f 0.15 65.35 f 0.42 42.93 f 0.44 '5.40 f 0.61 53.54 f 0.17

'2.00 2 0.18

Crosses bs'x 2f.s';) X 2)+ (W in Figure 3 ) W 3 7 5 X FC1 3 '0.0056 360 X FCI 6 '0.63 f 0.05 X511 X FCI 12 31.31 f 0.15 HE122 X F C I 25 53.22 f 0.08 W 3 7 5 X ac19 27 '4.00 f 0.44 FC302 X FCI 28 '3.20 f 0.25 opUV357 X F C I 31 62.65 f 0.25 FC21 X F C 1 33 '3.47 f 0.34 X511 X ac19 36 74. 13 f 0.45 375 X FCI 48 43.95 f 0.41 tiV357 X acl9 55 '5.44 f 0.60 375 X ac19 72 '6.25 f 0.65 FC55 X ac19 73 76.15 f 0.41

I n all crosses presented at least one of the parents carried a nonrepairable mutation (LR marker). Average frequencies are shown; the bold-typed superscripts are the numbers of crosses upon which the average is based; SD designates standard deviation of the average; bs nlems base substitution mutation; 2bs, a mutation with two bases substituted;fs, either deletion or insertion of 1 bp,fs+ and 2&+ designating one- and two-nucleotide insertions andfs- deletion of 1 bp.

-

mutants were shown earlier to be LR-markers distances were much less than the hDNA length: The (SHCHERBAKOV et al. 1982b). The maximum physical mean value for the hDNA length in T4, as measured distance between the markers was 90 bp, i e . , all the by different methods, was found to be around 400 bp


6 t ‘L

/

FIGURE 3.-Marker effects in two-factor crosses. The following crosses are presented: bs X f s - (0), bs X 2bs orfs X 26s (O), bs xfs’ (U), bs X 2fs’ orfs X 2fs+ (W), and bs X FC47 (A); bs designates base substitution mutation; 26s means a mutation with two bases substituted; fs means a frameshift mutation,fs’ and 2fsf being designd- tions for one- and two-nucleotide insertions;fs- means deletion of one base pair. The straight line was taken from Figure 2 and corresponds to the basic recombination. Other designations are as in Figure 2. Data are from Table 1.

(BERGER 1965; BROKER 1973; TOOMPUU and SHCHER- BAKOV 1980). Recombination in such crosses should reflect the behavior of some basic mechanism dependent essentially on strand exchange.

As is shown in Figure 2, this homogeneous group of two-factor crosses iJ X Zj demonstrates an excellent linear relationship between the wild-type recombinant frequencies RV and the physical distances S,, except three points from crosses of markers separated by very short distances. Because of marker interference (see below), these deviate significantly from the regression line, drawn through the experimental points by means of the least-square method in accord with the equation

Rq = (23 + 3.8S,)1OT5. (1)

The observed linearity may mean that within the rZZB gene segment under study (the 122 bp of the HE122-N24 interval), recombination events are dis- tributed uniformly. It means also that most of the patches of the main subpopulation are longer than 90 bp or even longer than 120 bp, judging from the data in Figure 3. (The recombination contribution of the patches which are shorter than the marker separation is distance-independent.)

An interesting feature is that the empirical straight line does not run through the origin of coordinates; the cut off segment of the ordinate, which is equal to (23 & 2)10T5, while being small by absolute value, differs significantly from zero. Three origins of this minor distance-independent recombination compo- nent may be considered: residual repairability of the mismatches formed by the LR-markers; operation of these mismatches as weak terminators of hDNA prop-

agation; and existence of a subpopulation of very short (below 20 bp) patches. We discuss this phenomenon in detail elsewhere (V. D. SHCHERBAKOV, L. A. PLU- GINA and E. A. KUDRYASHOVA, in preparation).

Marker-dependence: In Figure 3, using the same coordinates as in Figure 2 (recombinant frequency versus physical distance), we plotted the data obtained in crosses of mutants with various changes in the mutated sites (Table 1). The straight line in Figure 3 is the same as that in Figure 2 (basic recombination).

An inhibition of recombination down to the level below the basic one (straight line) at ultrashort distances is also seen in this figure. We saw such reduction only at the distances below ten bp. All the other crosses gave frequencies expected for LR markers (basic) or higher (marker-dependent recombination). The comparison of the marker structures and the corresponding recombination data enables one to ob- serve the following correlations. (1) All mutations with two base substitutions, forming mismatches with two noncorrect pairs, recombine with a heightened frequency; we conclude that they are susceptible to mismatch repair. (The nucleotide structures of some of the relevant mismatches are shown in Table 2.) (2) Mutations resulting from two base pair insertions (ac19 and F C I ) are frequently corrected to the corresponding wild-type alleles. (3) Some of the markers with single base pair insertions (e.g. FC301) are corrected to the wild-type allele at an appreciable rate, while other mutations of similar structure (FC302 , FC55) behave as LR-markers. Deletions of one base pair (P53, FC21) are not corrected to the wild-type alleles. (4) The FC47 mutation, resulting from substitution of CTG for T T , behaves as an LR-marker.

A simple way to measure mismatch repairability: In the previous paper (SHCHERBAKOV et al. 1982b), we have described several purely genetic methods to measure the contribution of mismatch repair to recombination frequency, termed “repairability.” Tak- ing advantage of the empiric plot for distance-related recombination (Figure 2), and provided the physical position of a given mutation is known, one can easily find the value of repairability by the equation

K,-J = R , - ~ i - 1 - (23 k 3.8s~) lo-‘, (2)

where K,,, and K~+J are repairabilities of i and j mutations to the corresponding alleles Z and J . If i is a mutation of the LR-type, i . e . , Ki-1 is Close to zero, Kj-J

is the difference between the measured recombinant frequency and its expected value. The distance i-j should be an “indicator” one. At indicator distances, which are short when compared to the mean length of the hDNA, but exceed the length of repair tracts, mismatch repair mechanism makes its maximum contribution to the recombination frequency. The notion


IO za 30 40 sa 5ij l b f )

FIGURE 4,"Frequencies of recombinants obtained in crosses with the mutant F C I . Line I corresponds to basic recombination (Figure 2). Curve I I was drawn through the points (X) obtained in two-factor crosses of FCI against rIIB mutants of low repairability (1.R-type). The upper, linear part of the curve I I was drawn by the least-square method in parallel with line I . See text for the meaning of other points.

FIGURE 5.-Frequencies of recombinants obtained in crosses with the mutant aclY. Line I corresponds to basic recombination (Figure 2); line I I was drawn through the points (X) obtained in two-factor crosses of ac19 against YIIB mutants of LR-type by the least-square method in parallel with line I . See text for the meaning of other points.

of indicator distances was substantiated by SHCHER- BAKOV et al. (1 982b).

Examples of repairability determinations are given on the plots of Figures 4 and 5. Curve ZZ in Figure 4 (X) shows the frequencies of wild-type recombinants R, as a function of physical distance in two-factor crosses iJ X Zj, in which j = F C l (insertion of two base pairs), while i markers are rZZB mutations of LR type. The straight line Z reflects basic recombination. In the crosses presented, the apparent recombinant frequencies quickly increase as the distance increases up to 20 bp (the inferred average repair tracts) (SHCHERBAKOV et al. 1982a) and then they run approximately in parallel with the basic frequencies. The ordinate difference between the parallel part of the curve ZZ and the basic line is 1.9 - and this is the value of contribution of mismatch repair F C l + wt to the recombinant frequency. Note that the initial part of curve ZZ demonstrates marker interference: zero frequency values at nonzero distances.

Similarly, in Figure 5, the repairability ac19 + wt was found to be 2.9- Some other repairability

values along with the corresponding mismatch structures are shown in Table 2.

Asymmetry of mismatch repair: Generally, a mismatched region is structurally asymmetric. It is reasonable to expect that repair of mismatches in two alternative directions may go with different rates. The promotor proximal segment of rZZB gene is not essen- tial for T 4 growth on X-lysogenic E. coli (CHAMPE and BENZER 1962; BARNETT et al . 1967). Within this gene segment, mutations producing frame shifts in opposite directions suppress each other with high efficiency, so that the double mutants have fully wild-type pheno- types, and both reciprocal recombinants in crosses between such mutants produce plaques on X-lysogens. In some special cases (see Figure 6 ) the reciprocal recombinants can be determined separately as follows:

From the cross FC302 X F C l (Figure 6A) two recombinants arise: wild type and the double mutant FC302-FCI. Both recombinants produce plaques on E. coli CA167(X) bearing an ocher suppressor, while the double mutant cannot grow on 5 9 4 ( X ) because of the terminating ocher triplet TAA (barrier b , ) gen- erated by the FC302 phase shift. The following frequencies of recombinants (the mean value with respect to five determinations and the standard deviation of the mean) were obtained:

On CA167(X): R, + R, = (4.9 k 0.3)10-'; On 594(X): R, = (3.2 k 0.3)10-';

hence

Rg = (1.7 f 0.4)10-3.

It is evident that the FC302-FCI double is formed in this cross at about one half of the frequency of the reciprocal wild-type recombinant. The difference R , - Rij = (1.5 f 0.5)10-3 is significant. The observed value R,j (shown as the first open square in Figure 4) does not differ significantly from the corresponding basic value predicted by the line I. We interpret this result to mean that there are no significant mismatch corrections wt + FC302 and wt + F C l , while the opposite ones make a significant contribution to the recombination frequencies.

The observed asymmetry of repair of the mismatch F C l l w t , suggesting preferential removal of the longer DNA strand, was confirmed in several other cases. For example, in the cross F C l X FC55, both reciprocal recombinants grow on X-lysogenic bacteria. The sum of frequencies R, + R, was found to be equal to 5.7. lo-'. Subtraction of the known value for K ~ ( : l - l , equal to 1.9. (Figure 4) gives the frequency 3.8. that, when divided by two, is close to the corresponding basic frequency (second open square in Figure 4).

In the three-factor cross F C l X a m W 3 7 5 - F C 5 5 (Figure 6B), the allele F C l is situated just opposite the sequence a m W 3 7 5 on the chromosome of the mating


TABLE 2

Structures and repairability values for some of the mismatches formed in the crosses of rZZ mutants

Mismatch structure’ Repairabilityb

(mean & SD) (X 103) Mismatch structure’

Repairabilityb

(mean f SD) (XlOS)

SD12Y‘ - CGCTAAAAA - -GCGA-TTTT-

Wl

opX504 - ATGTGAAATA -TACATGTTAT-

wt

ac19 -GTACCACAATA- -CATGC-TTAT-

HE122

FC30 J - ACAAATATT - -TGTT-ATAA-

wt

FC I -AAACACGAACAAG- -TTTG-ATCGTTC-

amlJV375

am360 -CGAATAGGCTG- -GCTTGTTCGAC-

wt

0~360 -CGAATGAGCT- -GCTTGTTCGA-

wt

FC40 - ATTCAAAGT- -TAAG-TTCA-

wt

FC21 -TTCA-GTGG- - AAG’JTCACC -

wt

FC55 -TACACCCCA- -ATGT-GGGT-

Wt

1 .o

m = 4

0.5 f 0.1 n = 4 m = 19

4.3 ? 0.7 m = 6

1 .o n = 2 m = 10

1.4 m = 4

0.6 f 0.1 n = 7 m = 38

0.9 f 0.1 n = 7 m = 28

Low (m = 8)

Low n = 3 m = 9

Low n = 6 m = 35

FC302 ? - ATGTTACAA - Low Low

-TACA-TGTT- n = 4 w t m = 25 m = 5

Low m = 6

?

Low m = 4

?

Lowd (n = 2) (m = 8)

Low

m = 4

Low

m = 3

ac19 -GTACACAATA- 3.0 f 0.1 LOW -CATG-TTAT- n = 5

wt m = 38 m = 6

FC 1 - AAACACGAAC - -TTTG-CTTG-

wt

a m W 3 7 5 - AAACTAGCAAG - -TTTGCTTGTTC-

wt

am360‘ -GAATAGGCTG- -CTTACTCGAC-

op360

amW357 -TATTAGAGTG- -ATAAGTTCAC-

wt

FC40 -TATTCAAAGT- -ATAAC-TTCA-

o p W 3 5 7

1.9 f 0.2 n = 5 m = 24

Low n = 4 m = 20

0.5

2.8 k 0.2 n = 4 m = 15

4.1‘

Low, n = 2 m = 8

Low

?

3

FC21 -TATTCA-GTGG- Low 1 .O -ATAACTTCACC-

o p W 3 5 7 m = 8 m = 8

FC47 -TCGTCTGTTTG- Low Low -AGCACA-AAAC- n = 7

wt m. = 41 m = 4

Mismatched and nonmatched nucleotides are bold-typed; wt means wild-type sequence. ” Term “repairability” (SHCHERBAKOV et al. 1982b) designates the absolute contribution of mismatch repair to the recombination frequency;

j -+ J or J -+ j show direction of correction of the j / J mismatch. Repairability value for a given mismatch j / J may be estimated in a single cross i X j , provided the distance i-j is an indicator one and the i marker is of the LR-type (single-point determination). To have more reliable determination, i t is better to use several different i markers (multipoint determination). Here, we used “n” to designate the number of different crosses or number of i markers in multipoint determinations, while “m” shows total number of crosses made. The SD values are shown only for multipoint estimations made in the present paper. We designated as low the repairability values that do not differ significantly from zero, while ? means that the corresponding value was not measured.

The SDJ2Y mutation is located at the distal part of rIlA gene. Its repairability was measured in crosses with the closely linked rIIA mutations N21 and C6 (our unpublished data).

“ T h e value was measured in crosses against rIZB frameshift mutants FC9 and pJ0, which are intragenic suppressors of the FC40 (our unpublished data).

“ The data are from SHCHERBAKOV et al. (1982b).


F C 3 0 2 bl wt A T ”

X

I wt F b l

F C l wt I I

X

a c l 9 Ib wt -TGA-

X

7 - wt F b 5 5

a c l 9 lb wt -TGA-

X

I

HE1 22 F b 5 5

opuv357 FC4 7 I I

X

I 1 F C 2 1 wt

FIGURE 6,”Structure of the crosses used to demonstrate asymmetry in reciprocal recombinant formation. I b and bl are out-of- phase translation terminators.

partner, which differs from that of wild type in two nucleotides (see Table 2). As a result, a rather complex mismatch is formed, which includes two nonmatched nucleotides along with two mismatched nucleotide pairs. The correction F C l + a m W 3 7 5 makes a contribution to the frequency of recombinants of amber phenotype, while the opposite correction a m W 3 7 5 + FCI contributes to the frequency of double mutants FCl-FC55 of wild-type phenotype. On E. coli C A 2 6 5 , which is permissive for both recombinants, and on E. coli C A 2 4 4 , which is permissive for only FCl-FC55, the recombinant frequencies were (4.7 k 0.5)l 0-3 and (1.4 f 0.2)10-’, respectively (four determinations). The latter frequency is even somewhat lower than the corresponding basic frequency, as is shown by the open circle in Figure 4. Hence, there is no correction of a m W 3 7 5 to F C l , while the opposite correction F C l + a m W 3 7 5 has the rate (see black circle in Figure 4) similar to that of the F C l + wt correction. It is evident that in this case too, the longer strand is preferentially removed during mismatch repair.

The same regularity was found for another mutant with the 2-bp insertion ac19 (Figure 6, C and D). In the cross ac19 X FC55, true wild type and double mutant ac19-FC55 are produced as recombinants. The double cannot grow on E. coli 594(X) because of the TGA barrier I b (see Figure I), but it grows on the opal suppressor strain K 2 2 3 . The frequency of wild- type recombinants (R,) producing plaques on 5 9 4 ( X )

was (6.2 f 0.4)10-3 (black circle in Figure 5), while the sum of R, + R, (growth on K 2 2 3 ) was (9.5 f 1.3)10-3. Hence, R, = 3.3. This value does not differ from the corresponding basic frequency (open circle in Figure 5), so there is no detectable correction in the direction wt 4 ac19 or wt + FC55, while the opposite correction ac19 + wt is very high, the corresponding repairability being 2.9- lop3 (line ZZ in Figure 5). Again, the asymmetry results from preferential removal of the longer strand in the mismatched region.

In the cross ac19 X HE122-FC55, the heteroduplex with two nonmatched nucleotides and one mismatched pair is formed (Table 2). From two reciprocal recombinants, one, namely H E 1 2 2 , has the amber phenotype and grows on E. coli C A 2 6 5 , while the double ac19-FC55 has the opal phenotype and grows on E. coli K 2 2 3 . The frequency of H E 1 2 2 recombinants, to which ac19 + H E 1 2 2 correction contributes, was (7.2 k 0.7)10-3, while the reciprocal recombinants ac19-FC55 arose with a frequency of (3.6 f 0.2)10-’ (six determinations). These values are plotted in Fig- ure 5 (black square and open square, respectively) to show that they are similar to the corresponding frequencies found in the cross ac19 X FC55, represented by black and open circles, respectively. We conclude that the addition of the base substitution H E 1 2 2 to the mismatch a c l 9 / w t did not significantly change the correction pattern, though it somewhat enhanced the frequency.

One more example of the correction asymmetry is demonstrated by the cross FC21 X o p W 3 5 7 - F C 4 7 (Figure 6E), in which homoalleles FC21 (deletion of one nucleotide) and o p W 3 5 7 (base substitution) form a complex mismatch, with the structure shown in Table 2. The frequency of recombinants FC21-FC47 of wild-type phenotype determined on E. coli 5 9 4 ( X ) was (3.8 f 0.1)10-3 and the sum of the frequencies of the reciprocal recombinants ( o p W 3 5 7 and FC21- F C 4 7 ) , both of which grow on K 2 2 3 , was estimated as (6.6 f 0.3)10-3 (eight determinations). The observed frequency of o P W 3 5 7 recombinants (2.8. does not exceed the expected basic value, while reciprocal recombinants arise much more frequently. Since the correction wt + FC47 is infrequent (see below), the difference between the two reciprocal recombinant frequencies must be attributed to the contribution of correction o P W 3 5 7 + FC21, the correction FC21 + o p W 3 5 7 being negligible. Again, this correction asymmetry suggests preferential removal of the longer DNA strand. The repair proficiency of some of the markers with single base pair insertions is also in accord with this evidently general rule.

The double FC21-FC47 was shown to have fully wild-type phenotype (our unpublished data), so in the cross FC21 X FC47 both reciprocal recombinants


grow on X-lysogens. The combined frequency of the recombinants was found to be (5.6 +. 0.3)10-3 (four determinations). Since this value is just double the expected basic frequency corresponding to this marker separation (67 bp), we conclude that both mismatches, FC2l lw t and FC47/wt , are poorly repaired in either direction.

Factors of mismatch repairability: Based on the analysis of the data presented in this paper, one can make several quite definite conclusions on the nature of the marker discrimination by the mismatch repair systems operating during T 4 crosses.

First, heteroduplexes with one mismatched base pair, either transitions or transversions, are not repaired with notable frequency. We have investigated 14 such markers and all of them were of LR-type [SHCHERBAKOV et al. (1982b) and the present data], while all heteroduplexes susceptible to mismatch repair either have more extended base substitutions or are frameshifts.'

Second, among repairable mismatches those with two or more contiguous mismatched or nonmatched nucleotides are the most effectively repaired, while insertion of one correct pair between two mismatched ones reduces the repairability.

Third, heteroduplexes containing frameshift mutations are repaired asymmetrically, the longer strand being preferentially removed.

Fourth, among other factors, the sequence environ- ment is important for repair of the mismatch. Inspec- tion of the sequences flanking mismatches shows that runs of A:T pairs directly neighboring the mismatches greatly promote repair. Mutations ac19 and FCI both arose as AC insertions but repairability of ac1Y to wild-type allele exceeds twofold that of F C l . Note, however, that the mismatch FCl lw t is surrounded by G:C pairs (Table 2) while a rather long A:T run adjoins the aclY/wt mismatch. Single base insertion mismatches SD12Y/wt and FC?Ul/wt are flanked by A:T runs and they both are repairable, while other single base insertion or deletion markers (FC?U2, FC21, FC40, FC55) have no long A:T sequences adjacent to the corresponding mismatches and they are poorly repairable.

The structures of mismatches formed by mutations located near the W 3 5 7 site are especially demonstra- tive. (1) Mismatch a m W 3 5 7 / w t (two contiguous base substitutions) adjoins an A:T block and it is highly repairable. (2) Mismatch FC2l lw t is not repaired in either direction, but addition of a base substitution in this mismatch (see F C 2 l / o p W 3 5 7 ) leads to a structure with rather high repairability in the direction opW?57 - FC21. Mutant FC4U has not been sequenced di-

;,scribed to the well repairable mismatch amW357/wt the structure G/A ' 111 the previous paper (SHCHERBAKOV el al. 1982b), we erroneously

I,er;wse of mislocation of the W 3 5 7 site on the map by PRIBNOW et al. (1981). Its correct structure is shown in Table 2.

rectly but its structure can be inferred. It is a frameshift mutant of sign plus (BARNETT et al. 1967), non- recombining with FC21, but recombining with W ? 5 7 . The mismatch FC4U/wt is not repaired in either direction (SHCHERBAKOV et al. 1982b and unpublished data), so FC40 should be a single-base insertion. Such insertions arise usually as nucleotide duplications (of 12 such mutants sequenced by SHINEDLINC et al. (1987) none contradicted this rule), so it is very probable that FC40 is an insertion of A at the AA sequence just to the right of W 3 5 7 . The mismatch FC4U/wt is transformed into a highly repairable one after a single base substitution in the strand of DNA opposite to FC4U (see mismatch FC40/opW?57 in Table 2). If we have correctly determined the position of FC40, then the change of the wild-type allele for opW?57 leads simultaneously to two important changes in the mismatch, endowing it with the structure most favorable for repairability: two contiguous mismatched nucleotides become adjacent to a long A:T sequence. Ac- cordingly, the repairability FC40 + o p W 3 5 7 was found to be the highest of all we have studied.

An apparent exception to the above rule is the mismatch opX504/wt , which is repaired only moder- ately to wild type, despite its most favorable structure. This mismatch, however, may be efficiently repaired to the opX5U4 sequence, though we did not check this possibility. Mismatches with strands of equal length may be repaired asymmetrically. For example, mismatch am?60/op36U is repaired exclusively to op?6U (SHCHERBAKOV et al. 198213). The principles of the strand preference in these cases are not understood yet.

The mismatch FC47/wt is poorly repaired in both directions despite the fact that it includes one nonmatched nucleotide and one mismatched pair. The presence of one complementary base pair between two incorrect ones may be a factor hampering recognition of the mismatch by the repair system. Such discontinuity of mismatches seems always to reduce repairability. Thus, the mismatches amW?75/wt and am36U/wt are repaired rather infrequently, and the mismatch FC21/opW?57 is repaired much less efficiently than the similar but continuous mismatch FC4U/opW?57. Besides, we have the impression that an A:T run adjoining a mismatch should have some minimum length (say more than three) to be able to promote repair. The possible effects of some other structural peculiarities cannot be excluded as well.

DISCUSSION

Linearity of basic frequencies over short intervals: With respect to the models of recombination via hDNA, the basic recombination reflects the rate of the hDNA formation and the pattern of distribution of its end points. As Figure 2 and equation 1 show,

Mismatch Repair in Phage T4 68 1

the basic recombination within the proximal rZZB segment linearly depends on the physical distance with the ratio 3.8. per bp. We interpret the linearity demonstrated by Figure 2 and equation 1 as evidence for the uniform distribution of the points of hDNA initiation and termination within the rZZB gene segment under study.

The observed frequency of recombinants per bp, 3.8. is in accord with the prediction of “the best” mapping function of STAHL, EDGAR and STEINBERG (1964) for similar range of distances, some minor difference being reasonably accounted for by the mismatch contribution in the data used by STAHL, EDGAR and STEINBERG.

In the crosses with mutants within the lysozyme gene (RAVIN and ARTEMIEV 1974) and within the gene for glutamine tRNA (COMER 1977) of T 4 phage, linearity between the recombination frequencies and physical distances was also observed, but recombination frequency per bp exceeded twice that found in ou r crosses. One may speculate that in crosses in an rZZ- background (used also for “the best” mapping function construction) recombination is somewhat inhibited. Involvement of rII proteins in recombination seems probable (MOSIG, SHAW and GARSIA 1984). The general congruence between genetic and physical distances in T 4 phage has well known exceptions: there are chromosome regions with a heightened recombination ability (MOSIG 1968; BECKENDORF and WILSON 1972; Wu, YEH and EBISUZAKI 1984).

Linear distance/frequency relations were observed also in crosses of phage X (BLATTNER et al. 1974; GUSSIN, ROSEN and WULFF 1980; LIEB 1981, 1983). RecBCD- and RecF-mediated recombination between homologous cloned segments in phage X and plasmid pBR322 in E. coli cells also gave linear distance/ frequency relationships (SHEN and HUANG 1986). The absolute values of the observed frequencies per bp were different depending on the cross system and genetic background.

Mismatch repair pathway: Here we have shown that the main factor determining repairability of recombinational mismatches is the length of a contiguous heterologous region. DRAKE (1966) found an inverse correlation between the recovery frequency of a marker from heteroduplex heterozygotes and its apparent dimension. Some of the mutations have been characterized both by DRAKE and by us. General congruence between the data is apparent: the mutations UV375, X511, FC9, FCO, FC40, 375, N 2 4 , which we found to be nonrepairable, have been shown by DRAKE to exhibit high recovery frequency, whereas the readily repairable FCI was recovered less frequently. There were, however, two exceptions: the nonrepairable mutations FC21 and FC47 were mod- erately recovered from heterozygotes. The reason for

this discrepancy is unknown. Deletions larger than 10-20 bp were not found in heteroduplexes. There is evidence for efficient repair of heteroduplexes formed by large deletions in T 4 phage (BENZ and BERGER 1973; DOERMANN and PARMA 1967; Mosrc and POWELL 1985), most probably via removal of the single-stranded loop.

The observed absence of repair of single-base substitutions and the features of the most readily repairable mismatches suggest a rather simple principle of marker discrimination by the mismatch repair mechanism. It is evident that local single-strandedness of DNA plays the main role in the mismatch recognition. If so, a single-strand specific endonuclease should be a key enzyme in this process. Endonucleases that recognize minor distortions in DNA double helix structure are known and were thought to be involved in mismatch repair in fungi (AHMAD, HOLLOMAN and HOLLIDAY 1975; SHENK et al. 1975; WIEGAND, GOD- SON and RADDING 1975; DODGSON and WELLS 1977; PUKKILA 1978; HOLLIDAY et al. 1979). The most thoroughly studied enzymes S1-nuclease from Asper- gallus oryzae and DNase I from Ustilago maydis demonstrate activities that are very suitable for mismatch repair of the sort we found in T 4 phage. They recognize single-base mismatches poorly but recognize distortions in the DNA helix produced by supercoiling or by more extended mismatches. Moreover, the DNA cleavage reaction was shown to be promoted at AT-rich regions facilitating local DNA melting (BEARD, MORROW and BERG 1973). In vitro, an initial cut in one strand is followed by breaking of the other strand, but in vivo the second (evidently harmful) reaction may be prevented.

The properties very similar to those of single-strand specific DNases were observed also with T 4 endonuclease VI1 (endo VII) encoded by gene 49 (KLEFF and KEMPER 1988). Heteroduplex DNA with single- stranded loops of 51 or 8 nucleotides was cleaved by endo VII. This enzyme makes double-stranded breaks by introducing pairs of staggered nicks flanking the loop. The nicking sites fall exclusively at the 3’-side of the loop on both strands. The nicks are introduced in a stepwise fashion. These data are in accord with those of MOSIG and POWELL (1 985) who demonstrated packaging of heteroduplexes with loops under 49“ conditions in crosses between rZZ deletion mutants and wild-type phage. In our experiments with 49”rIIB mutants it was found that the probability for the mismatch ac l9 /w t to be repaired decreased at least 50-fold under 49- conditions (V. P. SHCHERBAKOV, L. A. PLUGINA, S. GREBENSHCHIKOVA and M. NESH- EVA, in preparation). This suggests that gp49 (endo VII) recognizes and cuts mismatches.

Allele tsL42 of gene 43 (ALLEN, ALBRECHT and DRAKE 1970), encoding an antimutator T 4 DNA po-


P a B

i e n d o n u c l e a s e VI1 .i (9P49)

a B

j 3 ’ + 5 ’ e x o n u c l e a s e J.

( g p 4 3 )

B a

: DNA p o l y m e r a s e ( g p 4 3 ) i p l u s DNA l igase ( g p 3 0 )

a B

FIGURE 7.-A pathway for mismatch repair in T4 phage. A hybrid region with two mismatches, Ala and b/B, is shown. Double and single lines indicate DNA of the parents Ab and aB, respectively. Arrows mark 3’ ends of DNA strands. Note that the correction b -P B gives an AB recombinant strand.

lymerase, was shown to change significantly the length of mismatch repair tracts in rZZB crosses, whereas repairability values were kept the same as under 43+ conditions (SHCHERBAKOV, KUDRYASHOVA and PLU- GINA 1980). Taking into account that T 4 antimutator DNA polymerases have a heightened activity of their 3’+5’ exonuclease (MUZYCZKA, POLAND and BESS- MAN 1972; Lo and BESSMAN 1976), it was concluded that the exonuclease activity of T 4 DNA polymerase is directly involved in the removal of a mismatched DNA strand during mismatch repair. Figure 7 illus- trates the proposed pathway for mismatch repair in T 4 phage: ( 1 ) A mismatch is recognized by endo VI1 making a nick at the 3‘-side of the mismatch with 3’OH and 5’P04 ends. (2) The 3’-5’ exonuclease activity of gp43 attacks the nonmatched 3’OH end and removes noncomplementary nucleotides along with several (about 20) complementary ones. This event determines the length of a repair tract. (3) The DNA polymerase switches from hydrolysis to DNA synthesis and fills the gap. (4) DNA ligase repairs the nick.

Our conclusions on the direct participation of endo VI1 and DNA polymerase in mismatch repair are

A B e-

I 1 >

A b

i 3 ‘ + 5 ‘ e x o n u c l e a s e .i

A e A <

1 1 + A b

i DNA polymerase j p l u s DNA l igase .i

A b c I 1

I 1 f

A b

FIGURE 8.-Hypothetical pathway leading to inhibition of recombination at ultrashort distances. The designations as in Figure 7. Note that the nearly completed recombinant (AB) strand is con- verted to the parental type Ab.

fairly self-consistent. Endo VI1 cuts loop-containing heteroduplexes exclusively at the 3’-side of the loop (KLEFF and KEMPER 1988), while 3’+5’ exonuclease attacks specifically single-stranded 3’OH ends of DNA (BRUTLAG and KORNBERG 1972). The observed in vitro second nicking on the opposite strand may be forbidden in vivo. The postulated behavior of T 4 DNA polymerase in this pathway is the same as in DNA synthesis when the proofreading exonuclease removes erroneously included nucleotides, and then polymerization is resumed.

The proposed mismatch repair pathway facilitates understanding molecular events underlining well known severe inhibition of recombination in crosses at ultrashort distances, one to three bp, in T 4 phage (TESSMAN 1965; KATZ 1968; KATZ and BRENNER 1969; RONEN and SALTS 197 1) as well as in E. coli (YANOFSKY et al. 1964; ZIPSER 1967) and in phage X (GUSSIN, ROSEN and WULFF 1980). This inhibition is equivalent to the map expansion phenomenon and reflecs interference of the markers themselves with the recombination process (FINCHAM and HOLLIDAY 1970).

Our data confirm those earlier observations. As can be seen in Table 1 and Figures 2, 3 and 4, the recombination is strongly inhibited at ultrashort distances. In fact, at distances of one to three bp the observed recombinant frequencies did not differ from zero.

Possible events leading to the marker interference are shown in Figure 8. T o form a recombinant, a DNA break must occur between the markers. In the case of ultrashort distances, the mismatched nucleotides occupy positions adjacent to the break. The ligation of such a break may be a very slow process, while single mismatched strands should be very sen-


sitive to nuclease attack. As a result, nearly completed recombination is canceled and the parental form reap- pears.

Note that the cancellation of exchanges at ultrashort distances is in fact mismatch repair in its late stage, so we propose for it the same pathway and postulate that T 4 DNA polymerase (gp43) removes a mismatched strand by its 3’+5’ exonuclease and then fills the resulting gap.

Relation to the host mismatch repair systems: It is evident that the inferred mismatch repair mechanism related to marker-dependent recombination in T 4 phage differs qualitatively from the known bacterial systems of heteroduplex repair (for review see CLAVERYS and LACKS 1986; RADMAN and WAGNER 1986; MODRICH 1987; SMITH 1988). In E. coli, multiple repair pathways (partially overlapping) have been described. The general methyl-directed or Mut-dependent repair system, very similar to the Hex system of Streptococcus pneumoniae, requires functions of genes mutL, mutS, mutH and mutU, as well as ssb. It is active in postreplication DNA correction and seems to be specifically aimed at the most frequent errors of replication, recognizing most efficiently transition mismatches with minimum distortions of the double helix structure. The Mut and Hex systems readily recognize also single-base deletions and insertions. Both systems produce long (about 3000 bp) repair tracts.

A sequence-specific, very short patch (VSP) repair system was observed in E. coli (LIEB 1983, 1985; JONES, WAGNER and RADMAN 1987), which specifically corrects G/T mismatches resulting from transitions CAGG - TAGG or CCAG - CTAG. A biolog- ical sense for such refined repair lies in the observation that the sequence d(CC$GG) is modified by dcm methylase at the internal cytosine and such sequences are hot spots for mutations due to deamination of the methylated cytosine (DUNCAN and MILLER 1980). The length of repair tracts for the VSP system is less than ten nucleotides and can be as short as two nucleotides (LIEB, ALLEN and READ 1986). This system depends on the function of the E. coli gene v s r and, to a lesser extent, on mutL and mutS (LIEB 1987; RAPOSA and FOX 1987; SOHAIL et a l . 1990).

An additional short patch repair system dependent on the mutY function of E. coli corrects specifically A/ G mismatches to C/G (Au et al. 1988; NGHIEM et aE. 1988; LU and CHANG 1988; RADICELLA, CLARK and FOX 1988). The MutY system does not require any functions of the methyl-directed repair system. Local- ized, sequence-specific mismatch repair very similar to VSP repair of E. coli was observed in S. pneumoniae (SICARD et al. 1985).

None of these bacterial repair systems is similar in specificity to that described in the present paper.

While the Mut, Hex, VSP or MutY repair systems may manifest themselves in general recombination, their direct function is clearly related to reduction of mutagenesis. In contrast, the repair system under study here does not recognize mismatches equivalent to the most frequent spontaneous mutations (single- base substitutions and single-base insertions or deletions), so its antimutagenic role is doubtful. One may wonder if E. coli mismatch repair systems make an antimutagenic or a recombinational contribution during T 4 multiplication. The low repairability of single- base substitutions in T 4 suggests that short patch repair systems of the host did not contribute to recombination in our crosses. The rZZ mutations we used did not have the context sequences 5’CCAGG3’ or 5’CCTGG3’, recognized by the VSP system and the effects of the systems with long repair tracts (either host- or phage-specified) could not be detected in our crosses because of joint repair. Apparently the sequence-independent MutY system was also inefficient despite the presence in our crosses of A/G mismatches, e.g. , ocX504/wt or W 3 5 7 / w t , the correction of which to C/G would produce the corresponding wild-type sequences.

It may be argued, however, that T 4 DNA does not undergo postreplication repair. The average mutation rate in T 4 phage, about per bp, can be fully accounted for by the observed fidelity of DNA replication without additional accuracy-enhancing steps (SINHA and GOODMAN 1983). On the other hand, T4 DNA undergoes complex processing before packaging, which includes resolution or repair of branched and loop-containing structures. We believe that T 4 marker-dependent recombination is related to such processing aimed to restore distorted areas of the double helix. It is meaningful that one enzyme, endo VII, is involved both in the above processing (MINO- GAWA et d . 1983; MIZUUCHI et al. 1982; JENSCH and KEMPER 1986; MOSIG and POWELL 1985) and in mismatch repair.

Mutagenesis via sequence conversion: In other work using T 4 rZZB mutants (DEBOER and RIPLEY 1984), evidence was obtained for in v ivo production of frameshift and base substitution mutations via met- abolic processing of misaligned quasipalindromic DNA sequences. Since the mismatched regions in the stem-loop or hairpin structures formed by quasipalindromic sequences are similar to those in recombination heteroduplexes, it is reasonable to think that they are processed by the same mechanism. The mu- tagenic processing is similarly asymmetric: in mismatches with strands of unequal length the longer one is preferentially removed. In addition, the size of the region involved in “repair” of quasipalindromes is also similar to that in recombination-related mismatch repair: DEBOER and RIPLEY (1 984) observed both joint


and separate processing of multiple mismatches set only several base pairs apart on the same stem-loop structure.

This mismatch repair system may also operate in mutagenesis via sequence conversion which is related to direct sequence repeats with partial homology (RIPLEY, CLARK and DEBOER 1986; SHINEDLING et al. 1987). Transient heteroduplexes are probably formed at the initial step of general recombination, when a recombinogenic DNA structure (e.g., a complex of single-stranded DNA and recA-like protein) searches for a homologous region (GONDA and RADDING 1983). Mismatch repair in these transient heteroduplexes will lead to mutation in the “corrected” strand.

We are very grateful to BRITTA SWEBILIUS SINGER for generously supplying us with the T 4 r l l strains and for providing the corresponding sequence data obtained in L. GOLD’S Laboratory prior to publication. We are indebted to TANYA KOLYSHEVA, LENA PAR- VITZKAYA and IRA SERGEYEVA for technical assistance.

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Communicating editor: G. R. SMITH

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