asymmetric effects of deletions and substitutions on … · all cases the lefthand marker was a bio...

Copyright 0 1982 by the Genetics Society of America

ASYMMETRIC EFFECTS OF DELETIONS AND SUBSTITUTIONS ON HIGH NEGATIVE INTERFERENCE IN COLIPHAGE LAMBDA

G. J. VANCE MAKIN' AND W. SZYBALSKI'

McArdIe Laboratory for Cancer Research, University of Wisconsin, Modison, Wisconsin 53706

F. R. BLATTNER

Deportment of Genetics, University of Wisconsin, Madison, Wisconsin 53706

Manuscript received June 24, 1980 Revised copy accepted August 5, 1982

ABSTRACT

Experiments have been performed to help clarify the role of nonhomologies in phage h recombination. Three-factor crosses were carried out, and the frequencies of single and double recombinants in the two adjoining intervals were compared when the central marker was either a double point mutation (vlv3) or deletion (rex-cl deletion) or nonhomologous substitution (imm434). In all cases the lefthand marker was a bio substitution (Fec- phenotype, which does not permit plating on recA-), and the righthand marker was an amber mutation in gene 0. Experiments were performed in all four possible arrangements of the central and rightward markers, while selecting for the Fec' phenotype on the recA- host. As anticipated, high negative interference (HNI) was observed with point mutations, but when the central marker was a substitution nonhomology, HNI was reduced about tenfold. Surprisingly, when the central marker was a simple deletion, a dramatic asymmetry in results was observed, with HNI being exhibited only when the central deletion marker was acquired by the double recombinant. These results indicate that under normal conditions (red', gam', ret+) and with noninhibited DNA replication, recombination in coliphage h entails a highly asymmetric step that could be at the level of strand transfer or mismatch repair.

HE mechanisms of general recombination have proved to he a particularly T intractable problem in molecular biology, although numerous possibilities have been proposed (MOSIG 1970; RADDING 1973, 1978; MESELSON and RADDING 1975; BROKER and DOERMANN 1975; HOTCHKISS 1974). Most of the proposed mechanisms are of the break-and-rejoin type and may he subdivided into two mechanistic types: those in which the exchange of genetic information occurs through the transfer of a single-stranded portion of one parental chromosome into the other, and those in which the genetic information, as duplex D N A , is reassembled from the parental chromosomes into a recombinant chromosome. Strand transfer has been postulated by several workers [see, for example,

Present address: Department of Clinical Neurological Sciences, University of Western Ontario, London.

To whom reprint requests should be addressed. Ontario, Canada N6G 2B2.

Genetics 102: 299-317 November, 1982

300 V. MAKIN, W. SZYBALSKI AND F. R. BLATTNER

WHITEHOUSE 1963; HOLLIDAY 1964; Fox 1966; MESELSON and RADDING 1975), and has been shown to be a physical possibility by BROKER and LEHMAN (1971), SIGAL and ALBERTS (1972) and THOMPSON, CAMIEN and WARNER (1976)l.

The two formal break-and-rejoin mechanisms (with and without strand transfer) can be refined by considering the possible roles of isomerization of crossed-strand exchanges (EMERSON 1969; SIGAL and ALBERTS 1972; SOBELL 1973) and of repair of mismatched base pairs by cellular repair functions. Thus, the break-and-rejoin model could involve (1) isomerization of a crossed-strand exchange, followed by cleavage of the crossed strands or by cleavage of the uncrossed strands, (2) cleavage of the uncrossed strands in the absence of an isomerization (see e.g., BLATTNER et al. 1974a), or (3) by the less likely route of reassembly of double strand fragments of parental DNA (see SZYBALSKI 1964; THOMAS 1966). In the case of strand transfer models, observed patterns of recombination would depend upon the length of the strand transfer, the pres- ence of associated isomerization of crossed-strand intermediates and mismatch repair of heterozygosities.

To be successful a model of recombination must account for the phenomenon of high negative interference (HNI), in which multiple recombinants appear at much higher frequency than would be expected as the product of independent single crossover frequencies. This may suggest that single and multiple recombinants are both formed from some common precursor molecule, such as a heteroduplex. HNI occurs only within rather small genetic intervals and has been observed in phage h (AMATI and MESELSON 1965), phage T4 (CHASE and DOERMANN 1958) and fungi (FOGEL, HURST and MORTIMER 1971; MORTIMER and FOGEL 1974). Break-and-rejoin models without strand transfer (see 3 in the previous paragraph) would likely require an independent molecular event for each genetic crossover. Thus, multiple recombinants would be infrequent, occurring perhaps according to a statistical distribution such as the Poisson distribution (HALDANE 1919), possibly modified by such factors as high local recombination-enzyme concentrations or pre-alignment of chromosomes after the first crossover. Therefore, it seems unlikely that such a model could adequately account for HNI, and this model will not be further discussed. Strand transfer models, on the other hand, are able to explain such high- frequency multiple events on the basis of a single rate-limiting molecular event, the strand transfer itself. Two possible consequences of strand transfer may be postulated. First, the transfer may be long and cover several markers; mismatch repair of heterozygosities would then result in frequent single and multiple recombinants. Such a mechanism has been favored by WHITE and Fox (1974) as the probable cause of HNI in phage A, at least under conditions of blocked DNA replication. Under these conditions they, as well as Russo (1973), have found that strand transfers are frequently very long in relation to the interval over which the HNI effect is observed. In addition, WILDENBERG and MESELSON (1975) and WAGNER and MESELSON (1976) have observed significant levels of mismatch repair in transfection experiments with heteroduplex h DNA (int-red-) and recA- E. coli. Thus, reduction to homozygosity of extensive heteroduplex tracts seems a likely mechanism of importance in general recombination.

HIGH NEGATIVE INTERFERENCE I N LAMBDA 301

The second possible mode of strand transfer resulting in HNI is one producing a heteroduplex region covering only the central marker of the classical three- factor cross. Such a heteroduplex would segregate double recombinants upon replication without requiring any associated mismatch repair (see, for example, FOX 1966; BLATTNER et al. 1974a). An important prediction of such a model is that HNI should operate mainly in the case of double recombinants, with the frequency of triple and higher order recombinants not significantly higher than the product of double and single recombination frequencies, unless there is additional mismatch repair. This appears to be the case for recombination in phage h carried out under wild-type (Red', Reef, Gam+, normal replication) conditions (J. D. BOREL, T. M. SHINNICK, V. MAKIN, W. SZYBALSKI and F. R. BLATTNER, unpublished data). This mechanism is not in conflict with the long strand transfers observed by WHITE and Fox (1974) under replication-deficient conditions since, as recognized by them, short insertion heteroduplexes may simply not mature into phage particles under their conditions (see also STAHL and STAHL 1974).

It might be predicted that a strand transfer process, whether symmetric (HOLLIDAY 1964) or asymmetric (MESELSON and RADDING 1975; see also DISCUS- SION), would be hindered by major genetic nonhomologies (BROKER and LEHMAN 1971; Fox, DUDNEY and SODERCREN 1979). To test this prediction, three-factor crosses have been performed in which the central marker could be either a point mutation, simple deletion or substitution nonhomology. This was done with all possible orientations of markers in order to detect any possible coop- erative effects (positive or negative) of particular marker combinations upon recombination or subsequent recombinant recovery. As hypothesized, the con- siderable abundance of double recombinants observed with the point mutation crosses was reduced by an average of tenfold when the substitution nonhomology served as the central marker. In the case of the simple deletion, a dramatic asymmetry in results was observed, HNI being exhibited by only one subset of marker orientations.

All of the experiments reported here have been carried out under conditions in which the Red system of h is operating and the Rec system of E. coli is partially inhibited by the Gam function of h (KARU et al. 1974). In addition, replication is normal so that there is no major constraint upon emerging phages to be in some way recombinant in order to be maturable (STAHL and STAHL 1974; SKALKA 1974), nor is there any reason to suppose that the error-prone recombination enzymes (SOS system) have been activated (WITKIN 1976).

MATERIALS AND METHODS

Media and buffers: NZB/Mg medium consists of 10 g/liter N-Z-amine A (Humko-Sheffield), 5 g/ liter NaCl and 0.01 M MgS04. Tris-MgS04 (TMS) buffer is 0.1 M Tris HCl, 0.1 M NaCl and 0.01 M

Plastic Petri plates, containing 40 ml of a mixture of 10 g/liter N-Z-amine A, 11 g/liter Bacto-Agar (Difco Laboratories) and 5 g/liter NaCl, were well dried before use. Top agar was a mixture of 10 g/ liter N-Z-amine A, 6.5 g/liter Bacto-agar and 5 g/liter NaCl.

Propagation of phages: For phages with clear phenotype, a single plaque was impaled upon a sterile Pasteur pipette and blown into 0.1 ml of a fresh overnight culture of permissive cells, usually

MgS04, pH 7.6.

302 V. MAKIN, W. SZYBALSKI A N D F. R . BLATTNER

E. coli Ymel, grown in NZB/Mg. Phages with turbid phenotype were treated identically except that three to five plaques were suspendcd in 1 ml chloroform-saturated TMS and allowed to stand a few minutes at room temperature; the chloroform was driven off in a stream of filtered air and 0.1 ml of the resulting suspension was used to infect 0.1 ml of cells. In each case, 15-min incubation at 37' was followed by the addition of 10 ml NZB/Mg, and the culture was shaken at 37" until lysis was complete. Chloroform was then added and bacterial debris removed by centrifugation. In the case of the XcZ'nin5 used here, which grows poorly, a secondary lysate was prepared by infecting 10 ml of stationary phase Ymel cells in NZB/Mg with the primary lysate, at multiplicity of infection (m.0.i.) of IO-", incubating 15 min at 37", and diluting into 1 liter of NZB/Mg for further growth at 37" with shaking until lysis was complete. After removal of debris, the phage were sedimented by 3 hr centrifugation at 18,000 rpm in a Beckman rotor 19 and resuspended in 5 ml TMS.

Plaque assays: Phage diluted in TMS were adsorbed in 0.1 ml of stationary phage indicator cells and incubated for 15 min at 37". Top agar, 2.5 ml, at 50" was added and the suspension was gently vortexed and poured over bottom agar in a plastic Petri plate.

Construction of phage strains employed in crosses: The origin of bacteria and of phage strains employed in these crosses is specified in Table 1. Construction crosses were done as plate crosses (BLATTNER et al. 1974b) or in the same manner as the crosses for quantitative analysis. Although only phages in which the N gene was deleted required the nin5 deletion for their growth (COURT and SATO 1969; FIANDT et al. 1971), we incorporated this deletion into all parental phages to ensure that both single and double recombinants from any one cross were uniform with respect to this marker. Crosses used in construction of the nin5 phages with all required arrangements of the three markers are given in Table 2. Structure of all strains was verified as follows (see also MAKIN 1976). Efficiency of plating for all Xbio phages was less than on recA- bacteria, for all hOam29 less than on homoimmune lysogens. The measured buoyant densities of the phages (Table 3) were in good agreement with predicted densities. Several of the phages were also checked by electron microscopy of appropriate DNA heteroduplexes (BLATTNER et al. 1974a; M. FIANDT, unpublished data).

Physical properties of the phages and positions of markers: guoyant densities of the phages, which served for the verification of their physical structure, are listed in Table 3. Positions of markers used in this study, expressed in %A units 1485 base pairs (bp)] measured from the left terminus, along with references, arc shown in Table 4.

Crosses for quantitative analysis: Phage particles, 5 X lo", of each parent, as calculated from the lowest of three independent titerings to ensure a minimum m.0.i. of 5, were adsorbed to 10' cells of late log phase Ymel cells growing in NZB/Mg, for 15 min at 37" in a total volume of 0.3-0.8 ml. NZB/Mg, 10 ml, was then added, and cultures were shaken at 37' in 125-1111 Erlenmeyer flasks for 75 min more, followed by addition of chloroform and storage at 4'. Detailed analysis of the crosses was completed within 5 wk.

Quantitative analysis of crosses 1-12: Total output was determined for each cross by plating on Ymel cells (Table 5, column B). Parental (Table 5, columns C and D) and recombinant (Table 5, columns E and F) outputs were also determined by plating on appropriate bacteria wherever possible. Crosses 1-8 were plated on N100(+80am2 su3) to near-confluent lysis and incubated at least 18 hr at 37O to allow maximum visibility of turbid plaques. Clear plaques from crosses 1 and 5 were transferred by toothpick to lawns of N100(+80am2) and N100(@80am2 S U ~ ) , respectively. Appearance of a clear or sectored area of lysis on N100(+80am2) implies the cI- phages were O+. The validity of this assumption has been confirmed in reconstruction experiments. Turbid plaques from crosses 3 and 7 were similarly analyzed. Clear plaques from crosses 2 and 4, and turbid plaques from crosses 6 and 8, were analyzed similarly except for an initial purification procedure designed to separate them from the confluent background of Fec+am+ phages. This was accom- plished by transferring the plaque by toothpick onto a lawn of N100(+80amZ su3) and then streaking out with a strip of paper; lawns were poured 24 hr previously and stored at 4" (this results in smaller plaques). Approximately six plaques per plate can be purified in this way. Crosses 9 and 11 were plated on N100(+80am2 su3)(XOam29) and N100(@8Dum2 su3)(himm434 Oam29), respectively, at a concentration sufficient to give about 25 plaques per plate, and these were then transferred by toothpick to N100(+80am2)(hOam29) followed by N100(@80amZ su3)(XOam29) in the case of cross 9 or N100(@80amZ)(Ximm434 Oam29) followed by N100(+80am2 su3)(Ximm434 Oam29) in the case of cross 11 Crosses 10 and 12 were plated on the appropriate indicators as for crosses 9 and 11, hut

on s u o (W3350) bacteria and for all himmX and himm434 phages less than

HIGH NEGATIVE INTERFERENCE IN LAMBDA

TABLE 1

Sources of bacterial, lysogenic and phage strains"

- Relevant phenotype

Bacteria and lysogens Ymel

W3350

su3, suppresses Oam29, not

su O

Pam3

NlOO

N100(+8Oam2) N100(+80am2 su3) N100(+3Oam2)(hOom29) N100(+80am2)(himm4340

N100(+80am2 su3)(hOam29) N100(+3Oam2 su3)(Ximm4340

Ymel(himm21 clts4 Pam3) CA266(Aimm434 Pam8O)

W3350(h) Bacteriophages"

Ymel(h)(PZ) Ymel(himm434)(P2) C600(himm434)(P2) Ymel(P2) NlOO(Ximm434) W3350(P2) AclAKH54 Pam80

am29)

am29)

hcZAKH54 nin5 Xb515b519bioKH341 c1857

Ximm21 clts4 nin5 himm434 cl- Oam29Pam3 hbioN2-1 imm434 nin5 hvl v3 Oam29 hbio30-7 Pam3 nin5 Xbio30-7 nin5

nin5'

suo, recA

sul, suppresses Oom29, not Pam80

su2, suppresses Oam29

rex-cl deletion

Sourceb and/or reference

303

J. S. SALSTROM

J. S. SALSTROM;

J. S. SALSTROM; GOTTESMAN

W. F. DOVE W. F. DOVE This study This study

This study This study

CAMPBELL (1961)

and YARMOLINSKY (1968)

J. S. SALSTROM This study

1. S. SALSTROM

This study J. S. SALSTROM J. S. SALSTROM J. S. SALSTROM J. S. SALSTROM

K. HASS; BLATTNER et al.

K. HASS K. HASS

(1974b)

J. S. SALSTROM 1. S. SALSTROM J. S. SALSTROM J. BOREL J. S. SALSTROM J. S. SALSTROM

" All donors from McArdle Laboratory, University of Wisconsin, Madison, WI. For phages constructed in this study see Tables 3 and 4. A new Abio isolated as hSpi- after induction of 594 (Ab515b519 xis6 cI857 nin5) lysogen. The bio

cndpoint lies between Nam7 and Nom53 as determined by marker rescue study, at 72.5 %A (see Table 4).

plaques were first purified on N100(+8Oam2 su3) by the paper strip technique and then toothpicked to N100(+80am2) followed by N100(+80om2 su3).

None of the procedures results in any significant level of false-positive or -negative results. In crosses 1-8, the original N100(+8Oam2 su3) plates were scanned carefully and only after at least 18 hr of incubation to guard against possible visual bias toward larger or more distinct recombinant plaques, which could have tended to be of one 0 genotype. Similarly, in crosses 9-12, all visible selected plaques were tested on each selection plate. As can be seen by comparing the frequencies

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TABLE 3

Buoyant densities of bacteriophages

305

Buoyant density (g/cm") Phage

Anin5 AOam29 nin5 Av1v3 nin5 hvlv3 Oam29 nin5 AcIAKH54 nin5 hcIAKH54 Oam29 nin5 himm434 cI- nin5 himm434 cI- OamZ9 nin5 Abio30-7 nin5 hbio30-7 vlv3 nin5 Abio30-7 Oom29 nin5 hbio30-7 vlv3 OamZ9 nin5 hb515b519 bioKH341 nin5 hb515b519 bioKH341 Oam29 nin5 Ab525b519 bioKH341 clAKH54 nin5 hb515b519 bioKH341 cIAKH54 Oam29 nin5 Ab515b519 bioKH341 imm434 cl- nin5 Ab515b519 bioKH341 imm434 cl- Oom29 nin5

1.5002 1.5001 1.5000 1.5001 1.4944 1.4946 1.4969 1.4973 1.4855 1.4836

n.d. 1.4836 1.4967 1.4973

n.d. n.d.

1.4937 1.4939

~ ~

a Buoyant densities determined by analytical ultracentrifugation in CsCl gradient versus A+ (1.508 g/cm3) and Ab2 (1.491 g/cm3) (see SZYBALSKI and SZYBALSKI 1971). All of the phages used in crosses 1-12 are listed here. n.d. = not determined.

TABLE 4

Physical positions of markers and lengths of intervals

Marker Position Reference

bio30-7 (right end) bioKH341 (right end) vlv3 (point mutations) cIAKH54 (deletion ends) imm434 (substitution ends)

Oam29 (point mutation)

77.7 BLATTNER et al. (1974a) 72.5 M. FIANDT, unpublished data 78.55" 74.1-78.4 BLATTNER et 01. (1974b) 73.5-79.1

81.15b

BLATTNER et 01. (1974b); MANIATIS et al. (1975)

WESTMORELAND, SZYBALSKI and RIS (1969); BLATTNER et

BLATTNER et al. (1974a and unpublished data) al. (197413)

Intervals, %A units

d! di d I -d2 bio markers imm marker bio to imm imm to Oom.29 bio to Oam29

bio30-7 vlv3 bioKH341 cIAKH54 bioKH341 imm434

0.85 1.6 1.0

2.60 2.75 2.05

3.45 4.35 3.05

Combination of electron microscopic and sequencing data. 1 %A = 485 bp. Derived from genetic mapping data in relation to electron microscopic maps.

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m 0 3 a l Z e m --e: " 0 m z h C , s E s 8 2 g . g k . E + ~ C D G Z E ~

HIGH NEGATIVE INTERFERENCE IN LAMBDA 307

recombinant plaques might introduce such a bias since these might tend to be preferentially of one of the 0 genotypes. Each cross was analyzed several times, and the results are combined in Table 6.

RESULTS

Design of crosses Three-factor crosses were made to test the effect of various kinds of nonho-

mologies on the outcome of a recombinational event in bacteriophage A. The marker arrangements for the crosses are represented in Figure 1. The left marker in each case was a bio deletion-substitution with the Fec- phenotype (absence of growth on recA- hosts) due to the loss of the red and gam genes. The right marker was the Oam29 point mutation. The central marker was either a point mutation (actually a double point mutant v l v3) or a major genomic modifica- tion, i.e., deletion or substitution. Different bio substitutions were used to keep the intervals, denoted dl and dz, between the three markers reasonably constant (Table 4). This was done since previous studies of AMATI and MESELSON (1965) indicated that the magnitude of the high negative interference effect varied with the interval length. Thus, we were able to compare recombination frequencies directly without correction for interval length, as is done in Figure 1 and Table 6.

Recombinants in the dl (bio-imm) interval were selected as Fec+ phages with the desired immunity phenotype using indicator strains nonselective for the

TABLE 6

Frequency of double recombinants among total recombinants in dl interval"

A B c n E

Total recom- combinants % double Average per binants in d l in d , and dl among total Xh (interfer-

Cross imm marker interval intervals recombinants Average (?A) ence value)

Double re-

1 2 3 4 .

7 8 !

11 l l 12

144 164 126 vlv3

124

262 129 121 cIAKH54

120

194 99

260 imm434

119

25 113 32 24

65 76 1 3

17.4 68.9 25.4 19.4

32.8 9.5

24.8 58.9

41.9 9.6

1.7 0.4 0.83 2.50

2.58

1.1 4.05 2.30 5.04

3.5

Cross numbers refer to Figure 1. Column A represents total number of recombinants (plaques) in interval d, (bio-imm). Column B represents those recombinants in column A that were also recombinants in interval d2 (imm-Oam29) (see MATERIALS AND METHODS). Numbers in column E (interference values) are values in column D divided by physical length of the corresponding interval (d, + dn) in %A units (see Table 4).

V. MAKIN, W. SZYBALSKI AND F. R. BLATTNER 308

FIGURE 1.-Diagrammatic representation of crosses used in this study. The left marker was always a bio DNA substitution (represented as an open rectangle) and the right marker was always an OamZ9 point mutation. The central marker was varied to include a (double) point mutation v l v3 (crosses 1-4), simple deletion cIAKH54 (crosses 5-8) and substitution of imm region of X with nonhomologous imm DNA of phage 434 represented as a rectangle (crosses 9-12). The outside heavy lines represent the parental phage genomes (all carrying the nin5 deletion) and the inside lines the single and double Fec+ recombinants. Each column includes all four possible orientations of the central and right markers. The "percent doubles" for each cross are taken from Table 6, column C .

righthand markers. In crosses 1-8, this was done by plating the cross outputs on recA- su3 bacteria and choosing the appropriate clear or turbid plaques. In crosses 9-12, recA- su3 lysogens carrying either X or Ximm434 prophages were used as a lawn to select the appropriate immunity type among the Fec+ recombinants, and the plaques were subsequently purified on nonlysogens. In all cases the procedure was nonselective with respect to the righthand (Oam29) markers.

Recombinants in the dl interval were then scored for the 0 phenotype on recA- su O cells to determine whether a second crossover had occurred in the da interval. The percentage of double recombinants occurring in the populations selected for at least a single crossover in dl is summarized in Figure 1.

Results of the crosses The results of 12 experiments numbered as in Figure 1 are shown in Tables

5 and 6. All crosses were done on the same day using the same batch of YmeJ cells infected with each parent at a m.0.i. of approximately 5.

Characterization of crosses: Table 5 specifies the output titers of the parents and frequencies of various recombinants in intervals dl or dz or both. These data provide evidence that phage adsorption, overall recombination levels and phage growth were comparable in all 12 crosses. The total outputs of progeny phage were approximately constant (2.6-5 X 10s/ml; see column B) and outputs of parents (column C and D) are in agreement with the total outputs. The frequencies of recombination per %A length in the scored (dz) intervals are constant within reasonable limits (columns G and H), indicating that none of the phages employed exhibited any anomaly in the observed recombination


frequencies in this interval, i.e., an idiosyncratic increase (“hot spot”) or de- crease. This is of importance for analysis of data derived from Table 6.

The frequencies of crossover per %A length in the selected (dl) interval (Table 5, column G) tended to be somewhat lower when the interval was bounded by two major nonhomologies. However, this lesser degree of uniformity should not affect the conclusions derived from Table 6, because this was not the interval in which the second (unselected) recombination event was scored.

Analysis of crosses with respect to high negative interference: The results shown in Table 6 show a very high excess of double recombinants when the point mutations vlv3 serve as the central marker (columns C and D, crosses 1- 4). For example, in cross 2 more than half (68.9%) of the recombinants in inteval dl recombined also in interval dz, which is more than 100 times the expected frequency of single crossovers in interval dz [equal to about 0.6%,3 derived as the product of the interval length (2.6 %A, Table 4) and recombination frequency per %A of 2.17 X low3, Table 5, column H, line 2, and expressed as a percentage]. When the central marker is a substitution nonhomology, the double recombinant frequency is dramatically lowered by factors up to 30 with an average of ten (Table 6, columns C and D, crosses 1-4 vs. 9-12). When the central marker is a deletion, the recombination frequencies fall into two classes: one resembling the point mutant crosses 1-4, in cases in which the deletion is on the same strand as the bio substitution (crosses 5 and 6), and the other resembling the imm434 substitution (crosses 9-12), in cases in which the deletion is on the Fec+ strand (crosses 7 and 8). To normalize the data to the total interval length (as to generate the so-called

interference value), the values in column D were divided by the physical distance dl + dz and are listed in column E. These interference values are included mainly for historical interest, so as to permit a comparison with the results of AMATI and MESELSON (1965). However, we divided the data in column 3 by the total interval dl + d2 rather than by the measured single recombination frequency in the interval d2, as was done by AMATI and MESELSON (1965). Since recombination rates are generally proportional to distance, our values in column E should be proportional to their interference index. As stated before, our unadjusted data in columns C and D can be used directly, since the interval lengths dl and dz were chosen to be approximately constant (Table 4).

DISCUSSION

The principal result of this study is the observation that major nonhomologies within a recombining region exert a profound effect on HNI, measured as the excess of double recombinants in the three-factor crosses. In those cases when the central marker was a point mutation, about one-third of the preselected

”The value 0.65: is based on a model in which the second recombination event is independent of the first. The first crossover requires two chromosomes to enter a mating pool and subsequently undergo the necessary molecular events, whereas the second crossover requires only the latter factor. MAKIN (1976) has estimated that the probability of entering the mating pool is about 15% in this experiment, assuming that frequencies in crosses 7-12 are taken as representing the absence of any high negative interference, and percentage calculated by dividing the average of the single recombination frequencies in the same intervals (X100) by the average frequencies in crosses 7-12.


single recombinants in region d l were also recombinant in the dz region (Table 6, crosses 1-4, column D), whereas the frequency of such double recombinants was about tenfold lower in cases in which the central marker was a substitution (crosses 9-12, column D). This result is in agreement with the simple prediction of the strand transfer models, already discussed. Thus, we believe that recombination in phage X (under wild-type recombination and replication conditions: Red’, Gam+, Rec+) proceeds predominantly by a strand transfer mechanism.

However, in crosses in which the central marker is a simple deletion, we observed a profound asymmetry, with crosses 5 and 6 yielding more than a 20 times higher percentage of double recombinants than crosses 7 and 8 (Table 6, column D). This result might relate to the seemingly opposite results of DOERMAN and PARMA (1967), who observed no affect of central deletion on the recombination frequency, and of BERGER and WARREN (1969), who recorded a depression in recombination frequency in the case of central deletion (see also the discussion of these results by MOSIG 1970).

This asymmetry observed for the cI deletion should permit a more detailed examination of various strand transfer mechanisms of recombination. As shown in Figure 2, there are two main modes of strand transfer, symmetric (a) and asymmetric (b). The present asymmetric results could be explained by asymmetric strand transfer or by mismatch repair. If such enzymatic repair of heteroduplex intermediates formed by strand transfer were itself asymmetric, both symmetric and asymmetric models could explain our results. However, we shall first discuss only the asymmetric models and determine which of these could explain our data without invoking mismatch repair. Subsequently, we will also consider mismatch repair as an auxiliary mechanism.

A general formulation of all asymmetric models of strand transfer is depicted in Figure 2. Such models (2b) imply a mechanism of displacement and assimi- lation of one strand in any of several possible enzymatic ways. Displacement, as mediated by element D, must occur in general by breaking the hydrogen bonds between the strand to be displaced and its complementary strand. This could occur either by replacing the displaced strand with one newly synthesized by DNA polymerase (Zc), by removing the partner strand altogether with an exonuclease (2d), or by tying it up with a single strand DNA-binding protein (Ze), such as the coliphage T4 gene 32 protein (ALBERTS and FREY 1970), or one of the DNA helicases (KUHN, ABDUL-MONEM and HOFFMANN-BERLING 1979). Similarly, either polymerase, exonuclease or a single-strand-binding protein could remove the appropriate strand on the recipient parent to allow assimila- tion as shown in Figure Zf , g and h. As there are two strands, two strand polarities and three possible displacing or assimilating elements, it is possible to propose 2 X 32 formal models for asymmetric strand transfer, as listed in Table 7. Note that all of these models would result in nonreciprocal recombination, as has been shown to be the case for the X Red system (JACOB and WOLLMAN 1954; SARTHY and MESELSON 1976).

Consideration of the features of these 18 models allows several more likely possibilities to emerge. Models 1 through 8, including model 2 (Figure 2i) ascribed to CASSUTO and RADDINC (see SIGAL and ALBERTS 1972), lead to the formation of a so-called 5’ + 5’ overlap. WHITE and Fox (1974) have shown that


symmetric swondmnsfer AsymMn'cnmnd Transfer

bplacing Jcmnt D

A (0) x tb) Mimim r&t

Dimtbnof trwofcr D;rutbn +"

311

FIGURE Z.-Strand transfer mechanisms for genetic recombination. (a), Symmetric strand transfer according to HOLLIDAY (1964) and SIGAL and ALBERTS (1972). (b), Asymmetric strand transfer as proposed by MESELSON and RAnDrNC (1975) requiring displacing (D) and assimilating (A) elements. Diagrams c-h illustrate all possible roles of the DNA polymerases, exonucleases and single strand DNA-binding proteins (SSDBP) acting as displacing c, d and e or assimilating f , g and h elements, with DNA strand polarities not specified. Three specific examples, which correspond to models 2, 5 and 14 (see Table 7) with DNA strand polarities indicated, are represented in diagrams i, j and k. Note that in models 2 and 5 the 5' end of the DNA strand is displaced (5' + 5' overlap) and in model 14 the 3' end is displaced (3' + 3' overlap; see Table 7).

in h recombination, albeit under nonreplicating conditions, the inserted strand of DNA begins with a 3' end (3' + 3' overlap: see e.g., Figure 2k). As a 3' += 5' DNA polymerase most probably does not exist (KORNBERG 1974), models 10, 11, 12, 13 and 16 are unlikely. Of the remaining models, only models 14 and 15 utilize an exonuclease of the same polarity as X exonuclease (LITTLE 1967; RADDING 1969), which is known to be involved in Red-mediated recombination (SIGNER 1971). Model 15 requires a hitherto undiscovered single strand binding protein of 3' -+ 5' polarity, whereas known examples are either nonpolar (phage T4 gene 32 protein) or migrate in a 5' -+ 3' direction (DNA helicases; KUHN, ABDUL-MONEM and HOFFMANN-BERLING 1979). On this basis, we favor model 14 for Red-mediated recombination in phage X (Figures 2k and 3). Model 14 would also appear to present less of a topological problem, there being less than four strands in the recombining region. However, if the product of X gene bet, which at present has no known biochemical activity, indeed is a 3' --+ 5' DNA-binding

312 V. MAKIN, W. SZYBALSKI A N D F. R. BLATTNER

TABLE 7

Enzyme combinations capable of driving an asymmetric strand transfer reaction“

Model Displacing element Assimilating element

1 5’+3’ polymerase 5’+3’ polymerase 2 5 i 3 ’ polymerase 5 1 3 ’ exonuclease 3 5’+3’ polymerase 5 1 3 ’ SSDBP 4 3’+5’ exonuclease 5’+3’ polymerase 5 3’+5’ exonuclease 5’+3’ exonuclease 6 3’+5’ exonuclease 5 1 3 ’ SSDBP 7 3’+5’ SSDBP 5’43’ polymerase 8 3’+5’ SSDBP 5’+3’ exonuclease 9 3’+ 5’ SSDB P 5’+3’ SSDBP

10 3’+5’ polymerase 3 1 5 ’ polymerase 11 12 13 14 15 16 17 18

3’+5’ polymerase 3’+5’ polymerase 5’+3’ exonuclease 5’-+3’ exonuclease 5 1 3 ’ exonuclease 5 i 3 ’ SSDBP 5’+3’ SSDBP 5’+3’ SSDBP

3’45’ exonuclease 3’+5’ SSDBP 3’+5’ polymerase 3’+5’ exonuclease 3 ’4 5’ S S D BP 3’+5’ polymerase 3 1 5 ’ exonuclease 3 1 5’ SSDBP

Displacing and assimilating elements are diagrammatically represented in Figure 2, where also the specific model 2 (attributed to CASSIJTO and RADDING by SICAL and ALBERTS 1972), model 5 and model 14 (favored by this study; see Figure 3) are shown. The symbol SSDBP represents a single strand DNA-binding protein, e.g., the product of phage T4 gene 32 (ALBERTS and FREY 1970) or one of the DNA helicases (KUHN, ABDUL-MONEM and HOFFMAN-BERLING 1979); the latter could be considered as the 5’+3’ SSDBP. Note that models 9 and 18 are equivalent if the SSDBP exhibits no polarity. In models 1-9, a 5’ end of the DNA strand is displaced (5’45’ overlap), and in models 10- 18, a 3’ end is displaced (3’+3’ overlap, suggested by the results of WHITE and Fox 1974).

protein, then model 15 may represent the Red-mediated mechanism. Alterna- tively, bet protein may act as a cofactor of the host 3’ + 5’ exonuclease or of h exonuclease of model 14 or of a host DNA-binding protein.

Figure 3 shows in detail several stages in the proposed mechanism of model 14 as it applies to the crosses involving the deletion nonhomology (see Figure 1, crosses 5-8). The first two columns represent the crosses productive of double recombinants (crosses 5 and 6) and the last two columns the nonproductive crosses (crosses 7 and 8). It is possible to rationalize this asymmetry in several ways (MAKIN 1976), e.g., by an inability of the 3’- 5’ exonuclease to wait at the base of the cl+ loop (Figure 3, columns 3 and 4, line 2) for the 5’- 3‘ exonuclease to complete its excursion, while the opposite combination is functional (columns 1 and 2, line 2).

The asymmetry observed in crosses 5-8 could also be due to destruction of heteroduplex intermediates of the type shown in Figure 3 (fourth line) in spite of their successful formation. Such intermediates could be formed either by symmetric or asymmetric strand transfer process. If the cl+ single strand loops of such heteroduplexes were enzymatically converted to the deletion genotype, then the excess of double recombinants in crosses 5 and 6 (in relation to crosses 1-4) and corresponding deficiency in crosses 7 and 8 (relative to crosses 9-12) would be observed. Indeed, a slight trend in this direction may be discerned in


3't"inat strand d i r p h d (s'-~'~onuc(uucuedispla~th~ element and ~'-5'nmnudavc os arimilatity &me)

-- --- F u + Ad

313

Dirpbcingdemcntm rm-dactiondxomoronw ,-. .

c)' '. r

FIGURE 3.-Detailed mechanism of model 14 (5' + 3'-exonuclease as displacing element and 3' + 5'-exonuclease as assimilating element; see Table 7 and Figure 2) for crosses 5-8 as represented in Figure 1. The first and second columns (displacing element on deletion chromosome) represent crosses 5 or 6, whereas the third and fourth columns represent crosses 7 or 8 (location of 0- marker not specified). The orientations of strands 1 and r are 5' + 3' and 3' + 5', respectively. The DNA of the bio parent is represented by thin lines and of the Fec+ parent by thick lines. The top line of the figure represents an early stage of the transfer process with a 3' end of a DNA strand of the bio parent being transferred to the Fec+ parent, the joint being represented by the solid square. The second line shows the progression of the transfer across the deletion nonhomology with the loop representing the cIc DNA. The third line formally illustrates the termination of the transfer process, with the short horizontal slash representing the required endonucleolytic cleavage of the displaced strand. The fourth line shows the end products of the transfer process, with the inserted strand bonded by two solid squares. The bottom line shows the double recombinant chromosome segre- gated during the first round of DNA replication. Note that only the Fec+AcI double recombinants (bottom line of first two columns) were observed at high frequency (see crosses 5 and 6 in Figure 1 and Table 6, column C).

Table 6 (column D: 41.9% doubles vs. 32.8%, and 1.7% doubles vs. 3.5%). Such a phenomenon would be consistent with the evidence supporting a role for mismatch repair cited in the introduction. Although repair must remain a mechanism to be considered, preliminary data to test this possibility under our wild-type experimental conditions have tended to negate it (see MAKIN 1976), especially since no obvious asymmetry was observed for the single crossovers (ratio of values in columns F and E of Table 5 are .140 and .167 for crosses 5 and 7, respectively). Also, no appreciable deficiency in the cI+ recombinants was

3 14 V. MAKIN, W. SZYBALSKI A N D F. R. BLATTNER

observed in crosses 5-8. Such deficiency, if seen in Table 5, might have indicated a ‘‘repair’’-mediated loss of the cI+ “loops” in the cI+/KH54 heteroduplexes, the mechanism that was invoked by BENZ and BERGER (1973) to explain the loss of the rII+ allele in crosses with rII deletion mutants of phage T4. Such repair in T4 was observed only when DNA synthesis was reduced and the products of T4 genes v and x were present. This is expected, since v and x are thought to be required for repair.

If there is no significant mismatch repair, as is considered likely, the asymmetry in HNI is consistent with an asymmetric initiation of recombination. We favor the asymmetric, X exonuclease-mediated strand transfer model described by BROKER and LEHMAN (1971) (Figure Zk), as it successfully reconciles the 3’ + 3’ overlap observed by WHITE and Fox (1974), and the nonreciprocity for the Red system observed by JACOB and WOLLMAN (1954), WOLLMAN and JACOB (1954) and SARTHY and MESELSON (1976), with known enzyme polarities. This mechanism probably predominates under our wild-type conditions when A replicates freely and the RecBC system of the host is suppressed by the X Gam function (KARU, ABDUL-MONEM and HOFFMAN-BERLING 1974). However, when X DNA replication is artificially blocked, as is the case for most other analogous published data (e.g., WHITE and Fox 1974; Russo 1973; WEINSTOCK 1974; WIL- DENBERG and MESELSON 1975) mismatch repair may be a predominant process, especially since under such conditions the error-prone repair functions of the SOS pathway are known to be induced (WITKIN 1976). Moreover, the results of Fox, DUDNEY and SODERGREN (1979; see also MOSIG et al. 1979) raise the question of whether heteroduplexes across nonhomologies are generated by the same mechanism as heteroduplexes involving extensive and perfect homologies. We realize that several recombinational systems are operational in the E. coli-X system, and depending on the conditions one or another of those seems to predominate.

The experimental studies, which were carried out in years 1974-1976, were supported by the National Cancer Institute Program-Project grant CA-07175. We would like to acknowledge the critical reading and comments of DRS. T. BROKER, A. H. DOERMANN, R. HOLLIDAY, C. M. RADDING, H. M. SOBELL and F. W. STAHL. We would also like to thank our colleagues J. S. SALSTROM, A. HONIGMAN, S.- L. Hu, E. ROSENVOLD and M. FIANDT for their help and advice. The excellent technical assistance of M. METZDORFF and K. HOLT is gratefully acknowledged.

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asymmetric effects of deletions and substitutions on … · all cases the lefthand marker was a bio...

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