Defined oligomeric SV40 DNA: A sensitive probe of general recombination in somatic cells

Defined oligomeric SV40 DNA: A sensitive probe of general recombination in somatic cells

Cell, Vol. 21. 141-148. August 1980, Copyright 0 1980 by MIT Defined Oligomeric SV40 DNA: a Sensitive Probe of General Recombination in Somatic C...

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Cell, Vol. 21. 141-148.

August

1980,

Copyright

0 1980 by MIT

Defined Oligomeric SV40 DNA: a Sensitive Probe of General Recombination in Somatic Cells Claire T. Wake Marrs McLean Baylor College Houston, Texas

and John H. Wilson Department of Biochemistry of Medicine 77030

Summary We have constructed well defined oligomeric molecules of simian virus 40 (SV40) DNA as probes for investigating mechanisms by which cultured somatic cells recombine DNA. Restriction enzyme fragments from different temperature-sensitive mutants were joined in a head-to-tail orientation to create partial dimers 1.84 genome lengths in size. These molecules are too large to fit into a viral capsid. Therefore an assay that depends on production of progeny virus after infection with oligomerit DNA is a selective measure of precise conversion of oligomers to monomers. By constructing oligomers from appropriate combinations of temperature-sensitive DNAs, we have been able to study the conversion process in several defined regions of the SV40 genome. Our results indicate that conversion of oligomers to monomers occurs uniformly throughout the genome and is not dependent on normal viral DNA replication. These data indicate that conversion occurs primarily by general, homology-dependent recombination. At least one secondary mechanism that generates a low level of wild-type progeny was also detected. Studies with heteroduplex molecules indicate that repair of mismatched bases may be the secondary mechanism. Introduction Genetic recombination in eucaryotic cells occurs in both germ line and somatic cells. Recombination in germ line cells probably functions in the evolution of organisms by rearranging genes for the next generation. The function of recombination in somatic cells must differ, however, because genetic information in somatic cells dies with the organism. Several types of recombination have been observed in somatic cells. General recombination, or recombination between homologous DNA duplexes, has been studied in yeast, Aspergillus, Drosophila and maize. Recombination of DNA sequences with limited homology has been suggested as an essential component of several diverse cellular processes. Perhaps the most notable are the genetic rearrangements of immunoglobin gene sequences that occur during development of B lymphocytes (Brack et al., 19781, and the regulation of mating type interconversion in yeast (Hicks, Strathern and Herskowitz, 1977; Hicks, Strathern and Klar, 1979). We have used the extremely well defined DNA of

simian virus 40 to investigate specific questions about somatic recombination in cultured mammalian cells. Our strategy begins with construction of genetically marked, oligomeric molecules of SV40 DNA. Oligomerit molecules are too large to be encapsidated and therefore must be converted to monomeric molecules by the cell in order to be packaged and detected in viruses. Because our oligomers contain temperaturesensitive mutations, we can assay selectively for production of viruses containing wild-type monomers. Our previous experiments with heterogeneous mixtures of oligomeric and monomeric molecules suggested that conversion of oligomers to monomers occurs by homology-dependent, intramolecular recombination (Wake and Wilson, 1979). We have refined the SV40 system by constructing and purifying precisely defined oligomeric molecules. These oligomers are composed of complete or partial genomes from two different temperature-sensitive mutants linked together in a particular arrangement. Using defined partial dimeric molecules, we demonstrate in this paper that the principal mechanism for generating monomers occurs uniformly throughout the genome and is not dependent on normal viral DNA replication. These observations are strong evidence that oligomers are converted to monomers primarily by general, homologous recombination. Results Construction of Defined Oligomeric Molecules In our earlier experiments we created oligomers of SV40 DNA by the ligation of Eco RI-cleaved linear monomers (Wake and Wilson, 1979). As a result, each oligomeric size class in the heterogeneous ligation mixture actually contained several types of molecules. The linear dimer fraction, for example, consisted of heterologous dimers composed of two different DNAs and homologous dimers composed of only one of the parental DNAs. In addition, because the parental monomers were ligated at a symmetric restriction enzyme cleavage site, half the dimers were made of monomers joined tandemly in a head-to-tail orientation, and the other half were joined in a head-to-head or tail-to-tail orientation. Of the variety of possible combinations, we anticipated that only heterologous oligomers joined head-to-tail would be useful substrates for our recombination assay since they could generate viable molecules with a genotype (wild-type) distinguishable from the parental genomes. To create oligomers containing only head-to-tail junctions, we used the restriction endonuclease Bgl I, which is a member of the class of restriction enzymes that cuts outside its recognition sequence. Bgl I recognizes the boxed nucleotides in the SV40 DNA sequence shown below and cleaves at the arrows to leave complementary three-base single strands (Shor-

Cell 142

tle and Nathans,

1979).

DNAs were cleaved with either Hpa II or Taq I, then treated with bacterial alkaline phosphatase to prevent ligation of these ends at a later step. After the removal of the phosphatase, the linear monomers were cut with Bgl I, mixed together and ligated. The head-totail, heterologous partial dimers were identified readily on agarose gels and purified as described (Wake and Wilson, 1979). The structure of purified 1.84 partial dimers was verified by restriction enzyme analysis (Figure 3).

q-q&yg$g:;: Normal pairing of these ends is possible only in the head-to-tail orientation. This expectation was verified experimentally, as described in Figure 1. A particular kind of heterologous oligomer, a partial dimer that is 1.84 genome units in length, was constructed by the procedure outlined in Fig. 2. The partial dimers were designed so that the small segment from the Taq I site (0.90) to the Hpa II site (0.06) was present only once per molecule, whereas the remainder of the genome was repeated. Circular

BglI

EcoRl + HpoII Digestion

junctions

ECORI ,., .T e-m,..we--

Markers I

lipoIl EcoRI mt

Recombinational Potential of Oligomeric DNA We had assumed that head-to-tail, heterologous oligomers were the principal substrate detected in our

Frogmerits 2

EcoRI EcoRI + +DNA + BgII llgaae tipon 3 4 5

6011 t lipon. 6

Frogmen,* 7

0.27

Bgll T.-r

H.H

EcoRI tipan. lips1 EcoRI -.q !,! r ___ BglI

m+

0.27

x

EcoRl

ECORI ---e---

11.34] 6011

NC L EcoRI

,,.T

T.T

BglI +

HpoII Digestion

junctions

EglI Itpan .-5: z; EcoRl

SC

9gII I---

m+

0.06 0.43 0 33

8911

BglI ---l-l---

11.341

0 27

EcoRI H.,.,

Figure

Bgl I HpoU HpoIt 6glI ._--w EcoRI

1. Orientation

I 00 0 86 073 067 0 57

mt

ot Bgl I- and Eco RI-Cut

0.06 0 I4

Fragments

Ligated

in Vitro

SV40 circular DNA was cleaved into two fragments with Eco RI and Bgl I and then incubated with T4 DNA ligase to form an array of molecules containing ligated Bgl I and Eco RI junctions. All junctions arose from the intermolecular association of fragments with complementary ends. Half the ligated mixture was treated with Eco RI and Hpa II in order to analyze the Bgl I junctions. The other half was treated with Bgl I and Hpa II to examine the Eco RI junctions. (A) Diagram of Bgl I- or EGO RI-cut fragments religated in three possible orientations: head-to-tail (H.T). tail-to-tail (T.T) and head-to-head (H .H). Predicted products of subsequent digestions with either Eco RI + Hpa II or Bgl I + Hpa II are given in fractional genome units. The boxed fragments are diagnostic for each possible orientation. As measured from the origin of replication, Bgl I, Hpa II and Eco RI cleave at 0.0, 0.06 and 0.33 fractional genome units, respectively. (6) Agarose gel demonstrating the orientation of ligated junctions. (Lane 1) Supercoiled (SC), linear (L) and nicked circular (NC) SV40 DNA (3.3 X 1 O6 daltons); and h DNA (30 X 10’ daltons). (Lanes 2 and 7) Size markers of unit-length linear SV40 DNA and a mixture of fragments generated by cleaving circular SV40 DNA with pairs of one-cut restriction endonucleases. (Lane 3) Fragments produced by treating circular DNA with Eco RI and Bgl 1 (note that some partially cleaved monomer linears remain). (Lane 4) Productsof lane 3 after incubation with T4 DNA ligase. (Lane 5) Products of digestion of the ligated mixture with EGO RI and Hpa II. (Lane 6) Products of digestion of the ligated mixture with Bgl I and Hpa II. The resulting fragments in lanes 5 and 6 verify that Bgl l-generated ends rejoin only in the head-to-tail orientation, whereas EGO RI-cut ends rejoin in all oossible orientations.

fqe3neral Recombination

in Somatic

Cells

earlier recombination assays using heterogeneous mixtures of ligation products. Therefore we predicted that our measured frequencies of genetic recombinants would increase substantially upon infection with defined oligomers that were uncontaminated with nonproductive species. The procedure in Figure 2 was used to join the Taq I/Bgl I fragment from tsB4 (designated TB4) to the Hpa ll/Bgi I fragment from tsB228 (designated HB228) to construct TB4.HB228 partial dimers. In addition, a variety of full-length oligomers containing these same mutations were constructed as described in the legend to Table 1. The frequencies of genetic recombinants produced by these oligomeric species are compared with results using several mixtures of tsB4 and tsB228 DNAs in Table 1. The results with mixtures 1-3 are consistent with our observations using other pairs of mutant DNAs (Wake and Wilson, 1979). The mixture of untreated circular DNAs produced a very low fraction of wildtype progeny. We also observed recombination between Bgl l-cut linear monomers. The heterogeneous ligation mixture of tsB4 and tsB228 DNAs produced a 17 fold increase in the proportion of genetic recombinants. As predicted, the defined oligomers yielded the largest increases in genetic recombinant& up to 500 fold over the mixture of untreated circular DNA. The partial and full-length heterologous oligomers thus constitute very sensitive probes for the detection of genetic recombinants.

Comparison of Genetic and Physical Distances Conventional genetic analysis assumes that homologous recombination occurs approximately uniformly throughout the genome (Radding, 1978). This assumption was verified for +X1 74 when the physical distances determined by DNA sequencing were compared to the genetic map (Warner and Tessman, 1978). In Table 1 the observed recombination values for the SV40 oligomers, especially the partial dimers,

A. Fragments

1.84 Partial Dimer +TaqI

I2

+HpaII +BamHI

345

Fragments

6

I.1 0. 0, ii

Toq I

Toq I TaqI

lipdl I

0 0

BAP

I

@PII ToqI HpolI

HpqU I

nq1 I

I’

HP0n

0

BAP

To41 &$I Hpd

Toql Bgll HwU wp-

-ppi

0. .I4

Tqql

‘--jyGd

B. HpaII t .06

I, I

I

1.0Mo”omer

lg-

1.0 Monomer 0.16 Pmrm Monomsr

*

Figure

2. Procedure

for Constructing

1 .S4 Partial

Dimeric

DNA

In separate reactions circular DNA was cleaved either with Hpa II or Taq I, then treated with bacterial alkaline phosphatase (SAP) to prevent ligation of the ends at a later step. After inactivation of the phosphatase. the linear molecules were digested with Bgl I. then mixed together and ligated. Joining of the 0.94 Hpa II/Bgl I fragment with the 0.90 Taq I/Bgl I fragment generated a 1.84 partial dimer.

Figure

8amHI 1 .4? 3. Verification

Bgll HpaII I 1 I .90 .06 0

TaqI

of the Structure

BamHI I I .47

TaqI 1 .90

of 1.84 Partial Dimeric

DNA

(A) Agarose gel showing the results of restriction enzyme analysis of the 1.84 partial dimer. Restriction endonuclease cut sites are marked on the structural map in (6). (Lanes 1 and 6) Size standards of unitlength linear SV40 DNA and a mixture of fragments generated by cleaving circular SV40 DNA with pairs of one-cut restriction endonucleases. (Lane 2) Purified 1.84 partial dimer. (Lanes 3. 4 and 5) Products from Taq I, Hpa II and Barn HI cleavage of the partial dimer. Products of these digestions are consistent with the structural map of the 1.84 partial dimer. shown in (B), and inconsistent with head-tohead or tail-to-tail junctions. (B) Structural map of the 1.84 partial dimer.

Cell 144

are in rough agreement with theoretical expectations that assume conversion of oligomers to monomers occurs with equal probability throughout duplicated regions of the genome. In addition, our work with heterogeneous mixtures of full-length oligomeric molecules suggested that the production of recombinant monomers was proportional to the distance between markers (Wake and Wilson, 1979). To extend these observations we constructed partial dimers from all Table 1. Genetic Crosses with tsB4 and ts.6228 Purified Oligomeric Molecules Genetic Recombinant@ %R

DNA Species” (1)

Circular untreated

DNA Mixtures

Factor Increase

monomers, 0.025

1

(2)

Linear monomers,

unligated

0.098

(3)

Linear

ligated

0.43

17

(4)

monomers,

4

mB4.8228

L-2

3.8’

150

(5) mB4.8228

L-3

2.6’

100

(6) mB4 .B228

L-4

2.5’

100

(7) mB4.8228

C-2

4.3

170

12.1

480

(8)

and

TB4.HB228

L-l .84

“The circular monomers of tsB4 were 40% supercoiled and 60% relaxed circles: tsB228 monomers were 10% supercoiled and 90% relaxed circles. Linear monomers of tsB4 and tsB228 prepared by Bgl I digestion were mixed together and assayed before (mixture 2) and afler ligation (mixture 3). The purified oligomers are indicated by L (linear) or C (circular) followed by the number of genomes per molecule. To construct full-length linear dimers, trimers and tetramers. a mixture of tsB4 DNA (m). methylated at the EGO RI site with EGO RI methylase. and excess tsB228 DNA was cut with Bgl I, ligated and then cut with EGO RI endonuclease. and the linear oligomers were purified from agarose gels (our unpublished observations). Circular dimers were created in vitro by ligating linear dimers. The TB4. HB228 1.84 partial dimer was constructed by joining the Taq I/Bgl I fragment of tsB4 to the Hpa ll/Bgl I fragment of tsB228. b CVl monolayers on 60 mM plates (2 x 10’ cells) were infected with 16.8 ng to 168 pg of mixtures l-3 or 1 ng to 10 pg of species 4-8 and then incubated at 41” or 33°C. %R was calculated using the relationship %R = 200% (41°C titer/33’C titer). Monomers and oligomers constructed from wild-type DNA were assayed at 41’ and 33°C to obtain a correction factor for differences in plaquing efficiencies at the two temperatures. lnfectivities of the DNA species at 33’C were: (mixture 1) 2.2 X 1 O3 pfu/ng; (mixture 2) 130 pfu/ng; (mixture 3) 520 pfu/ng: (mixture 4) 210 pfu/ng: (mixture 5) 220 pfu/ng; (mixture 6) 240 pfu/ng: (mixture 7) 270 pfu/ng; (mixture 8) 50 pfu/ ng. %R values for mixtures l-3 are the average of duplicate plates; %R values for mixtures 4-7 are the average of eight assays each performed in duplicate or triplicate; the %R value for mixture 8 is the average of one assay performed in quadruplicate. ’ The linear dimers. trimers and tetramers yielded wild-type genomes in the expected relative proportions. As a result of the method of construction, trimers contained two tsB4 genomes and tetramers contained three tsB4 genomes. Because monomers derived from adjacent tsB4 genomes would have the parental tsB4 genotype, it was expected that the recombination value for the trimer would be two thirds, or 67%. that of the dimer and that the recombination value for the tetramer would be 50% that of the dimer. The proportion of wild-type recombinants produced by the linear trimers and tetramers was 68 and 66% of the dimer value, respectively, which was in reasonable agreement with expectation.

pairwise combinations of five DNAs containing temperature-sensitive markers-tsB228, tsB4, tsA58, tsA28 and tsA30-whose approximate physical locations are known from marker rescue experiments (Lai and Nathans, 1975). There are two possible ways to link two mutant DNAs into a partial dimer, depending on which DNA is cut with Taq I and which is cut with Hpa II. One such pair of reciprocal partial dimers is diagrammed in Figure 4A. TA28.HB4 partial dimers (type I) can recombine anywhere in the shaded region to generate wild-type monomers. However, because of the small deletion at the ends, TB4.HA28 partial dimers (type II) cannot yield wild-type monomers in a single homologous recombination event. The grid in Figure 4B is arranged so that recombination values with type I partial dimers are displayed above the diagonal and those with type II molecules are shown below the diagonal. The diagonal itself represents self crosses and is thus a measure of reversion frequencies. In Figure 4C the recombination values for type I partial dimers are compared to the physical distances between the mutations. The physical separations, which are shown in parentheses, are expressed as a fraction (percentage) of the total size of the duplicated segment between the Hpa II site and the Taq I site. The excellent agreement between measured genetic distance and known physical distance indicates that conversion of partial dimers to monomers occurs uniformly throughout the region of homology. This result, along with the distinct asymmetry across the diagonal, strongly suggests that the mechanism of conversion involves general recombination. Other Pathways That Produce Genetic Recombinants Although recombination values for type II molecules are generally much smaller than those for their reciprocal type I molecules, they are still 1 O-100 fold above reversion levels (Figure 4B). The mechanism(s) by which these genetic recombinants are produced is unknown. One possibility is that multiple rounds of homologous recombination eventually produce wildtype from type II molecules. (At least three distinct recombination events would be required, and one of them would have to occur in the same interval as that required to produce wild-type from type I molecules.) However, for crosses of tsA28 by tsA58, whose mutations lie in the same restriction fragment (Lai and Nathans, 1975), there was no significant difference in recombination values between the reciprocal partial dimers (Figure 4B). To account for these results solely by homologous recombination, one would need to argue that multiple rounds of recombination occur as frequently as one round of recombination in A28. A58 reciprocal partial dimers, but not in other pairs of reciprocal partial dimers. Consequently, it seems unlikely that multiple rounds of homologous recombina-

General 145

Recombination

in Somatic

Cells

~A28*~84

tion are the only mechanism by which wild-type are produced from type II molecules. Another possibility is that heteroduplexes are formed during recombination and sometimes include the mutant sites. If such heteroduplexes included both mutant sites, then repair of the resulting mismatched bases in type II molecules could lead to wild-type. Cellular repair of mismatched bases in SV40 and polyoma heteroduplexes has been demonstrated previously (Lai and Nathans, 1975; Miller, Cooke and Fried, 1976; Wilson, 1977). To test whether heteroduplexes involving the mutations in Figure 46 would be repaired efficiently in our CV1 cells, we constructed and purified fully heteroduplex circular molecules, as described in Experimental Procedures. The high infectivities of these heteroduplexes at restrictive temperature (Figure 5) suggest that the mismatches are repaired very efficiently. Thus, heteroduplex formation during recombination, followed by mismatch repair, is a plausible mechanism for the production of genetic recombinants from type II molecules.

~A28

T8‘

Taq I 8228

84

A58

A28

A30

HpalI

d 6.1

0.01

C .06

8228

HpaII

84

A28 A58 A30

Taq I

410.9

37

4

(46) 45 (52)

1 31 ’ (38) Figure

4. Genetic

Crosses

.90

with 1.84 Partial Dimeric

’ I DNA

(A) One pair of reciprocal partial dimers. The 0.94 Hpa II/Bgl I fragment is denoted by a thick line and the 0.90 Taq I/Bgl I fragment is denoted by a thin line. The shaded area in the type I molecule indicates the region in which homologous recombination would generate wild-type monomers. (B) Genetic crosses with reciprocal pairs of partial dimers. The mutation contained in the Taq I/Bgl I fragment is shown across the top of the grid. The mutation contained in the Hpa II/Bgl I fragment is shown at the side of the grid. Self crosses, that is, partial dimers constructed from a single mutant DNA, are marked by heavy boxes along the diagonal. One such self cross was not tested (N.T.). 1 and 5 ng amounts of each ts partial dimer were assayed at 41 “C and compared to the plaquing of a partial dimer constructed from wildtype DNA according to the equation %W = 100% (41 “C titer of ts L-

Independence of Recombination and Viral Replication An additional consideration in an investigation of conversion of oligomers to monomers is the possible role of viral DNA replication. We have examined this question by studying the effects of mutations in the SV40 A gene, whose product is required for initiation of DNA replication (Tegtmeyer, 1972). At the restrictive temperature (41 “C) the functional A gene product is at a minimum in cells infected with tsA mutants. Under these conditions a variety of partial dimers containing two tsA mutations yielded recombination frequencies proportional to the physical distances (Figure 48). In these cases creation of a wild-type A gene must have occurred before DNA replication could proceed normally. We have extended these observations by studying recombination of partial dimers under permissive con1.84/ng)/(41 “C titer of wild-type L-l .84/ng). The grid is arranged so that results with type I molecules are above the diagonal and results with type II molecules are below the diagonal. It is not clear which of the tsA28 by tsA58 partial dimers is type I and which is type II. The physical order of tsA28 and tsA58, which map in the same restriction enzyme fragment, is unknown and cannot be determined unambiguously from these data. The results with this pair of partial dimers have been placed on the grid arbitrarily. Five plaques that formed at 41°C were picked from each cross. All but three were verified to be wild-type by reassay at 33°C and 41 ‘C. Three complementation plaques were detected: two in TB4.HA58 and one in TB228.HA58. (C) Comparison of the physical map with the genetic map generated by the 1.84 partial dimers. The top line represents 84% of the SV40 genome (4390 bp) extending from the Hpa II site c.06) to the Taq I site c.90). Open boxes indicate the regions in which the ts mutations have been mapped by marker rescue (Lai and Nathans. 1975). %W values, indicated below the structural map, are taken from the grid in (B). Physical separations calculated for mutations lying at the midpoint of each box are shown in parentheses (96 of 4390 bp).

Cell 146

A58

crosses under conditions favorable for replication was not significantly different from that produced under nonpermissive conditions (Table 2). In another experiment, cells separately infected with all the partial dimers indicated in the grid in Figure 48 were incubated at the permissive temperature for one round of infection (96 hr). After disrupting the cells, the progeny were assayed at 41’ and 33°C. The recombination frequencies, as judged by the 41X titer/33’C titer ratio, showed the same asymmetry observed in Figure 4 when reciprocal DNA pairs were assayed directly at the restrictive temperature (our unpublished observations). Thus the relative difference between reciprocal partial dimers in the production of wild-type monomers is also not dependent on normal replication.

A28

Discussion

A30

We constructed specific oligomeric molecules of SV40 DNA to investigate the mechanism of somatic cell recombination. Selective use of several enzymes enabled us to link restriction fragments from two temperature-sensitive mutants in a head-to-tail orientation to create partial dimers 1.64 genome units in length. Because these oligomers were too large to fit into a viral capsid, we could selectively assay for conversion of oligomers to packageable, unit-length monomers. Specific questions about the conversion process were answered by constructing partial dimers from selected pairwise combinations of temperature-sensitive DNAs. Because these mixed oligomers produce genetic recombinants about 500 times more frequently than do mixtures of circular monomers, they were ideal probes for studying the fate of various forms of DNA molecules undergoing recombination. Our earlier work suggested that formation of wildtype monomers from a heterogeneous mixture of oligomers occurred at many sites throughout the genome (Wake and Wilson, 1979). To confirm and extend that work a series of partial dimers was constructed from all pairwise combinations of DNA from five different ts mutants. For type I partial dimers the fraction of wild-type formed was equal to the fractional distance separating the mutations (Figure 6). This result indicates that conversion of oligomers to monomers occurs with uniform probability throughout the genome. Because this behavior is its hallmark, we conclude that general recombination is the primary mechanism for monomer formation. A secondary mechanism for production of genetic recombinants was detected using type II partial dimers, which cannot produce wild-type by a single homologous recombination event. In contrast to type I molecules, recombination values for these partial dimers showed no evident pattern with physical separation, but rather varied up and down about a mean of 0.7% (Figure 6). The significance of this value, if any, is not clear. The identity of this secondary mech-

ditions where DNA replication is unrestricted. Cells infected with five partial dimers containing defective A genes were incubated at 33°C for up to three days to permit replication to occur, then shifted to the nonpermissive temperature. The proportion of wildtype recombinants produced from type I and type II

Taq I

A. I3228

84

A58

A28 A30

WT

8228 84

HpaII

WT

B. 8228

84

A58

A28

A30

WT

8228 84 A58 A28 A30 WT Figure

5. Repair

of Mismatched

Bases

in Circular

Heteroduplexes

(A) CVI monolayers were infected in duplicate with 0.3 ng of purified circular heteroduplex DNA and incubated at 41 “C. Circular heteroduplexes were prepared from mixtures of Taq I and Hpa II linears as described in Experimental Procedures. Values shown are the sum of six 41 “C plaque counts (three assays performed in duplicate). Wildtype heteroduplexes prepared from a mixture of Taq I and Hpa II wildtype linears yielded 480 plaques per 1.8 ng. Self heteroduplexes are along the diagonal. Reciprocal heteroduplexes above and below the diagonal contain the same mismatched bases but differ in the exact relationship of the mismatched bases to the nicks at the Taq I and Hpa II sites. (B) Plaque counts for reciprocal heteroduplexes were averaged and expressed as a percentage of the plaque counts of wild-type heteroduplexes (shaded boxes above the diagonal). Numbers below the diagonal in this grid are the ratio of plaque counts of reciprocal heteroduplexes in grid (A): plaque counts above the diagonal/plaque counts below the diagonal. Because these ratios are near 1 .O (average 1 .O), we infer that the relationship of the single-strand nicks at the Taq I and Hpa II sites relative to the mismatched bases, which is the only difference between corresponding heteroduplexes above and below the diagonal, does not bias the repair.

f;;eral

Recombination

in Somatic

Table 2. Recombination

Cells

as a Function

of Viral Replication Hr at 33°C

Cross

Type

Experiment

0 %W

24 %W

48 %W

72 %W

Average

25

25

33

15

25

TA28.HB4

I

1

T84.HA28

II

1

0.94

1.3

0.97

1.1

TA58.HA28

1

0.44

0.51

1.4

0.34

0.69

TA28. HA58

1

0.65

0.54

1.1

0.88

0.82

2

0.65

0.88

0.54

1.3

TA30.HA58

I

1

1.2

2.2

3.2

2.8

2

2.1

2.3

1.5

4.4

2.5

CVl monolayers were infected with 1 or 5 ng of the indicated partial dimers (in triplicate) or with 0.2 ng of wild-type partial dimers (in quadruplicate) and then were incubated at 33°C for 0.24,48 or 72 hr before being shifted to 41 “C. The recombination values are expressed as %W, which was calculated as described in the legend to Figure 4. In all cases %W was calculated by comparing plaque counts of mutants and wild-type partial dimers that had been treated identically. Because the physical order of tsA28 and tsA58 is unknown, reciprocal crosses with these mutants cannot be designated as type I or type II.

anism is unknown. One possibility is that during recombination heteroduplexes form which span the mutant sites and the resulting mismatched bases are sometimes repaired to wild-type. This process is thought to underlie the phenomenon of gene conversion commonly observed in meiotic recombination (Hastings, 1975; Radding, 1978). Whatever its explanation, this secondary mechanism appears to approach the primary mechanism in importance at recombination values of about 1%. To test whether mismatch repair was a reasonable possibility, we studied the infectivity of purified circular heteroduplexes formed from all pairwise combinations of five ts mutations. Their very high infectivities at 41 “C suggested that our CVl cells repair mismatches quite efficiently. The lower infectivity of heteroduplexes containing the closely spaced mutations, tsA28 and tsA58, appears to be characteristic of close mutations (W. Ft. Lacewell and J. H. Wilson, unpublished observations). A mismatch repair mechanism, which could account for such observations, has been treated theoretically by Warner, Fishel and Wheeler (1979), who have suggested it as the mechanism for genetic recombination of bacteriophage S13. Neither the primary nor the secondary pathway seems particularly sensitive to the level of viral DNA replication. At the restrictive temperature (41 “C) cells infected with tsA mutants produce very little functional T antigen, an essential viral product required for SV40 DNA replication (Tegtmeyer, 1972). Under these conditions type I partial dimers containing two tsA mutations recombined as expected from their physical separation (Figure 4). This level of recombination, as well as the low level of recombination with type II partial dimers, was not increased by preincubating the infected cells for up to three days at 33°C conditions under which viral DNA replication proceeds unrestricted (Table 2). Thus our recombination values show no dependence on functional T antigen over the

Physical Distance (%I Figure tance

6. Correlation

between

Physical

Distance

and Genetic

Dis-

%W values for 1.84 partial dimers. type I (0). and type II (0). are taken from Figure 48. The line is drawn to illustrate the expected correlation if genetic distances equal physical distances.

range we have been able to test. However, because of unanswered questions about the “leakiness” of tsA mutants, analogous experiments with deletion mutants are required to settle finally the question of whether recombination can proceed in the complete absence of functional T antigen. The apparent lack of a requirement for viral DNA replication indicates that the recombination detected with partial dimers is different from another kind of “recombination” involving SV40 DNA, namely, the formation of free viral genomes from SV40 DNA integrated into the chromosomes of transformed cells. At 41 “C fusion of permissive cells with nonpermissive cells transformed by the tsA28 mutant produced at

Cell 146

least 100 fold fewer free SV40 genomes than those produced by fusion with cells transformed by the wildtype virus (Botchan, Topp and Sambrook, 1979). A similar dependence on T antigen was observed for the excision of integrated genomes of the closely related virus polyoma (Basilic0 et al., 1979). In contrast to the recombination of partial dimers, these events involving integrated genomes are extremely sensitive to the level of functional T antigen. The data presented in this paper demonstrate that specific oligomeric molecules of SV40 DNA generate wild-type recombinants primarily by a mechanism that has properties consistent with the expectations of general recombination. These molecules therefore constitute sensitive probes for investigating the details of general recombination in cultured mammalian somatic cells. Experimental

Procedures

Cells and Viruses Procedures for growth of the established monkey kidney CVl cell line have been described (Wilson, DePamphilis and Berg, 1976). The temperature-sensitive 8s) mutants of SV40 were obtained from P. Tegtmeyer and R. Martin. Mutants used in these experiments were tsB226. tsB4. tsA28. tsA30. hrBCl906tsA28, hrBCl906tsA30 and hrBCl906tsA58. Double mutants containing the host-range mutation hrBCl906 (Wilson et al., 1976) and a tsA mutation were constructed previously. The host-range mutation does not alter plaquing efficiency on CVl cells and its presence was disregarded in these experiments. The wild-type strain was Rh911 a. Construction of 1.84 Partial Dimeric DNA Circular wild-type and ts DNAs were digested for 30 min either with Hpa II (at 37°C) in 20 mM Tris-HCI (pH 7.5), 7 mM MgClr. 2 mM fimercaptoethanol, or with Taq I (at 68°C) in the same buffer adjusted to 0.1 M NaCI. The linear DNAs were subsequently treated with bacterial alkaline phosphatase after adjusting the NaCl concentration to 50 mM. The phosphatase was inactivated by heating twice to 90°C for 2 min in the presence of 10 mM EDTA. The DNAs were then digested with Bgl I under the same conditions as those for cutting with Hpa II. Equimolar amounts of pakwise combinations of mutant DNAs were mixed and ligated overnight at 0°C using T4 DNA ligase in 66 mM Tris-HCI (pH 7.9). 10 mM MQCI.. 10 mM dithiothreitol and 0.2 mM ATP. Agarose (Bethesda Research Laboratories) gel electrophoresis on horizontal slabs was performed in Tris-acetate buffer [40 mM Tris, 5 mM NaOAc, 1 mM EDTA (pH 7.9)] containing 1.7 pg/ml ethidium bromide. Extraction of the 1.84 partial dimers from the gel was performed as described (Wake and Wilson, 1979). Construction of Heteroduplexes Supercoiled DNAs of wild-type and ts mutants were isolated from preparative agarose gels. About 80% of the purified molecules were supercoiled and 20% were nicked circles. Half of each DNA sample was cut with Taq I and the other half was cut with Hpa II. The Taq Icut linear form of each DNA was mixed in pairwise combinations with each Hpa II-cut linear form. The mixtures, which ranged in concentration from 5 to 12 pg/ml, were denatured in boiling water for 10 min. then reannealed at 66°C for 4 hr. All mixtures yielded roughly equal amounts of circular heteroduplexes and linear homoduplexes (Wilson, 1977). Circular heteroduplexes were isolated from agarose gels. Recombination Assays DNA plaque assays ware performed as described The amounts of DNA assayed per plate are given figure legends.

(Wilson, 1978). in the table and

Acknowledgments We acknowledge the technical assistance of Kathleen Marburger. We thank Henry Epstein, Ray Fenwick and Mike Mann for critical comments on this manuscript. This work was supported by research grants from the U. S. P. H. S. and the National Foundation-March of Dimes. J. H. W. is a Research Career Development Awardee. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

May 1, 1980

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