Intrachromosomal recombination mediated by papovavirus large T antigens.

JOURNAL OFVIROLOGY, June 1990,p. 2958-2966 Vol. 64, No.6 0022-538X/90/062958-09$02.00/0

Copyright © 1990, American Society for Microbiology

Intrachromosomal

Recombination

Mediated

by

Papovavirus

Large T

Antigens

LUC ST-ONGE, LOUISE BOUCHARD, SYLVETTE LAURENT,AND MARCEL BASTIN* Department ofMicrobiology, University ofSherbrooke, Sherbrooke, QuebecJJH5N4, Canada

Received 8December1989/Accepted 18 March 1990

Toinvestigate the mechanism by whichthelargeTantigen(T-Ag) ofpolyomavirusandsimian virus 40 can promote recombinationin mammaliancells,weanalyzed homologousrecombinationeventsoccurringbetween two defective copies of the polyomavirus middle T (pmt) oncogene lying in close proximity on the same chromosome in a rat cell line. Reconstitution of a functionalpmt geneby spontaneousrecombinationoccurred at a rateof about2 x 10-7per cellgeneration. Introduction of thepolyomavirus largeT (plt) oncogene into the cell line by DNAtransfection promoted recombination veryefficiently, with rates in therange of10-1 to 10-2percell generation.Recombination was independent of anyamplification of viral sequences andcould even be promoted by the large T-Ag from simian virus 40, which cannot activate polyomavirus DNA replication. To explain the role of large T-Ag, we proposea novel mechanismofnonconservative recombination involving slipped-strand mispairing between the twoviral repeatsfollowedby gaprepair synthesis.

Intrachromosomal recombination, that is, recombination between sister chromatids or between repeated sequences on a single chromatid, can play an important role in both gene expression and genome evolution in eucaryotes (30, 51). It is thought to involve both reciprocal and nonrecipro-cal modes ofexchange, although rigorous proof is lacking because of the inability to recover all products of recombi-nation (e.g., see Bollag and Liskay [6]). Gene conversion, one of the nonreciprocal modes of exchange, could be

responsible for maintaining sequencehomogeneity in multi-gene families and for transferring information between re-lated genes, at least when viewed on an evolutionary time scale (3, 18). It is also the process that operatesduring B-cell ontogeny to generate diversity within the chicken immuno-globulinlight-chain gene (24, 35, 49). In mammals,

recombi-nationinimmunoglobulin genes could involve another form ofexchange, such asunequal crossing over between sister chromatids (50, 52).

Despite substantial progress in understanding recombina-tion in bacteria, little is known about the process in eucary-otes, especially about cellular factors that promote

high-frequency intrachromosomal recombination. Recent studies onpapovavirusDNAreplication have suggested that one of the virally encoded proteins, the large T antigen (T-Ag), is associated with a DNA helicase activity and that the protein isresponsible for the duplex-unwinding activity that is likely to initiate replication (13, 42). Large T-Ag is known to promote phenomena such as amplification and excision of integrated viral sequences (7, 9, 27, 31), but the mechanism

leading to homologous recombination is still unclear.

Ac-cordingto the "onionskin" model (7), initiation of replica-tionat agiven proviral locus could result in multiple rounds of DNA synthesis so as to form a localized onionskin of

amplified sequences. This aberrant, polytenic structure could then represent afavorable substrate for homologous recombination, leading to excision or amplification.

Alterna-tively, large T-Ag could have a recombination-promoting

activity per se,independent of replication (8, 10, 12, 19, 33). To address this question, we have developed a system to

analyze homologous recombination events occurring

be-*Correspondingauthor.

tween two copies of the polyomavirus middle T (pmt) oncogenelying in close proximity on thesame chromosome in cultured ratcells. We reporthere that the polyomavirus large T-Ag can promote recombination between the viral sequences veryefficiently when the latter carry a functional origin of viral DNA replication. Furthermore, the simian virus 40(SV40) large T-Ag,which does not activate replica-tion at the polyomavirus origin, promotes recombinationas

efficiently as does the polyomavirus large T-Ag. We show that, contrary to the onionskin model, homologous recom-bination canoccurindependently of any amplification of the viralinsert, and we suggest that the role of large T-Ag isto melt and unwindthe DNA at the viralreplication origin, so as to create afavorable substrate forhomologous recombi-nation.

MATERIALS AND METHODS

Plasmids and mutants. pMT97.484 (see Fig. 1) encodes two defective copies of the pmt gene. The first copy was obtained by producinga 30-base-pair (bp)deletion between nucleotides (nt) 1366 and 1397 in the middle T-Ag-coding sequenceso as toinactivateitstransformationproperties (2). The second copy isaPstI-HindIIIfragment (nt 484 to1656) lacking thepolyomavirus origin of replication, the promoter, and the sequence coding for the first 104 amino acids of middle T-Ag. Bothcopies were separated by 3.5 kilobases

(kb)of pBR322 DNA(HindIII-PstIfragment,nt29 to3608). pneo-LT1 (seeFig. lb) carriesbothneoand the plt gene (8). Thisplasmidwasobtained by inserting the BamHI insert of pPyLT1, thelargeTplasmid constructed by Zhuet al.(54), intopSV2neo (41). pneo-LT97 carries bothneoandLT97,a pltmutantgenewitha30-bpdeletion (nt 1367to1396). This mutant is deficient in the initiation of viral DNA synthesis (2). pneo-SV2 carries both neoand the SV40 largeT gene. The sequences coding for large T-Ag were provided by mutantA2005, which does not produce small T-Ag. Details onthis constructhave beenpublished previously (1).

Cells and cultures. All cells were grown at 37°C in Dul-beccomodified Eagle medium supplemented with 10% fetal calfserum. ThepMT97.484 construct was transfected with

hygro (CG214 x SVHy; agift from C. Gdlinas) into mono-layers of FR3T3 cells, and colonies of hygromycin-resistant 2958

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HOMOLOGOUS RECOMBINATION BY LARGE T-Ag 2959

(a)

PstI

(b)

Pvull _neo BamHI on Ssti

I

I

V

I

PI,

Sstl BamHI

I

I

pneo-LT1

FIG. 1. (a) Structure of the plasmid used in the construction of the Hy2 cell line. pMT97.484 contains two defective copies of thepmt oncogene(boxes) in the same orientation. The first copy (pmt-dll) has a 30-bp deletion (dll) around theSstIsite at nt 1373. The second copy

(pmt-d12)lacks the 5'portion of pmt up to thePstIsite at nt 484. Notethe presence of the intron inpmt-dl2. Polyomavirus sequences (thick linesandboxes) are separated by 3.5 kb of pBR322 DNA (pBR) on one side and by 3.3 kb of pAT153 DNA (pAT) on the other side. pBR and pAT sequences arein opposite orientations in the plasmid. Homologouspmtsequences are represented by black boxes. (b) Structure of theplt plasmid. pneo-LT1carriesboth theneogene and the genecoding for large T-Ag. The structure shown is inserted between theBamHI andPvuII sitesof pAT153 (not shown). The position of the deleted intron inpltis indicated by the triangle.

cells, designated Hyl, Hy2, Hy3, etc., were isolated. All of the cell lines isolated in this manner exhibited a normal

phenotypeindistinguishable from that of the parental FR3T3 cellline. After multiple passages in the absence of hygromy-cin, the cells retainedresistancetothe drug, indicating that the transgene wasstably integrated into the cell genome (37).

About half of the cell lines were able to reconstitute a

functionalpmt geneby homologous recombination following retransfection with plt. The rate of spontaneous transforma-tion was determined by the fluctuation test of Luria and Delbruck (23). Parallel clonal populations were grown to

confluence in either 6- or 15-mm Linbro microplates, i.e.,

sufficientlysmallpopulations so that no transformants would be observed in a significant proportion of cultures. The

mutation rateis given by the expression a =

(-lnPO

ln2)/

(Nf-Nd)

per cellgeneration, where

P0

is the probability that nomutationwill occur and Nf and

Ni

arethefinal and initial numbers of cells, respectively.

Tointroducelarge T into Hy2, we retransfected the cells withplasmids of the pneo series (e.g., LT1 and

pneo-SV2) and carried outneo selectionas describedpreviously

(8).

Analysis of middle and large T-Ags. The procedures for radiolabeling of cells with[35S]methionine,T-Agextraction, andimmunoprecipitation have beenpublishedin detail

pre-viously (38). The T-Ags were released from protein A-Sepharose by boiling for2min in 50

ALl

of dissociation buffer andwere purifiedfurtherbya secondimmunoprecipitation.

Analysis ofintegrated sequences. The arrangement of the

plasmidsequenceswithin thecellularDNA wasanalyzed by

Southernblotting(40). The DNAwasdigested byrestriction endonucleases and fractionatedbyagarosegel

electrophore-sis. The fragments were transferred onto nitrocellulose pa-perandhybridized to 32P-labeled probes.

RESULTS

Strategyofdetecting homologousrecombination. The strat-egyweusedtoanalyze homologousrecombination involved theconstruction ofratcelllinescarryingtwomutatedcopies

of the pmt oncogene lying as direct repeats on the same chromosome. The recombination substrate, pMT97.484, is shown in Fig. la. It was designed such that homologous recombinations across some ofthe repeated sequences re-constitute a functional transforming gene and convert the cells from the normalto the transformed state. pMT97.484

was introduced into ratFR3T3 cells by cotransfection with

hygro, and colonies of hygromycin-resistant cells,

desig-nated Hyl, Hy2, Hy3, etc., were isolated and established into cell lines. All of the cell lines established in thismanner exhibitedanormal phenotypeindistinguishablefrom that of FR3T3. Three ofthem, Hy2, Hy3, and Hy5, were selected for furtheranalysis. Characterization of both Hy3 andHy5

will be presented elsewhere (L. Bouchard, unpublished data). In Hy2, the recipient cell line used in the present

study, thepmt-dll copy was interrupted between theAvaI site (nt 1016) and the AccI site (nt 1500), presumably as a result of the integration of pMT97.484 into the cellular genome. However, as shownbelow, Hy2was able toform an intactcoding region for the

polyomavirus

middle T-Ag,

and the particular structure of its insert facilitated the detection ofhomologous recombination by Southern

blot-ting.

Effectofpolyomavirus large T-Ag. Toassess whetherthe

m

0

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2960 ST-ONGE ET AL.

TABLE 1. Activity ofrecombinant

Plasmid Coding

transfected capacity

pSV2neo neo

pneo-LT1 neo+plt

pneo-LT97 neo+plt(d197 mutant)

aColonies of G418-resistant cellswerepicked andt

Linbro microplates.The cultures wereobservedevery

changes. About half of the transformants appeare reached70%confluence (16 to 17 cellgenerations). T within the next 10days.

polyomavirus large T-Agcouldinduce rec viralinsert, we retransfected Hy2 with the neo and examined the resultingG418-resi morphological changes. The cells hadnorn the time of selection in G418. However, appeared during propagation of the col

About half of the lines establishedbytram (neoand plt) became transformed within confluence in 15-mm Linbro microplate

spontaneoustransformants appeared unde tions of transfection with pSV2neo (ne pneo-LT97, a plt mutant defective in the DNA synthesis (2). Furthermore, no

trn

observed when thelargeT construct wasit

originalFR3T3 cell line.

Toverify that the transformants expres wepicked six foci at random, subcloned th them by immunoprecipitation after lal

[35S]methionine.

The 56K middleT-Ag

al

allof thetransformants (Fig.2). The parer produced apolypeptide of about 53,000 d recognized by the polyomavirus anti T-remainedvisible in the Hy2transformants tide has not been detected before in F derived cell lines. It is, presumably, a t middle T-Ag produced by the resident pm

CY) LO OM C\M C\

>1 >11 >1 3

i-1 I

T-C\M C\M C\

'I- >-, >1

im

Kd

92

68

slilm.o...

Wlt-43

28

18

FIG. 2. Analysisof middle andlarge T-Ags

Cell lines of the Hy2LT series were isolate colonies by transfecting Hy2 with pneo-LT]

middle T-Ag (MT), large T-Ag (LT), and tl

indicated. Kd, Kilodaltons.

plasmids Although thesixcelllinesexpressed G418resistance, the

No. of transformed

100K

large T-Ag was barely detectable in only two of them. cultures/no. of A possible explanation is that large T could reconstitute a culturesa(%) functional pmt gene evenwhenexpressedatverylow levels

0/75(0) orthat, in theabsence ofselection,the cellstended to lose 40/77 (51.9) the

ability

to express the gene. We verified that the trans-0/52(0) formants had stably integrated the transfected

plt

gene into the genomic DNA (see Fig. 3C). Furthermore, all of the transferred into15-mm representative transformants analyzed showed the

proper-48 h formorphological

d before the cultures ties characteristic ofpolyomavirus transformation, such as 'he other halfappeared growth in soft agar and

production

oftumorsin animals(data

notshown).

Recombination in the viral insert. To

analyze possible

ombination in the changesin thestructureof theinsert,weisolated DNA from plt genelinkedto a number oftransformants that had presumably sustained istant colonies for recombination and examined itwith Southern blots

hybrid-nalmorphologyat ized to apmtprobe. The DNA from transformantsexhibited transformed cells major changes in restriction patterns (Fig. 3B and C). The [onies in culture. most obvious one was an amplification of the viral insert sfecting pneo-LT1 which occurred in 10 of 27 different cell lines analyzed. This 10 days following amplification was not surprising because it is well known s (Table 1). No that large T-Ag can initiate DNA synthesis at the viral origin

-rthe samecondi- evenwhen the DNA is

integrated

and when thecellsare

only

o without

plt)

or semipermissivefor viral DNA

replication

(7, 31). Allof the

initiation of viral transformants analyzed exhibited a 2.2-kb

BamHI-HindIII

ansformation was fragment which was

produced

as a result of

homologous

ntroduced into the recombination in the insert (see map in Fig. 4). Digestion by

;sed middleT-Ag, SstI

yielded

a

742-bp fragment

which was not detected in

iem, and analyzed

Hy2,

the

parental

cell line

(Fig.

3C).

This

fragment

was beling cells with produced

by

reconstitution of the SstI site in the

pmt-dll

ppeared clearly in copy. Both 2.2-kb and

742-bp fragments

were

amplified

in

rital cell line, Hy2, some cell

lines,

suggesting

that

amplification

could occur

ltalt cellhline

H afterrecombination. Some transformants had sustained

re-serums andh

which combination without any evidence of

amplification

(data

not

Such a polypep-

shown).

However, one could argue that the

amplified

se-'R3T3 or FR3T3- quences were lost following homologous recombination.

runcated form of

Effect

of

SV40

large T-Ag. To determine the role of large

t-dll

copy. T-Ag in recombination, we attempted to obtain evidence of

recombination

by using

a

large

T

totally

defective in viral DNA

replication.

dl97, a

polyomavirus large

T mutant unableto initiate viralDNA

replication,

was also unableto

J _J promoterecombination in Hy2 (see

above).

However,

sur->~

>

~

prisingresultswereobtained whenHy2wastransfected with I I a plasmid encoding the large T-Ag from

SV40.

Here, selec-tion for recombinaselec-tion couldnotbe

applied

because the cells

L-T

acquired

the

morphology

ofSV40transformants as soon as

they expressed T-Ag. Although the SV40 T-Ag does not activate thepolyomavirusoriginof

replication

(5),it recom-MT bined the

Hy2

insert so

efficiently

that the

products

of

53K

_recombination could be observed in the cell

population

53K without any selection. Figure 4A shows the appearance of new restriction fragments in a transfectant (SV5) grown to ca. 108cells, i.e., ca. 27 cell

generations

after the introduc-tion oflargeT. On the basis of band

intensities,

we deter-mined that the cells that had sustainedrecombination repre-sented about 25% of the total cell population. This value

corresponds to a recombinant rate of 1.5 x

10-2

per cell

generation

(M.

Bastin, unpublished

data). Ofeightdifferent SV40 transfectantsanalyzed,five exhibitedrecombination in the viral insert, while three did not. Similar results were intransfected cells. observed with

plt

(see above), since only half of the trans-d as G418-resistant fectants yielded foci of transformed cells. The reason for this l. The positions of is unclear. It is unlikely due to a lack of T-Ag expression

he 53K protein are because the SV40 transfectants

expressed

the transformed

phenotype. We are currently

investigating

the

possibility

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HOMOLOGOUS RECOMBINATION BY LARGE T-Ag 2961

A

H1.-`

0

B

!l!iC-~L: f{'.CLTI3r,! 4:

k

-= = _ _

,

o.

_ _ _ _

5 _ 1>;>

:

_

''3

= _ _

-__

E _ _

-rrx

! t.

__

_

PS

SH

P

L..LI

1

B PS

oriW

HS

II

1-804- I-1229 t

-2430---1.9 1 3.6

1.11. -F-I.1 1

FIG. 3. Southernblotanalysis of Hy2 cells(top)andmapof theviral insert (bottom). (A)Parental Hy2 cell line. The 1,229-bp fragment

isproduced by cutting the BamHI site atthe pAT-polyomavirusjunction (nt4632) and the SstI site (nt 569) inpmt-dll. The804-bp SstI

fragmentoriginates from thepmt-d12copy.ThepresenceofaPstIfragment of 3.6 kb indicates that the pAT153sequenceis intactinHy2and

that thepBR322sequenceis interrupted. The 1.9-kb fragmentcontainspmt-d12andaportion of pAT. (B) Two of the transformants isolated

asG418-resistantcolonies by transfecting Hy2 withpneo-LT1. A similar analysis of Hy2 before transfection is shown in Fig. 4A. Note the

presenceofanamplified 2.2-kb BamHI-HindIII fragmentin both transformants. (C) Analysis of transformants bySstI.Reconstitution ofthe

SstI site(nt 1373) inpmt-dll by homologous recombination producesafragment of742bpinsteadof804bp becausepmt-dlllacks the intron

(seemapfordetails). The 2,968-bp fragmentcomesfrompneo-LT1 (nt 1373to4341)since ithybridizestoaspecificpltprobe (nt 1656to3918)

(notshown). Fragment sizesareindicated in basepairsorinkilobases. Abbreviations: B,BamHl;H, Hindlll;P,PstI, S,SstI.

thatonlyasubfraction of Hy2 cellscarrythe viral insert ina

configurationthatallows homologous recombination. To determine whether the viral insert could recombine spontaneously, we usedaprocedure baseduponthe

fluctu-ation test of Luria andDelbruck (23). Parallel clonal

popu-lations of Hy2 cells were grown in 15-mm Linbro

mi-croplatesandobserved formorphological transformation for

up to 4 weeks following confluence. Only 1 of96 cultures analyzed becametransformed, correspondingtoa

recombi-nationrate of about 2 x 10-7 percell generation.

To determine how T-Ag rearranged the viral insert, we

subcloned six differentSV40 transfectants after 30to35 cell generations and mapped the rearranged inserts in four dif-ferent subclonesfromtwocell lines aswellasinfourother

cell lines. In the parental cell line, BgIII, a noncutting enzyme,producedasinglefragmentof13 kb(Fig. 5A).After

recombination, the fragment was 19 kb. The DNA was

further analyzed with different restriction enzymes that cleaveonce(suchasBclI; Fig.5A)orseveraltimes(suchas

BamHI and HindIII; Fig. 4C) within the insert, and the structure depicted in Fig. 4 was obtained for all of the recombinantsanalyzed.Therearranged insertcontainedthe

entire viralsequencepresentinHy2withaduplicationof the

sequencebetween the tworepeats,resultinginthe addition of 5.6 kb of DNA. Astrikingfeatureof thisstructure isthat it can be obtained by unequal sister chromatid exchange. However, unequal recombinationbetweenchromatids(Fig. 6)shouldyieldtwodifferentproducts(panelsbandc), each segregating into different daughter cells. In the absence of selection fora particular product, onewould expect to find

aninsertwith adeletion foreveryinsert withaduplication.

The insert with a duplication was detected in Fig. 4A.

However, fragments characteristic ofarecombinant with a

deletionwere neverdetected inouranalyses (seethelegend

to Fig. 6 fordetails). We conclude, therefore, that reconsti-tution of thepmtgene in the Hy2cell line did not occurby a simple mechanism such as unequal sister chromatid

ex-change.

Propertiesofreplication-defective mutants. In view of the roleoflarge T-Aginviral DNAreplication,itwasofinterest to examine the effect of replication-defective mutants on

recombination. To thisend, Hy2cellswere transfected with fourSV40mutantsthat havesingle-amino-acidsubstitutions resulting in altered specific DNA binding, ATPase, and/or

C

* . ,

I

Im}4

probe

mn I I I i -~~~~~~~I

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2962 ST-ONGE ET AL.

Ar

TI

n= 11 1

be 0

B

-- sitIl--I -7 to

S%R SV4 SV4

C

kb

8 1]I--- 81- 11 8r 1 --11

i3

I---]1 T3W}I I 3) 1 1311 1 i 131 1

':

*b* %*

56 eIhm.

~~ ~' %#h b

Hy2

HB H

B

HB

I1...

,'

I.

I

.

I

} --- --- 4.9 - 7.4

W- 2.0 ---+--- 6.7

--1.6 --I 3.5

SVI

-SV7-SV3

HB

H

I

ru8 I

4.9

F- 2.0 5.5

F-1.6 -1

ixw x !

B

Iori

L

I

H

B

HB

742l

orisL

5.6

--

7.4 - I

t

6t7 i

FIG. 4. Southern blot analysis ofSV40 transfectants. Cell lines designated SV1, SV2, SV3,etc.,wereisolatedasG418-resistantcolonies

by transfecting Hy2 with pneo-SV2,aplasmid carrying bothneoand theSV40 large Tgene(1). (A)The DNA designatedSV5wasextracted fromapopulation ofca. 108 cells, i.e.,ca. 27cellgenerations after the introduction of large T into Hy2 (cell lineSV5). Note theappearance

offragments of 5.6, 5.5, and 2.2 kb whichwerenotdetected in Hy2. (B) Analysis bySstI. The 742-bp fragment is barelydetectable after 27 cell generations. However, after 62 cell generations, the 742- and 804-bp fragments are present in virtually equal amounts. (C) SV40 transfectantswereanalyzed after subcloning. Hy2,Structure of the insert inHy2before recombination.SV1-SV7-SV3, Structure of the insert afterrecombination. The duplication comprisesarepeataswellasthesequencebetween thetwo repeats.Inadditiontothefragments shown, thelocations of various sites, such asPstI, SstI,andAccI, wereconfirmedonthemaps(notshown).

unwinding activities of largeT-Ag (25, 26,43). The

proper-ties ofthe various mutants are listed in Table 2. Since the

mutants were transformation competent, about half of the G418-resistant colonies hada transformed morphology and

thus provided a source of Hy2 clones expressing mutant

T-Ags. DNA was extracted from a total of41 such clones

grown to 25 to 26 cell generations and was subjected to Southern blot analyses similar to those shown in Fig. 4. However, none of themutants wasableto promote

recom-bination in the viral insert (Table 2). This result indicated that, as with polyomavirus large T-Ag, recombination

re-quired an SV40 T-Ag functional in viralDNA replication.

Requirement for a functional origin of replication. To

determine whetherrecombination in the Hy2 insert required the polyomavirus origin of replication, we constructed a

plasmid similartopMT97.484 butwithanonfunctional origin

ofreplication. The replication defect in the construct was

introducedby the deletionof 23 bp in the origincoreand the

addition ofanXhoI linkerat nt 37 to 60 (4). Although this mutation inactivated the replication origin, it did not affect expression from the polyomavirusearly region. Six indepen-dent cell lines carrying one copy or several copies ofthe

construct were established by cotransfection with hygro. However, in no case did retransfection with polyomavirus

largeTandneoleadtothereconstitution ofafunctionalpmt

geneby homologousrecombination(datanotshown).These resultsindicate that the large T-Agcanpromote recombina-tion between the viral sequences only in thepresence ofa

functional originofreplication.

DISCUSSION

Homologous recombination promoted by large T-Ag. We havedevisedasystem to analyze intrachromosomal recom-bination between two copies of the pmtoncogene lying in

close proximity on the same chromosome in cultured rat cells. The advantageofrecombiningpmtsequences instead

ofneo(36, 48) or tk(22) is that oncogenically transformed

cells are readily detected in culture and can be isolated

without selectivekilling by cytotoxicdrugs. Thissystemhas enabledustoanalyzethestateoftheviral insert in the whole population oftransfected cells and to detect recombination events in the absence of any selection. Recombination

promoted by large T-Agoccursatveryhigh frequencies.The

0.0

Oka 6hw

'k:*

* I*

%.46 *lW

wz'o

-i:A 4'A

.4:

B: BamHI

H:

Hindill

:

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HOMOLOGOUS RECOMBINATION BY LARGE T-Ag 2963

(a)

~~~~I 1

A

i 1;

I11- isI

I3-11

K.%v0

(b)

JML

B

(c)

13(1-I Bcll 13cil Buill

L1~J

-1(-ilI

L-LII)iCltion

13c11 BclI

I ,

1.

I~

-FIG. 6. Diagram showing how unequal sister chromatid

ex-change (a)producesachromatid withadeletion (b) andachromatid

withaduplication(c).The small vertical barsrepresentHindlllsites

inthe cellular DNA.Chromatid b should yieldaHindlIl fragmentof

3.2kb.

Bcll Bglll

.0--- 4

FIG. 5. (A)Analysis of SV40 transfectants by BglII(anoncutting enzyme) and BclI (a single-cut enzyme). The cell lineswere

sub-clones after 30cell generations. (B) Arrangement of the viral insert inHy2 asdeduced from panel A. Top, Hy2 before recombination.

Bottom, Hy2 after recombination. Black boxes represent thepmt

repeats.Thesingle BcIl site is locatedat nt5051onthe polyomavi-rusmap.

appearance ofnewrestriction fragments in the experiment

shown inFig. 4A iscompatible witharecombinationrateof 1.5 x 10-2percell generation. Considering that the rate of spontaneousrecombination intheparental cell line is about 2 x

10',

recombination is promoted by large T-Ag by

severalorders ofmagnitude. Another study has shown that the transfer ofplt into ratfibroblasts induces anincreased

frequency of homologous recombination events and the

appearanceof abnormalkaryotypes(19). Thesame phenom-enonhas beenobserved withc-mycandfunctionally related oncogenes, and it has been suggested that immortalizing

oncogenes may contribute to the transforming process by

increasing genomic instability (10).

Theeffect ofSV40 onthepolyomavirus insertwas

unex-pected. Although the large T-Agsfrom both polyomavirus and SV40 recognize and bind to the same DNA sequence

motif in vitro(34, 39), theycannotfunctionallysubstitute for

oneanothertopromoteviralDNAreplication (5). Itislikely thatpolyomavirusDNAreplication requires species-specific permissive factors suchasDNApolymerase a-primasefrom

murine cells or proteins associated with this complex (28, 29). Interestingly, a recent studyhas shown that polyoma-virusDNAreplicationinmonkey cellsisnotunconditionally

limited. DNAs containing the polyomavirus origin replicate

efficiently in monkeycells which constitutively express the SV40large T-Ag (46).

Our results are incontradiction with several reports claim-ing that recombination within transfected plasmid DNAs does not depend on T-Ag expression (16) or that neither DNAreplication of the recombination substrate nor T-Ag is essential for recombination (44). The reasons for such dis-crepancies are not understood at present. They may arise from differences in the cell lines used or in the nature of the

experimental designs, such as recombination within trans-fected plasmids versus homologous sequences stably inte-grated into the host genome.

Theadvantage ofusing a heterologous system is that the SV40 large T-Ag does not amplify the sequences at the

polyomavirus origin of replication and the recombination products can be identified easily. All ofthe SV40 transfec-tants analyzed exhibited the same recombination product, i.e., a duplication of the repeat as well as the sequence between the tworepeats.Such aduplicationcanbeexpected

[image:6.612.79.280.80.419.2]

from anevent ofunequal sister chromatid exchange. How-ever, unequal recombination between chromatids should

TABLE 2. Properties of SV40mutants

Mutanta Amino acid Origin

Recom-change binding binationb

pneo-SV2 None + + 5/8

pneo-C6-2 Asn-153- Thr - + 0/4

pneo-C8A Lys-224- Glu + + 0/11

pneo-CllA Pro-522 Ser + - 0/16

pneo-T22 His-203- Gln - + 0/10

aGenomicDNAwascleavedbyBamHIand inserted in the BamHIsiteof pSV2neo. pneo-SV2 carriesneoand thewild-typeSV40largeT gene.

bColoniesofG418-resistant cellswerepickedand grownto25to26cell generations.The DNAwasextractedandanalyzed bySouthernblottingas

described in thelegendtoFig.4.

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[image:6.612.353.523.83.307.2] [image:6.612.318.558.615.689.2]

2964 ST-ONGE ET AL.

(a)

(d)

on_

(b)

L

ori

LargeT-Ag melts andunwinds the DNAatthereplication origin

(c) break 5

-7 7 break

(e)

Single-stranded loops are produced

by slipped-strand mispairing

[image:7.612.66.545.65.215.2]

repair synthesis

FIG. 7. Model explaining the role of large T-Ag in recombination. (a) AlthoughtheSV40large T-Agcannotinitiatepolyomavirus DNA replication, itrecognizessomeelements of thepolyomavirus replication origin. (b) Large T-Agmelts andunwinds thedouble-strandedDNA

attheorigin, allowing therepeats topair with each other byslipped-strand mispairing. (c)Twosingle-stranded loopsareproduced. (d)Breaks

occuronopposite strands, resulting in largegapsofsingle-stranded DNA. (e) Repairsynthesis producesthe structurewithaduplication. yield two different products, one with a duplication and

another with a deletion, each segregating into different

daughter cells. Since the productwith adeletion wasnever

detected in our analyses, we conclude that recombination

didnot occurby unequalsister chromatidexchange.

Recombination mechanism. Several studies havereported that intramolecular recombination during transfection in mammalian cells is anonconservativeprocess. When

mole-cules bearingdirect repeatsundergo intramolecular interac-tions intended to yield reciprocal exchange, the process

generates only one of the two expected products (11). Similar conclusions were reached by Lin et al. (21), who proposedamodel by which the removal of unpairedDNAat thejunction between the paired and unpaired regions

per-mitsagaprepairprocesstoreconstructan intactgene. Our

dataareinagreementwith such anonconservative mode of

homologous recombination. To account for the observed effect ofSV40 large T-Agonthe recombination of the viral

insert inHy2, wepropose themodel shown in Fig. 7. How the SV40 protein interacts with the polyomavirus origin of replication is not clear. In vitro studies have shown that SV40 T-Ag binds to multiple discrete regions on the

poly-omavirusorigin (34, 39). It doesnotunwindDNA containing thepolyomavirus originaswellasit unwindsitsownDNA.

However, it does exhibitpartial specificity for the sequence

atthepolyomavirus origin (E. WangandC. Prives, personal communication). Whethertheinteractionismore specificin vivo remains to be seen. We propose that the function of

T-Agistodestabilize thedouble-stranded DNA attheviral replication origin so as to create a favorable substrate for

recombination. Once the two strands are separated, the

repeats are free to pair with each other by slipped-strand mispairing. Such an event would produce two single-strandedloops, eachcontainingoneof therepeatsaswellas

thesequenceslying between thetwo repeats.Slipped-strand mispairing has already been invoked as a mechanism that can generate small deletions or duplications in the genome

(20). It can explain the generation of deletions in the lacI

geneofEscherichia coli(15) andinthe,B-globingene(14). A deletion isproducedwhen the single-stranded loop is

recog-nized by DNA repair enzymes which excise the loop and rejointheends of the brokenDNAstrand. However,breaks occurringontheopposite strands would produceastructure

with largegaps corresponding to the single-stranded loops. Filling of the gaps by repair synthesis would generate the structure observed in the cell lines after recombination.

Unequal sister chromatid exchange has been directly documented inyeast(32, 45)andDrosophilaspecies (47).In mammals, it has been implicatedin the evolution of

multi-genefamilies(3) and in the rearrangement of immunoglobu-lin genes (51, 52). However, cells carrying the putative reciprocal products cannot always be isolated (e.g., see

Tilley and Birshtein [50] and Wabl et al. [53]). In one

instance, it hasbeen shown that the switch rearrangement in the mouse heavy-chain locus occurs by a mechanism of looping out and deletion, thus excluding the model of

un-equal sisterchromatid exchange (17). Whether the mecha-nism proposed here for the large T-Ag applies to other recombinationaleventsinthecellulargenomeremainstobe investigated.

Spontaneousrecombination occursin theHy2 insertata

rate ofabout 2 x 10-7percellgeneration. Other

investiga-torshavedevised similarconstructsthatrecombineatarate

onthe order of 10-6percellgeneration (22, 36).It isunclear why some of these constructs, which include a functional

originofSV40DNAreplication, donotrecombineathigher frequencies when they are introduced into COS cells that

expresstheT-Agandwhythetypeofrecombinationproduct described here has neverbeenobserved in thesecell lines. Experimentsarepresentlyunderwaytodetermine whether the SV40 origin can substitute for that ofpolyomavirus in pMT97.484. However, itseemsthat thestructureor

config-uration of the insert is very critical in determining the mechanismbywhichhomologoussequenceswill recombine inagivencell line. Since theinceptionof thiswork,wehave constructed and characterized five different cell lines

carry-ingtandemrepeats ofpolyomavirus DNA. They all

recom-bine under theaction ofpolyomavirus large T-Ag, although

notnecessarilyatthesamerateandbythesamemechanism.

In oneofthem, the insertsustains deletions either sponta-neously or in response to large T-Ag (8). In another line, whichdiffersfromHy2 by the position of thepolyomavirus origin with respect to the two repeats, the rearrangements involve both reciprocal (inversion) and nonreciprocal (gene conversion)recombinationalevents(Bouchardetal., unpub-lished data). The most striking feature of this line is that recombination can be promoted by SV40

replication-defi-cientmutants.Itseems,therefore,that differentfunctions of large T-Agmaybeinvolved inrecombination indifferentcell lines.

RoleoflargeT-Ag.Themodelwepropose mayshedsome

light on the role of large T-Ag in phenomena such as

ori

on

duplication

I I

on

m w

o.

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HOMOLOGOUS RECOMBINATION BY LARGE T-Ag 2965 amplificationand excision of integrated viral sequences from

chromosomes. Cells transformed by polyomavirus andSV40 can undergo a high rate of amplification or excision of the integrated genome, and both phenomena require a functional large T-Ag as well as the presence of homology within integrated sequences such as complete or partial tandem insertions of viral DNA (7, 27). Several mechanisms leading to excision or amplification have been proposed. Currently, the prevailing model is that proposed by Botchan et al. (7). Upon initiation of replication at a given proviral locus, multiple rounds of DNA synthesis occur to form a localized onionskin of amplified sequences. This aberrant, polytenic structure could represent a favorable substrate for homolo-gous recombination, leading to excision or amplification. In agreement with these studies, we find that recombination in Hy2 requires a large T-Ag functional in the activation of viral DNA synthesis as well as a functional origin. However, in contrast to the onionskin model, recombination can occur in our cell lines without any detectable amplification of se-quences. The relationship between recombination in Hy2 and the replicative function of large T-Ag is of special interest. If the function of T-Ag is simply to melt and unwind the DNA at the replication origin, one would expect repli-cation-deficient mutants with helicase activity (such as C6-2, C8A, and T22) to promote recombination. Our data show that they do not. Large T could also be implicated, by its replicative function, in the repair synthesis leading to the duplication of the viral insert (Fig. 7e). If repair is achieved through the interaction between T-Ag and polymerasea,it is not clear why the SV40 T-Ag is inactive in extrachromo-somal polyomavirus DNA replication and why it fails to amplify the viral insert as does its polyomavirus counterpart. An interesting possibility is that a functional T-Ag is required for the repair of large gaps and that its role is to activate polymerasea or other enzymes involved in repair synthesis. To the best of our knowledge, this possibility has not yet been investigated.

ACKNOWLEDGMENTS

We thank J. Toutant and C. Bergeron for excellent technical assistance, Y. Gluzman for SV40 mutants, C. Gdlinasfor the hygro construct, J. Hassell for the polyomavirus replication origin-nega-tive mutant, B. Schaffhausen for the gift of polyomavirus anti-T serum, and P. Bourgaux, D. Gibson, and C. Prives for fruitful discussions.

This work was supported by the Medical Research Council of Canada and by the National CancerInstitute of Canada. L.S. and S.L. were supported by the Cancer Research Society and by the Fonds de la Recherche enSantdduQudbec, respectively.

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