INDUCTION OF INTRACHROMOSOMAL RECOMBINATION IN YEAST BY INHIBITION OF THYMIDYLATE BIOSYNTHESIS

(1)

INDUCTION OF INTRACHROMOSOMAL RECOMBINATION

IN YEAST BY INHIBITION

OF THYMIDYLATE

BIOSYNTHESIS

B.

A. KUNZ,' G . R. TAYLOR AND R. H. HAYNES Department of Biology, York University, Toronto, Ontario, Canada M3J l P 3

Manuscript received October 28, 1985 Revised copy accepted July 1 , 1986

ABSTRACT

The biosynthesis of thymidylate in the yeast Saccharomyces cerevisiae can be inhibited by antifolate drugs. We have found that antifolate treatment enhances the formation of leucine prototrophs in a haploid strain of yeast carrying, on the same chromosome, two different mutant leu2 alleles separated by Escherichia coli plasmid sequences. That this effect is a consequence of thymine nucleotide depletion was verified by the finding that provision of exogenous thymidylate eliminates the increased production of Leu+ colonies. DNA hybridization analysis revealed that recombination, including reciprocal exchange, gene conversion and unequal sister-chromatid crossing over, between the duplicated genes gave rise to the induced Leu+ segregants. Although gene conversion unaccompanied by crossing over was responsible for the major fraction of leucine prototrophs, events involving reciprocal exchange exhibited the largest increase in frequency. These data show that recombination is induced between directly repeated DNA sequences under conditions of thymine nucleotide depletion. In addition, the results of this and previous studies are consistent with the possibility that inhibition of thymidylate biosynthesis in yeast may create a metabolic condition that provokes all forms of mitotic recombination.

NHIBITION of thymidylate (dTMP) biosynthesis in actively growing cells

I

results in thymine nucleotide depletion that can induce a variety of genetic

alterations

in

vitro

(for reviews, see KUNZ 1982; MEUTH

1984; HAYNES

1985;

HAYNES

and KUNZ 1986). Evidence that reduction of thymine nucleotide levels

may occur

in vivo

is based on the association between folate metabolism and

the synthesis of thymidylate. Mammalian cells cannot manufacture folate,

which is an essential vitamin and the precursor of a tetrahydrofolate cofactor

required for dTMP biosynthesis (BLAKLEY

1969; ERBE 1979; SHANE and

STOKSTAD

1985). Diminution of tetrahydrofolate pools leads to thymine nu-

cleotide depletion and produces various genetic, biochemical and clinical symp-

toms (COLMAN

1977; ERBE 1979; KUNZ 1982; KRUMDIECK 1983; SHANE

and

STOKSTAD

1985; HAYNES

and KUNZ 1986). Furthermore, folate and dTMP

'

Present address: Department of Microbiology, The University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2.

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376

B. A. KUNZ, G. R . TAYLOR AND R . H. HAYNES TABLE 1

Genotypes of strains

Strain Genotype"

BKSo-1A M A T a leu2-3,112 canl-52 his4-519 met8-1 SUP+ BKSo-1AT M A T a leu2-3,112 canl-52 his4-519 met8-1 SUP-o tup-

KT-1AD M A T a leu2-1 pBR322 leu2-3,112 c a n l - 5 2 his4-519 met8-1

KT-IATD M A T a leu2-1 pBR322 leu2-3,112 canl-52 has4-519 met8-I tup-

KT-IAS M A T a leu2-1 c a n l - 5 2 his4-519 met8-1

KT-IATS M A T a leu2-1 canl-52 his4-519 mst8-1 tup-

.,

T h e order of LEU2 alleles on chromosome 111 (Figure 1 ) is included in the genotype of the duplication strains and is as follows: centromere distal LEV2 allele; plasmid sequence; centromere

proximal LEU2 allele. As the tup- strains of the K T series are derived from BKSo-lAT, all tup- alleles are identical.

deprivation produce very similar, if not identical, types of

DNA

and chromo-

some damage

(KUNZ 1982; HAYNES

and

KUNZ

1986).

We have been using the yeast

Saccharomyces cerevisiae

as a model eukaryotic

system to study the genetic effects of antifolate drug-induced thymine nucleo-

tide

depletion. Previously, we found that inhibition of dTMP biosynthesis in

yeast is lethal, weakly mutagenic, induces

DNA

strand breakage and enhances

mitotic interchromosomal reciprocal (crossing over) and nonreciprocal (gene

conversion) recombination between homologous

DNA

regions on homologous

chromosomes

(KUNZ

et al. 1980; BARCLAY

et al. 1982;

KUNZ

and

HAYNES

1982;

KUNZ

et al. 1982;

ECKARDT,

KUNZ

and

HAYNES

1983).

More recently, we

demonstrated that thymidylate depletion also provokes mitotic unequal sister-

chromatid crossing over between reiterated ribosomal

DNA

genes and mating-

type switching between the MAT

and

HML

or

HMR

loci

(KUNZ

et al. 1984;

KUNZ, TAYLOR

and

HAYNES

1985).

These latter events are considered forms

of intrachromosomal recombination, a term applied to the transfer of genetic

information between repeated genes on the same chromosome or chromatid,

or

between sister chromatids

(PETES 1980; SZOSTAK

and

Wu

1980; JACKSON

and

FINK 1981; KLEIN

and

PETES

1981;

KLEIN 1984; HABER

and

HEARN

1985;

JACKSON

and

FINK 1985).

T h e data suggest that thymine nucleotide depletion

induces various types of intrachromosomal recombination.

To

explore such

phenomena more extensively, we have now examined the effect of inhibiting

dTMP biosynthesis by antifolate treatment

on genetic exchange between du-

plicated

LEU2

regions separated by

Escherichia coli

plasmid sequences. Recip-

rocal recombination and gene conversion, as well as unequal sister-chromatid

crossing over,

were

induced between the directly repeated

LEU2

sequences by

dTMP deprivation. We found that, although the majority of induced recom-

binants was d u e to gene conversion alone, the largest increase in recombination

frequency was observed for those events involving reciprocal exchange.

MATERIALS AND METHODS

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INTRACHROMOSOMAL RECOMBINATION

377

d T M P , a nucleotide permeable (tup-) derivative (BKSo-1 A T ) of BKSo-1 A was selected on YPDP medium containing antifolates plus dTMP, as described by LITTLE and HAYNES (1979). BKSo-1A o r BKSo-1AT were used for all constructions in this study.

Plasmids:

pYE16HI was obtained from T. D. PETES and carries the yeast leu2-1 allele. Other plasmids were constructed for this study. pGTlO consists of the pYE16HI BgZII fragment bearing leu2-1 inserted into the E. coli vector pBR322 (BOLIVAR et al. 1977) at the BamHI site. pGT12 has the YEp13 (BROACH, STRATHERN and HICKS 1979) XhoI-Sal1 fragment that carries the yeast LEU2 gene inserted at the Sal1 site of the E. coli vector pUC8 (MESSING and VIEIRA 1982).

Media:

YPDP and minimal omission media have been described previously (BARCLAY et al. 1982).

DNA

isolation:

Total yeast DNA was isolated by the mini-prep technique described by SHERMAN, FINK and HICKS (1983). Plasmid DNA was isolated by the alkaline extraction procedure of BIRNBOIM and DOLY (1 979).

Yeast transformation:

Transformation of yeast strains was carried out using lithium acetate, as described by ITO et al. (1983).

Hybridization analyses:

For colony hybridization, individual yeast colonies were suspended in 0.25 ml 0.9 M sorbitol, 0.1 M disodium ethylenediaminetetraacetate, pH 7.5, containing

0.5

mg/ml zymolyase 100,000 (Miles Laboratories) and incubated at 37" for 30 min t o induce spheroplast formation. T e n microliters of each spheroplast suspension was spotted onto a nitrocellulose filter that was air dried briefly. T h e filter was then treated with 0.5 M sodium hydroxide t o denature the DNA and was neutralized and dried, as described by SHERMAN, FINK and HICKS (1983). For DNA hybridization, total yeast DNA was digested with

BglII,

XhoI o r Hind111 endonuclease and was fractionated by electrophoresis on a horizontal

0.7%

agarose gel run at 1 V/cm. Electrophoresis was carried out in 40 mM Tris-acetate, 20 mM sodium acetate, 1 mM disodium ethylenediaminetetraacetate, pH 8.0. T h e gel was then soaked for 1 h r in 0.5 M sodium hydroxide t o denature the DNA, neutralized in 1 M Tris-hydrochloride, pH 8.0, for 1 h r and dried a t 60" under vacuum for 45 min (TSAO, BRUNK and PEARLMAN 1983). T h e dried nitrocellulose filters (for colony hybridization) o r gels (for DNA hybridization) were hybridized with "P-labeled pBR322 or pGT12 DNA, respectively. Nick translation, t o label the plasmid DNA, and hybridizations were performed according t o the methods of MANIATIS, FRITSCH and SAMBROOK (1 982). Following hybridization, the filters and gels were exposed t o Curix RP1 X-ray film at -70" using Dupont Cronex lightning plus intensifying screens.

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378

B. A. KUNZ, G . R . TAYLOR AND R . H. HAYNES

ixxB

Bocteriol vector DNA

0

leu 2 - 1 DNA

T

891 11

T

8gl I / / B a m H I

t s o 1 I

leu 2-3. i12 DNA

7

Hind Ill

-

Yeost chromosomal DNA

- Yeost wecror DNA I X h o 1

yeast leu 2-3.11zlocus

-A

Replacement w t h X h o / - S o l I frogmen? Directed integrotton ot

Xhol restrtction sife

k I I x + u

FIGURE 1 .-Formation of yeast strains having either directly repeated copies of Eeu2-I and leu& ?,112 or leu2-3,212 replaced by l e d - 1 . Following digestion with XhoI and transformation, the plasmid pGTlO integrates by homologous recombination at the XhoI endonuclease site 5’ to the leu2-3,IZZ locus on chromosome III of strain BKSo-1A or BKSo-1AT. Digestion of pGTlO with

XhoI and Sal1 before transformation results in replacement of the chromosomal XhoI-Sal1 fragment carrying leu2-3,llZ with the pGTlO XhoI-Sal1 fragment carrying leuZ-I. The structures predicted for the duplication and replacement strains were confirmed by DNA hybridization analysis, as described in MATERIALS AND METHODS. T h e XhoI and Sal1 endonuclease sites are centromere distal and centromere proximal to the LEU2 locus, respectively (DOBSON, KINGSMAN and KINCSMAN

198 1).

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INTRACHROMOSOMAL RECOMBINATION

1

2

3

4

5

0

23.7.

9.5.

6.7.

4.3.

2.3. 4 0 . 4

+

3.0

FIGURE 2.-Analysis of DNA insertions in duplication and lcu2-I replacement strains. Total yeast DNA was digested with BglIl and hybridized with a LEU2 probe, as described in MATERIALS

AND METHODS. DNA was isolated from the parental strain BKSo-1A (lane I), the duplication strains KT-IAD and KT-IATD. respectively (lanes 2 and 3), the leu2-I replacement strains KT-1AS and KT-IATS, respectively (lanes 4 and 5). T h e DNA size markers are in kilobases.

1

2

3

4 23.7-

9.5-

gl

6.7-

4.3 -

FIGURE 3.-Analysis of pGTIO integration in duplication strains. DNA was digested with Xhol and hybridized with a LEU2 probe, as described in MATERIALS AND METHODS. DNA analyzed was

pGTl0 (lane 1) or total yeast DNA isolated from the parental strain BKSo-IA (lane 2) and the duplication strains KT-1AD and KT-IATD, respectively (lanes 3 and 4). T h e DNA size markers are in kilobases.

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380

B. A. KUNZ, G. R . TAYLOR AND R . H. HAYNES TABLE 2

Segregation of spontaneous leucine prototrophs: frequencies and rates Frequency Rate" Strain Relevant genotype (X 106) (X 108)

KT-IAD l e d - 1 leu2-3,112 43.9 250 KT- 1 ATD leu2-I leu2-3,112 tup- 39.1 223

KT-I AS Eeu2-1 1.3 7.3

K T - 1 ATS leu2-I tup- 1.9 10.7

a Frequencies are expressed as events per surviving cell. Rates are expressed

as events per cell division.

had integrated at the XhoI endonuclease site adjacent to the LEU2 locus as depicted in Figure 1. However, these patterns also would have been observed if the positions of the leu2-I and leu2-3,112 alleles were switched, perhaps as a consequence of gene conversion during integration (ORR-WEAVER, SZOSTAK and ROTHSTEIN 198 1). T o es- tablish the orientation of the leu2 alleles relative to the centromere of chromosome ZZZ, total DNA from strains KT-1AD a n d KT-1ATD was digested with XhoI, ligated and transformed into E. coli strain JF1754 (TAYLOR et al. 1982) to retrieve plasmid pGTlO. Bacterial transformants were obtained using both yeast DNA samples, and plasmid DNA was isolated from these transformants, digested separately with XhoI and Hind111 and found to be 7.4 kb in size, as expected for pGT10. If the leu2 alleles were ordered in the duplication strains as shown in Figure 1, then the recovered plasmids should carry the leu2-1 allele and, consequently, be able to transform yeast strain BKSo-1 A to leucine prototrophy (see above). No Leu+ transformants would be expected if the plasmid carried the leu2-3,112 allele. T h e plasmid DNA retrieved from KT-1AD and KT-1ATD transformed BKSo-1 A to Leu+. Leucine auxotrophy in the transformants reappeared upon selection for loss of suppressor activity (see above), confirming that the plasmids carried the leu2-I allele. Given that the XhoI site is centromere distal to the LEU2 locus (DOBSON, KINGSMAN a n d KINGSMAN 1981), the order of the LEU2 alleles on chromosome

ZZZ

in the duplication strains is leu2-I, pBR322, leu2-3,112, centromere.

Antifolate treatment:

Yeast cells in the logarithmic phase of growth were suspended in YPDP broth (2 X l o 6 cells/ml) with or without d T M P (300 rg/ml) and incubated with shaking at 34" for 30 min (KUNZ et al. 1980). They were then transferred to an equal volume of YPDP broth, prewarmed to 3 4 " , containing methotrexate (100 pg/ ml) plus sulfanilamide (5 mg/ml) with or without d T M P (300 rg/ml) and incubated at 34" with shaking.

Determination of rates

of

leucine prototroph formation:

For each strain, ten tubes of YPDP broth (3 ml) were inoculated with approximately ten cells per tube and were incubated with shaking at 30" for 48 hr. T h e cells were harvested, washed three times with sterile, distilled H 2 0 ; diluted where appropriate; plated on complete minimal medium a n d leucine omission medium; and incubated for 72 h r at 30". T h e frequency of prototroph formation for each independent culture was then determined. T h e rates of leucine prototroph formation per round of replication were calculated according to the following expression (DRAKE 1970): rate = (0.4343 mean frequency)/(log mean final cell number-log mean initial cell number).

RESULTS

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381 BKSo-1

A T , having only the

leu2-3,112

allele, failed to produce Leu+ colonies

at a detectable frequency (the

Zeu2-3,112

allele has been reported to have a

spontaneous reversion frequency of

<1

Ow’*

(BROACH,

STRATHERN

and HICKS

1979)). T h e strains used in this study are isogenic except for the

LEU2

locus

and the presence or absence of the

tup-

mutation. In addition, all the nucleo-

tide-permeable strains carry the same

tup-

mutation. Furthermore, there is no

significant difference

(P

>

0.99) in the rates of leucine prototroph formation

when corresponding

TUP+

and

tup-

strains are compared. Finally, while both

the

leu2-1

and

canl-52

alleles present in the duplication strains are ochre

suppressible, the majority (585/604

=

95%) of the spontaneous KT-1 ATD

Leu+ segregants tested had not concurrently become canavanine-sensitive.

These various facts indicate that the

tup-

mutation did not influence the seg-

regation of leucine prototrophs and argue that the enhanced formation of

Leu+ colonies in the duplication strains was not due to reversion or suppression

of

leu2-1.

Intrachromosomal recombination also could produce Leu+ segregants. It has

been demonstrated in a similar system that spontaneous intrachromosomal

gene conversion occurs more frequently than events involving reciprocal ex-

change

(JACKSON

and FINK 1981), and hybridization analysis of DNA from

leucine prototrophs recovered here (see below) was consistent with this finding.

Thus, we conclude that the increased frequency of spontaneous Leu+ segre-

gants in the

LEU2

duplication strains was, in large part, a result of intrachro-

mosomal gene conversion.

Induction

of

leucine prototrophs by antifolate treatment:

The methylation

of dUMP to dTMP by thymidylate synthase in yeast (Figure 4) requires the

methyl donor

N5,N’o-methylene-tetrahydrofolate

(BISSON

and THORNER

1977;

TAYLOR

et

al.

1982). As

S .

cerevisiae

lacks the enzyme thymidine kinase (GRI-

VELL

and

JACKSON

1968) and the thymidylate synthase reaction constitutes a

significant drain on intracellular tetrahydrofolate pools (BLAKLEY

1969; ERBE

1979; SHANE

and STOKSTAD

1985), dTMP biosynthesis can be blocked by

limiting the supply of reduced folates. Treatment of yeast cells with the anti-

folate drugs methotrexate plus sulfanilamide effectively causes dTMP depletion

(KUNZ

et

al.

1984). When logarithmic phase cells of the

LEU2

duplication strain

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382

B. A. KUNZ, G. R . TAYLOR AND R . H . HAYNES

2-AMlN0-4-HYDRoX Y-

6-METHYLPERIN PARA-AMINOBENZOATE

GLUTAMATE

FIGURE 4.-A scheme for the inhibition of thymidylate biosynthesis by antifolates. Thymidylate synthase methylates dUMP to dTMP and NJ,N’o-methylene-tetrahydrofolate (FH4: tetrahydrofo- late) functions as the methyl donor in reaction 1 . In general, the dihydrofolate (FHz) analogue methotrexate inhibits dihydrofolate reductase (reaction 2), whereas sulfanilamide, a p-aminoben- zoate analogue, poisons de novo folate synthesis (reaction 3) (BLAKLEY 1969). In yeast, treatment with methotrexate plus sulfanilamide blocks dTMP biosynthesis presumably by inhibiting FH4 synthesis, so that N4,N”-methylene-FH4 consumed in reaction 1 is not replaced.

neously arising KT-1 ATD Leu+ colonies were treated with the folate antago-

nists. T h e survival after

9 hr exposure to the drugs was identical for both cell

types. Thus, leucine independence did not confer a selective advantage during

antifolate treatment.

Previously, we have determined that thymine nucleotide depletion in yeast

results in weak, allele-specific mutagenesis

(ECKARDT,

KUNZ

and

HAYNES

1983).

However, treatment of the

leu2-I

replacement strains K T - I

AS

and KT-1

ATS

with methotrexate plus sulfanilamide did not increase the frequency of leucine

prototrophs (Table

3).

In

addition, when Leu+ segregants induced by antifolate

treatment of KT-1 ATD were examined, the majority (>95%) was found to

have remained canavanine-resistant (Table

4),

although both

leu2-I

and

canI-

52 are ochre suppressible alleles. These data demonstrate that thymine nucleo-

tide depletion does not lead to reversion or suppression of

leu2-I.

This, plus

the fact that

leu2-3, I12

is not detectably reverted by physical or chemical

mutagens

(HINNEN,

HICKS

and

FINK

1978), indicates that the antifolate-induced

leucine prototrophs did not emerge as a consequence of mutation.

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383 I

c

/

_/o----*----o

c----. t dTMP

0--0

-

dTMP /

16 /

/O

/

I 3

L

/

/O

I

/

lot

/

TIME ( h o u r s )

FIGURE 5.-Induction of leucine prototrophs by dTMP depletion. Strain KT-1 ATD was treated with methotrexate plus sulfanilamide, as described in MATERIALS AND METHODS. At 3-hr intervals,

samples were withdrawn and were washed three times with sterile H,O; cell titers (A) were determined by Coulter Counter, and washed cells were diluted if necessary and were plated onto appropriately supplemented minimal media to detect survivors and leucine prototrophs. Surviving fractions (B: viable cells per milliliter divided by Coulter counts per milliliter) and the frequencies of leucine prototrophs (C) were determined after incubating the plates at 30" for 4 days. The data are expressed as the means (normalized to time zero) of four independent experiments: C---., with dTMP; 0- -0, without dTMP.

locus. To our knowledge, the exact positions of all three mutations have not

been established. In principle, three orientations are possible (Figure 6). Within

these orientations, specific recombination events can

be

characterized

by

loss

(pBR322-) or retention (pBR322+) of the pBR322

DNA

sequences.

In orientation

I

(Figure

6A),

a simple intrastrand reciprocal recombination

event between the mutant

leu2

alleles could

yield

a Leu+ recombinant and

result in excision of the pBR322

DNA

sequences on a circular

DNA

molecule

containing the three

leu2

mutations in a single allele,

leu2-3,112,1.

Following

(10)

384

B. A. KUNZ, G.

R.

TAYLOR AND

R.

H. HAYNES TABLE 3

Antifolate treatment of TUP+ and tup- strains"

Treatment Surviving Leu+ frequency Strain Relevant genotype period (hr) fraction (X 106)

KT-1AD leu2-I leu2-3,112 0

6 12 KT-IATD leu2-I leu2-3,IIZ tup- 0 6 12

KT- 1 AS leu2-I 0

6 12

KT-IATS leu2-I tup- 0

6 12

1

.oo

20

0.59 93

0.13 377

1

.oo

16

0.64 98

0.12 336

1

.oo

1.8

0.64 2.1

0.08 1.3

1

.oo

2.9

0.65 2.2

0.06 2.5

a Strains were treated with antifolates. as described in MATERIALS AND METHODS.

TABLE 4

Canavanine-resistant colonies among Leu+ segregants

Treatment Total no. of period (hr) colonies tested

0 200

3 400

6 400

9 400

12 400

Canavanine- resistant colonies 195 388 386 395 39 1

7% canavanine- resistant colonies 97.5 97.0 96.5 98.7 97.7

Leu+ colonies that emerged following antifolate treatment of strain KT- 1 ATD for specific periods were replicated to appropriately supplemented minimal medium containing canavanine (30 pg/ml), incubated at 30" for 48 hr and then scored for canavanine resistance.

centromere distal to the pBR322

DNA

sequences on the other chromatid also

could produce a Leu+ pBR322- recombinant having a single copy of the

LEU2

region. In orientations I1 and I11 (Figures 6B and C), no simple reciprocal

crossover could result in a Leu+ pBR322- recombinant. However, for all three

orientations, gene conversion of one

leu2

allele to

LElJ2,

in

G1

or

GP,

followed

by reciprocal recombination could generate a Leu' recombinant lacking the

pBR322

DNA

sequences. It might be suggested that, if intrastrand gene con-

version was initiated by double-strand gap repair (SZOSTAK,

ORR-WEAVER

and

ROTHSTEIN

1983), then the pBR322

DNA

sequences might be eliminated by

gap formation rather than by reciprocal exchange. If such elimination could

occur, generation of an intact chromosome, and

so

recovery of Leu+ recom-

binants, would still require reciprocal exchange. Thus, for these orientations,

we would classify the various events that could lead to loss of the pBR322

(11)

A. ORIENTATION

I

p B R 3 2 2

Q

B. ORIENTATION

GI

pWR322

/-c77

I

3 1-

+

C.

ORIENTATION

IU

FIGURE 6.-Formation of Leu+ recombinants by reciprocal exchange. A, Orientation I: In G I , an intrastrand event yields a Leu' pBR322- recombinant and a circular molecule carrying the pBR322 sequences and the three leu2 mutations. In G s , an unequal sister-chromatid event yields a Leu+ pBR322- recombinant and a Leu- recombinant carrying the three leu2 mutations, three copies of the LEU2 region and two copies of the pBR322 sequences, B, Orientation 11: In G I , an intrastrand event yields a leu- pBR322- recombinant carrying the three leu2 mutations and a circular DNA molecule carrying a Leu' allele and the pBR322 sequences. In Gs, an unequal sister- chromatid event yields a Leu+ recombinant with three copies of the LEU2 region and two copies of the pBR322 sequences and a leu- pBR322- recombinant carrying the three leu2 mutations. C, Orientation 111. In G I , an intrastrand event in any of the intervals A-D will not yield a Leu+ recombinant. In G P , an unequal sister-chromatid event in any of the intervals A-D will not yield a Leu+ recombinant.

(12)

386

B. A. KUNZ, G. R. TAYLOR AND R . H. HAYNES TABLE 5

Frequencies of gene conversion, reciprocal recombination and unequal sistershromatid crossing over among LEU2+ recombinants

Colony hybridization DNA hybridization

Treatment No. of % Leu+ % Leu+ No. of % single % pBR322

period (hr) colonies tested pBR322’ pBR322- colonies tested copy pBR322 duplication

0 100 95 5 18 83 17

12 100 73 27 20 85 15

Mean Frequency (X

Total recombination

Gene, conversion

Unequal Reciprocal sister-chromatid

exchange crossing over

0 1.15 0.91 (79)” 0.06 (5) 0.18 (16)

24.6 15.3 (62) 6.6 (27) 2.7 (11)

12

The mean frequency of total recombination is the arithmetic average of frequencies from four independent experiments. The mean frequencies of gene conversion, reciprocal exchange and unequal sister-chromatid crossing over were determined by multiplying the percentage of gene conversion, reciprocal exchange or unequal sister-chromatid crossing over by the mean frequency of recombination.

a The numbers in parentheses show the percentage of gene conversion, reciprocal exchange and

unequal sister-chromatid crossing over as determined by colony and DNA hybridization.

rocal exchange and could not be distinguished from each other in our system.

Colony hybridization using pBR322 DNA as the probe showed that antifolate

treatment caused a significant

(P

<

0.001) fivefold increase in the fraction of

KT-IATD Leu+ segregants that had lost the pBR322 DNA sequences (Table

(13)

387

1

2

3

4 23.7-

9.5

=

6.7-

4.3

9

FIGURE 7.-Quantitation of LEU2 regions in antifolate-induced Leu+ isolates. Total yeast DNA was digested with Hindlll and hybridized with a LEU2 probe, as described in MATERIALS AND METHODS. DNA was pGTlO (lane 1) or total yeast DNA isolated from untreated Leu- KT-IATD cells having a duplication of the LEU2 region (lane 2 ) and antifolate-induced Leu+ KT-1 ATD cells having a duplication and a triplication, respectively, of the LEU2 region (lanes 3 and 4). T h e DNA size markers are in kilobases.

hybridiiation bands, neither the size of pGT10, would

be expected if the Leu+

pBR322+ recombinant examined was the product of gene conversion alone.

T w o hybridization bands were detected for 15 of 18 spontaneous and 17 of

20 antifolate-induced KT-1 ATD Leu+ pBR322+ recombinants tested

by

such

an analysis; the remainder gave three hybridization bands (Figure 7, Table 5).

This indicates that the majority of recombinants examined resulted from gene

conversion. However, it should

be

noted that antifolate treatment caused sim-

ilar increases, 17- and 15-fold, respectively, in the mean frequencies of events

that could

be

characterized as intrachromosomal gene conversion

or

unequal

sister-chromatid crossing over, compared to the corresponding spontaneous

values (Table 5).

It is

well known that, for

diploid

yeast, different allelic combinations at a

particular locus exhibit different frequencies of induced mitotic interchromo-

somal recombination in response to the same treatment

(KUNZ

and

HAYNES

(14)

388

B. A. KUNZ, G.

R.

TAYLOR AND

R.

H. HAYNES

induction of intrachromosomal recombination

by

dTMP depletion might pro-

duce results quantitatively different from those reported here, but qualitative

differences would not be expected.

DISCUSSION

Antifolate treatment

to

inhibit thymidylate biosynthesis induced the forma-

tion of leucine prototrophs in a haploid strain of yeast carrying different mu-

tant

leu2 alleles directly repeated on the same chromosome. This induction of

Leu+ colonies was prevented by concurrent provision of dTMP. Appropriate

control experiments showed that induced mutation was not responsible for the

enhanced formation of leucine prototrophs. Hybridization analysis revealed

that the Leu+ segregants arose as a consequence of intrachromosomal recip-

rocal exchange and gene conversion, as well as unequal sister-chromatid cross-

ing over. Thus, the results presented here demonstrate that depletion of thy-

mine nucleotides in

S . cerevisiae

induces various forms of intrachromosomal

recombination between repeated alleles separated by plasmid

DNA sequences.

If, as it seems reasonable to assume (BARCLAY

et

al.

1982),

the genetic effects

of dTMP deprivation are dependent on DNA replication, then the reciprocal

exchange and gene conversion events may represent intrachromatid recombi-

nation. We have found previously that inhibition

of

dTMP biosynthesis en-

hances unequal sister-chromatid crossing over in the reiterated ribosomal

DNA

sequences of yeast (KUNZ et

al.

1984).

Here, it is shown that this type of

recombination also can be induced in other chromosomal regions.

Gene conversion was the major event detected; however, the fraction of

recombinants attributable to induced conversion alone was somewhat lower

than the fraction of spontaneous convertants. By contrast, the proportion of

events classified as reciprocal exchange was enhanced fivefold by antifolate

treatment. This resulted in a 110-fold increase in the frequency

of

reciprocal

recombination, compared to the

1 'I-fold increase observed for the frequency

of antifolate-induced gene conversion. Other evidence also suggests that en-

hanced intrachromosomal reciprocal exchange may be characteristic of thy-

mine nucleotide depletion. First, in an earlier study, we scored deletion of a

LEU2

insert from the ribosomal DNA gene cluster to assay the induction of

unequal sister-chromatid crossing over by inhibition of dTMP biosynthesis

(KUNZ

et

al. 1984). While this event produced Leu+/Leu- sectored colonies,

isolates that were entirely Leu- also were induced. T h e data suggest that

reciprocal exchange between the tandemly arrayed ribosomal DNA genes to

delete

the LEU2 insert led to the emergence of the Leu- colonies. Second,

preliminary experiments indicate that antifolate treatment also can induce ex-

cision of a transposon located 5' to the yeast gene for glucose repressible

alcohol dehydrogenase

(B.

A. KUNZ,

unpublished observations). This type of

event is believed to occur via reciprocal recombination between the homolo-

gous

delta sequences flanking the larger portion of the transposon (ROEDER

and FINK

1983).

Finally, thymidylate starvation of dTMP auxotrophs bearing

two directly repeated, partially deleted copies of the thymidylate synthase gene

(15)

INTRACHROMOSOMAL

389 panied by

loss

of the intervening plasmid sequences

(G.

R. TAYLOR,

unpub-

lished observations). The restoration of a functional

TMPl

gene appears to be

due to intrachromosomal reciprocal exchange between the duplicated defective

copies of

TMPl.

Not only are these various findings consistent with the induc-

tion of intrachromosomal reciprocal recombination by thymine nucleotide de-

pletion, but they also indicate that such induced exchange is not limited to

particular regions of the genome or to specific gene arrangements.

T h e orientation of the

leu2-1

and

leu2-3,112

mutational sites within the

LEU2

locus cannot be established from our data. However, certain of these

findings suggest that the

leu2

mutations are ordered as diagrammed

for

ori-

entation I (Figure 6A). First, following antifolate treatment, there was a large

increase in the mean frequency of Leu+ pBR322- recombinants (Table

5).

Only in orientation I can a simple reciprocal exchange in

G I

or

G2

produce

this type of Leu+ segregant. Second, although the frequency of events involv-

ing reciprocal recombination

is enhanced dramatically by dTMP depletion, the

frequency of identified unequal sister-chromatid crossovers is affected much

less markedly (Table

5).

Leucine prototrophs resulting from unequal sister-

chromatid crossing over in orientation

I

would lose the pBR322 DNA se-

quences, retain a single copy of the LEU2 region and so be characterized by

hybridization analysis merely as the product of a reciprocal exchange event,

rather than unequal sister-chromatid recombination. This could account for

the observed difference in the extent of induction of reciprocal recombination

and unequal sister-chromatid crossing over. In orientation I, identification of

a Leu+ unequal sister-chromatid recombinant as such would require that the

recombinant result from a gene conversion event associated with reciprocal

exchange. Thus, the relative paucity of characterized unequal sister-chromatid

recombinants also would be consistent with the finding that antifolate-induced

intrachromosomal gene conversion is largely unassociated with crossing over

(Table

5).

If the

leu2

mutations were in orientation 11, a much larger increase

in the frequency of identified unequal sister-chromatid recombinants would be

expected, as this orientation allows

a simple crossover to generate a leucine

prototroph with a triplication of the LEU2 region. Arrangement of the muta-

tions in orientation

I11

would necessitate formation of Leu+ pBR322- recip-

rocal recombinants and Leu+ pBR322+ unequal sister-chromatid recombinants

by crossing over following gene conversion. In this case, both types of recom-

binant would be expected to occur with similar frequencies. Although these

results argue in favor of orientation I, the order of the

leu2-1

and

leu2-3,112

mutations can be established unequivocally only by DNA sequencing. However,

knowledge of the precise orientation of the

leu2

mutations would not alter the

categories of intrachromosomal recombination as defined here, the fractions

of leucine prototrophs attributed to each category or the fact that dTMP

depletion preferentially induces intrachromosomal events that involve recip-

rocal exchange.

(16)

390

B. A . KUNZ, G. R. TAYLOR AND R. H. HAYNES

results of this investigation demonstrate that dTMP deprivation provoked by

folate antagonists also enhances various types of intrachromosomal exchange.

Taken collectively, these findings indicate that, in actively growing cells, inhi-

bition of dTMP biosynthesis may create a metabolic condition able to induce

all recognized forms of mitotic recombination. If intrachromosomal recombi-

nation plays a role in the evolution and stability of multigene families

(SMITH

1973;

TARTOFF

1973;

SZOSTAK

and

WU

1980;

BALTIMORE

1981;

NAGYLAKI

and

PETES

1982), our results also suggest that such phenomena may be influ-

enced by any factor(s) that causes thymine nucleotide deficiencies.

We thank D. C. HAWTHORNE for supplying yeast strains used to construct BKSo-IA, T. D. PETS for suggesting the use of leu2-I and providing plasmid pYE16H1, L. E. SPRACKLIN and L. T. JANDRISITS for technical assistance and I. FULTON for typing the manuscript. L. E. S. was supported in part by a grant from the E. I. Canada Career Access Program to B. A. K. This work was supported by the Natural Sciences and Engineering Research Council of Canada.

LITERATURE CITED

BALTIMORE, D., 1981 Gene conversion: some implications for immunoglobulin genes. Cell 24:

Genetic and biochemical

592-594.

BARCLAY, B. J., B. A. KUNZ, J. G. LITTLE and R. H. HAYNES, 1982

consequences of thymidylate stress. Can. J Biochem. 6 0 172-194.

BIRNBOIM, H. C. and J. DOLY, 1979

binant plasmid DNA. Nucleic Acids Res. 7: 1513-1523.

BISSON, L. and J. THORNER, 1977

myces cereuisiae are deficient in thymidylate synthetase. J. Bacteriol. 132: 44-50.

BLAKLEY, R. L., 1969

A rapid alkaline extraction procedure for screening recom-

Thymidine-5’-monophosphate requiring mutants of Saccharo-

The Biochemistry of Folic Acid and Related Pteridines. North Holland, Am- sterdam.

BOLIVAR, F., R. L. RODRIGUEZ, P. J. GREENE, M. C. BETLACH, H. L. HEYNEKER and H. W. BOYER, Construction and characterization of new cloning vehicles. 11. A multipurpose cloning

Transformation in yeast: development

1977

system. Gene 2: 95-1 13.

of a hybrid cloning vector and isolation of the CANZ gene. Gene 8: 121-133. BROACH, J. R., J. N. STRATHERN and J. B. HICKS, 1979

COLMAN, N., 1977

DOBSON, M. J., S. M. KINGSMAN and A. J. KINGSMAN, 1981

Folate deficiency in humans. Adv. Nutr. Res. 1: 77-124.

Sequence variation in the LEU2 region of the Saccharomyces cerwisiae genome. Gene 16: 133-1 39.

DRAKE, J. W., 1970 The Molecular Basis of Mutation. pp. 44-45. Holden Day, Oakland, California. ECKARDT, F., B. A. KUNZ and R. H. HAYNES, 1983 Variation of mutation and recombination frequencies over a range of thymidylate concentrations in a diploid thymidylate auxotroph of yeast. Curr. Genet. 7: 399-402.

ERBE, R. W., 1979

GRIVELL, A. R. and J. F. JACKSON, 1968

Genetic aspects of folate metabolism. Adv. Hum. Genet. 9 293-354.

Thymidine kinase: evidence for its absence from Neu- rospora crassa and some other microorganisms and the relevance of this to the specific labeling of deoxyribonucleic acid. J. Gen. Microbiol. 54: 307-31 7.

rad52-independent mitotic gene conversion in Saccharomyces cerevisiae frequently results in chromosomal loss. Genetics 11 1: 7-22.

Molecular mechanisms in genetic stability and change: the role of deoxy- HABER, J. E. and M. HEARN, 1985

(17)

39

1

ribonucleotide pool balance. pp. 1-23. In: Genetic Consequences of Nucleotide Pool Imbalance,

Edited by F. J. DE SERRES. Plenum, New York.

HAYNES, R. H. and B. A. KUNZ, 1986 The influence of thymine nucleotide depletion on genetic stability and change in eukaryotic cells. Curr. Sci. (Bangalore) 5 5 1 - 1 1 .

HINNEN, A., J. B. HICKS and G. R. FINK, 1978 Transformation of yeast. Proc. Natl. Acad. Sci. ITO, Y., Y. FUKADA, K. MURATA and A. KIMURA, 1983 Transformation of intact yeast cells

treated with alkali ions. J. Bacteriol. 153: 163-168.

Gene conversion between duplicated genetic elements in

Meiotic recombination between duplicated genetic elements

Lack of association between intrachromosomal gene conversion and recip-

Intrachromosomal gene conversion in yeast. Nature 2 8 9 USA 7 5 1929-1933.

JACKSON, J. A. and G. R. FINK, 1981 yeast. Nature 292: 306-31 1 . JACKSON, J. A. and G. R. FINK, 1985

in Saccharomyces cerevisiae. Genetics 109: 303-332. KLEIN, H. L., 1984

rocal exchange. Nature 310: 748-753. KLEIN, H. L. and T. D. PETES, 1981

144-1 48.

KRUMDIECK, C. L., 1983 Role of folate deficiency in carcinogenesis. pp. 225-245. In: Nutrition Factors in the Induction and Maintenance of Malignancy, Edited by C. E. BUTTERWORTH and M. L. HUTCHINSON, Academic Press, New York.

Genetic consequences of deoxyribonucleotide pool imbalances. Environ. Mu- tagen. 4: 695-725.

Induction of mitotic recombination in yeast by starvation for thymine nucleotides. Proc. Natl. Acad. Sci.

Thymineless recombination in

Saccharomyces cerevisiae is independent of the ability to undergo meiosis. Curr. Genet. 5: 29- 31.

Phenomenology and genetic control of mitotic recombination in yeast. Annu. Rev. Genet. 15: 57-89.

KUNZ, B. A. and R. H. HAYNES, 1982 DNA repair and the genetic effects of thymidylate stress in yeast. Mutat. Res. 93: 353-375.

Mating-type switching in yeast is induced by thymine nucleotide depletion. Mol. Gen. Genet. 1 9 9 540-542.

Inhibition of thymidylate biosynthesis induces mitotic unequal sister chromatid recombination in Saccha- romyces cerevisiae. Curr. Genet. 8: 2 1 1-2 17.

LITTLE, J. G. and R. H. HAYNES, 1979 Isolation and characterization of yeast mutants auxo- trophic for Z’-deoxyuridine 5’-monophosphate. Mol. Gen. Genet. 1 6 8 141-1 5 1 .

MANIATIS, T., E. F. FRITSCH, and J. SAMBROOK, 1982 Molecular cloning-A Laboratory Manual.

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

MESSING, J. and J. VIEIRA, 1982 A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. Gene 1 9 269-276.

KUNZ, B. A., 1982

KUNZ, B. A., B. J. BARCLAY, J. C. GAME, J. G. LITTLE and R. H. HAYNES, 1980 USA 7% 6057-6061.

KUNZ, B. A., F. ECKARDT, J. G. LITTLE and R. H. HAYNES, 1982

KUNZ, B. A. and R. H. HAYNES, 1981

KUNZ, B. A., G. R. TAYLOR and R. H. HAYNES, 1985

KUNZ, B. A., G. R. TAYLOR, B. KONFORTI, B. W. GLICKMAN and R. H. HAYNES, 1984

MEUTH, M., 1984 The genetic consequences of nucleotide precursor pool imbalance in mammalian cells. Mutat. Res. 126: 107-112.

(18)

392

ORR-WEAVER, T. L., J. W. SZOSTAK and R. J. ROTHSTEIN, 1981 PETES, T. D., 1980

ROEDER, G. S. and G. R. FINK, 1983 ROTHSTEIN, R. J., 1983

SHANE, B. and E. L. R. STOKSTAD, 1985 Vitamin BIZ-folate interrelationships. Annu. Rev. Nutr. SHERMAN, F., G. R. FINK and J. B. HICKS, 1983 Methods in Yeast Genetics. Cold Spring Harbor

Laboratory, Cold Spring Harbor, New York.

SMITH, G. P., 1973 Unequal crossover and the evolution of multigene families. Cold Spring Harbor Symp. Quant. Biol. 38: 507-513.

SZOSTAK, J . W., T. L. ORR-WEAVER and R. J. ROTHSTEIN, 1983 The double-strand-break repair model for recombination. Cell 33: 25-35.

SZOSTAK, J . W. and R. Wu, 1980 Unequal crossing over in the ribosomal DNA of Saccharomyces

TARTOFF, K., 1973 Unequal mitotic sister chromatid exchange and disproportionate replication as mechanisms regulating ribosomal DNA gene redundancy. Cold Spring Harbor Symp. Quant. Biol. 37: 491-500.

Isolation of the thymidylate synthetase gene by complementation in Saccharomyces cereuisiae. Mol. Cell Biol. 2: 437-442.

Hybridization of nucleic acids directly

Communicating editor: E. JONES B. A. KUNZ, G. R. TAYLOR AND R. H. HAYNES

Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA 78: 6354-6358.

DNA genes. Cell 1 9 765-774.

Genetic Elements, Edited by J. A. SHAPIRO. Academic Press, New York.

Unequal meiotic recombination within tandem arrays of yeast ribosomal

Transposable elements in yeast. pp. 299-328. In: Mobile

One-step gene disruption in yeast. Methods Enzymol. 101: 202-211.

5: 115-141.

cereuksiae. Nature 284 426-430.

TAYLOR, G. R., B. J. BARCLAY, R. K. STORMS, J. D. FREISEN and R. H. HAYNES, 1982