Sites of recombination are local determinants of meiotic homolog pairing in Saccharomyces

cerevisiae

Joshua Chang Mell*, Bethany L. Wienholz, Asmaa Salem, and Sean M. Burgess*

* Genetics Graduate Group

Section of Molecular and Cellular Biology

University of California, Davis

Davis, CA 95616

Genetics: Published Articles Ahead of Print, published on May 27, 2008 as 10.1534/genetics.107.077727

Running Head: Recombination stabilizes homolog interactions in cis

Keywords and phrases:

1 meiosis

2 homologous chromosomes

3 recombination

4 Saccharomyces cerevisiae

5 pairing

Corresponding Author:

Sean M. Burgess

Section of Molecular and Cellular Biology

Briggs Hall

University of California, Davis

One Shields Avenue,

Davis, CA 95616

phone: 530-754-5177

fax: 530-752-3085

email: smburgess@ucdavis.edu

ABSTRACT

Trans-acting factors involved in the early meiotic recombination pathway play a major

role in promoting homolog pairing during meiosis in many plants, fungi, and mammals. Here we

address whether or not allelic sites have higher levels of interaction when in cis to meiotic

recombination events in the budding yeast Saccharomyces cerevisiae. We used Cre / loxP site-

specific recombination to genetically measure the magnitude of physical interaction between

loxP sites located at allelic positions on homologous chromosomes during meiosis. We observed

non-random coincidence of Cre-mediated loxP recombination events and meiotic recombination

events when the two occurred at linked positions. Further experiments show that a subset of

recombination events destined to become crossovers increase the frequency of nearby Cre-

mediated loxP recombination. Our results support a simple physical model of homolog pairing

in budding yeast, where recombination at numerous genomic positions generally serves to

loosely co-align homologous chromosomes, while crossover-bound recombination intermediates

locally stabilize interactions between allelic sites.

INTRODUCTION

The pairing of homologous chromosomes during early meiotic prophase is crucial for

their appropriate segregation at anaphase I. In Saccharomyces cerevisiae (along with other

fungi, plants, and mammals), meiotic recombination—initiated by the formation of programmed

DNA double-stranded breaks (DSBs)—is required for the normal co-alignment and juxtaposition

of homologous chromosomes in early prophase I (WEINER and KLECKNER 1994, ROCKMILL et al.

1995; LOIDL et al. 1994b; NAG et al. 1995, PEOPLES et al. 2002; TARSOUNAS et al. 1999;

PAWLOWSKI and CANDE 2005; MAHADEVAIAH et al. 2001; TESSE et al. 2003).

The crossover recombination products (CRs) generated from a subset of meiotic DSBs, in

combination with sister chromatid cohesion, form locks between homologous chromosomes that

facilitate their appropriate segregation (PAGE and HAWLEY 2003; PETRONCZKI et al. 2003). The

remaining meiotic DSBs are repaired without reciprocal exchange into non-crossovers (NCRs)

or are repaired using sister chromatid templates. A number of studies have revealed an early

bifurcation in the meiotic recombination pathway, generating either CR products or

predominantly NCR products (MACMAHILL et al. 2007; DE LOS SANTOS et al. 2003; MERKER et

al. 2003; HUNTER and KLECKNER 2001; ALLERS and LICHTEN 2001; BORNER et al. 2004;

PAQUES and HABER 1999). Most DSBs destined for repair as CR products transit through a

series of progressively more stable joint molecule intermediates with extensive direct base-

pairing interactions between homologous chromatids involving both sides of the DSB. NCR-

bound DSBs appear to go through a synthesis-dependent strand annealing (SDSA) mechanism

that involves fewer steps and fewer base-pairing interactions in which the two homologs are

directly joined, and in which only one end of the DSB directly base pairs with the template

DNA.

Cytological evidence supports a role for meiotic recombination in co-aligning and

juxtaposing homologous chromosomes. In the budding yeast, “axial bridges” have been

observed between homologous chromosomes in the absence of the central element of the

synaptonemal complex (SYM et al. 1993). Similar axial bridges have been observed during

mouse spermatogenesis, and immuno electron microscopy has shown that these bridges contain

the strand-exchange proteins Rad51p and Dmc1p (MOENS et al. 1997; TARSOUNAS et al. 1999).

These data strongly suggest that recombination intermediates serve to connect homolog pairs at

discrete points along their lengths and indicate that the central element is dispensable for

homolog co-alignment in these organisms.

Exogenous site-specific recombination has been used to genetically dissect nuclear

architecture and meiotic homolog pairing in budding yeast (BURGESS and KLECKNER 1999;

PEOPLES et al. 2002), offering a quantitative measure of homolog associations between two

defined genomic positions independent of the endogenous meiotic recombination machinery

(FIGURE 1). Cre recombinase is sufficient and necessary to induce crossing over between two 34

bp loxP sites at a frequency that depends on the local concentration of the loxP sites in vivo and

in vitro (HILDEBRANDT and COZZARELLI 1995; MARTIN et al. 2003). We refer to these Cre-

mediated loxP crossover events as “loxP collisions” to distinguish them from endogenous

recombination events. Using a reporter activation assay to detect Cre-mediated loxP collisions,

we have previously observed aspects of vegetative nuclear structure in the budding yeast,

including the Rabl orientation, high levels of intrachromosomal interaction, and “somatic”

homolog pairing (BURGESS and KLECKNER 1999).

There is a strong meiosis-induced increase in the collision frequency of allelic sites

between homologs, which we have termed “close stable homolog juxtaposition” (CSHJ).

Measurement of the CSHJ phenotype in a series of mutants has shown that entry into meiosis

and meiotic recombination factors are required for achieving wild-type CSHJ (PEOPLES et al.

2002; LUI et al. 2006). Mutants with defects in recombination initiation or early stages of DSB

repair have severe defects in CSHJ, while those with defects in later stages of repair have

moderate or no defect. Epistasis analysis of mutants with intermediate CSHJ defects has

revealed that genes involved in the formation of specifically CR or NCR recombination products

make independent contributions to wild-type levels of meiotic homolog pairing (PEOPLES-HOLST

and BURGESS 2005; WU and BURGESS 2006).

Importantly, it is genes required for the formation of joint molecule intermediates, but not

for product formation per se, that are responsible for the elevated allelic loxP collisions observed

in meiosis (PEOPLES-HOLST and BURGESS 2005). This suggests that ongoing cellular processes

that pair homologous chromosomes—particularly meiotic recombination—increase the

frequency of allelic loxP collisions by bringing the loxP sites close together in cis, thereby

facilitating Cre-mediated recombination. These and other data (TESSE et al. 2003; SMITHIES and

POWERS 1986; CARPENTER 1987; LUI et al. 2006) suggest a relatively simple model for meiotic

homolog pairing, where the combined action of multiple recombination events initiated between

homologous chromatids serves—along with cis effects of limited diffusion and synapsis—to

bring segments of homologous chromosomes into juxtaposition. However, the pleiotropic

effects of many mutations on pairing, recombination, and synapsis—as well as the limited

resolution of current cytological methods— confound a statistical evaluation of the relationship

between meiotic recombination events and the juxtaposition of allelic sites in cis.

Whereas previous work using the Cre / loxP site-specific recombination system to

measure allelic interactions has focused on the role of trans-acting factors on meiotic homolog

pairing, in this study we focus on the cis-acting role of recombination events in locally pairing

allelic sites. We used heteroallele recombination reporters inserted at a series of three sites to

measure the coincidence of allelic loxP collisions and gene conversions occurring at different

genetic distances from each other. We found that collisions and gene conversions occur together

more often than expected when tightly linked. In contrast, they occur together less often than

expected when more distantly linked. We also performed a Cre induction time course to show

that meiotic recombination aids collisions rather than the converse. Finally, we show that CR-

associated, but not NCR-associated, recombination elevates nearby allelic loxP collisions. Our

results, along with previous mutant analyses, support a role for crossover recombination

intermediates in locally stabilizing allelic pairing interactions in the budding yeast.

MATERIALS AND METHODS:

Yeast strains: All S. cerevisiae strains analyzed in this study (TABLE 1) were derived in

the SK1 strain background (KANE and ROTH 1974). We constructed strains by standard methods

containing reporters of allelic loxP collisions and heteroallelic gene conversion at varying

distances from each other (TABLE 1, FIGURE 1). Cre recombinase is supplied from a galactose

inducible promoter, GAL1, at the LYS2 locus (BURGESS and KLECKNER 1999). Starting with

SBY1756 and SBY1074 (TABLE 1 caption), two constructs—one bearing LEU2:GPD1:loxP and

the other containing LEU2:loxP:ade2 (FIGURE 1b; amplified from pSB133 and pSB186,

respectively; PEOPLES et al. 2002)—were used to completely replace the ARG4 locus (SGD

coordinates: VIII 140004-141395). ARG4 was deleted to eliminate sequences homologous to the

plasmid-borne arg4 heteroallele constructs. The loxP constructs were inserted such that the

GPD1 promoter and ade2 ORF were oriented towards CEN8.

Next, URA3:arg4-bgl / URA3:arg4-nsp heteroallele pairs were inserted (FIGURE 1c and

1d) at the HIS4 locus on chromosome III and the centromere-distal THR1 locus on chromosome

VIII (~25 kb, ~20 cM from the arg4∆::loxP constructs) as previously described (WU and

LICHTEN 1995; GOLDMAN and LICHTEN 1996; arg4 heteroallele plasmids were generous gifts

from M. LICHTEN and A. GOLDMAN). For heteroallele insertions at the centromere-distal DED81

locus (3 kb, ~2 cM from the arg4∆::loxP constructs), PCR products from wild-type SK1

genomic DNA (SGD coordinates: VIII 143513-144010) were TopoTA cloned (INVITROGEN) and

subcloned into the EcoRI site of pMJ113 and pMJ115 to generate pSB397 and pSB398,

respectively (arg4-bgl and arg4-nsp), and the BstXI enzyme was used to linearize plasmids prior

to yeast integration. Heteroallele inserts at DED81 and THR1 were added in both linkage phases

relative to the loxP constructs (FIGURE 2a and 2b), such that the URA3 and arg4 ORFs were

directed away from the centromere . For convenience, we use GPD1-bgl and ade2-bgl to refer to

the two linkage phases, indicating whether the promoter-containing loxP construct is coupled to

arg4-bgl or arg4-nsp, respectively.

To generate strains bearing arg4∆::LEU2:GPD1:loxP:ade2 used in linkage control

strains, we sporulated SBY2186, SBY2185, SBY2257, and SBY2258 in the presence of 0.03%

galactose and identified Ade+ spore clones by tetrad dissection (TABLE 1, FIGURE 2c and 2d). To

distinguish crossover from non-crossover associated gene conversion at the DED81 locus, we

replaced the immediately centromere-distal YHL022c gene with a KanMx antibiotic resistance

marker by the method of WACH et al. 1994 to produce GenR haploids bearing arg4∆::loxP:ade2

alleles. Appropriate pairs of haploids were crossed to produce the experimental diploid strains

(TABLE 1).

Random spore analysis: Synchronized meiotic time courses were conducted in 10 mL

SPM cultures ± galactose induction of the Cre recombinase, as described (PADMORE et al. 1991;

PEOPLES et al. 2002). Galactose was added to a final concentration of 0.03% at t = 1, 2, 3, or 4

hrs in SPM for induced samples. Random spore analysis was performed after 24-48 hrs by

diluting zymolyase-treated sonicated spore suspensions and plating to appropriate media

(MCKEE and KLECKNER 1997). Dilutions were targeted to produce ~200-400 colonies per plate,

depending on media type. Arg+ prototroph formation (R) was determined by growth on SC-arg

plates relative to colony forming units (CFU) of spores on YPD plates. Ade+ prototroph

formation (C) was determined by the frequency of white colonies on YPD-ade plates. SC-arg

plates were replica-plated to YPD-ade to determine the frequency of Ade+ Arg+ spores (S and L).

Where applicable, spores were also replica plated to and from YPD+geneticin media to

determine the GenR or GenS spore genotype, i.e. the presence or absence of the KanMx resistance

cassette (for M, N, X, and O). We use subscripts bgl or nsp to refer to linkage phase GPD1-bgl

and ade2-bgl, respectively.

In order to determine whether or not there were specific biases in the measured frequency

of Arg+ Ade+ spores due to the direction of selection, we performed the reciprocal selection on

strain SBY2074 by screening and patching white Ade+ colonies from YPD-ade to SC-arg to

determine the frequency of Ade+ Arg+ spores. We were unable to directly select for Ade+ spore

clones, due to hypomorphic expression of the ADE2 gene when loxP sequences reside in the

5’UTR (unpublished observations). The frequency of coincident Arg+ Ade+ spores was in strong

agreement when selection (or screening) was carried out in either direction (data not shown), so

we report coincidence data normalized to Arg+ frequency to facilitate comparisons between

strains and reduce sampling error.

All heteroallele reporter strains were subjected to random spore analysis on at least two

different occasions from independent meiotic time courses. Errors were calculated as the

standard deviation of the mean from three independent cultures sporulated in parallel for each

experiment. Measured genotypic frequencies with a given strain were consistent between

independent experiments, and spore viability was consistently near 100% in all strains and

experiments (data not shown). Student’s t-tests (2-tails) on transformed frequency data were

used to evaluate differences in the data at a confidence threshold of p ≤ 0.05.

Calculation of collision frequency per meiotic gene conversion: We measured the

collision frequency among gene convertants as S(obs) = Ade+ / Arg+ spores in collision tester

strains. We calculated the expected frequency of Ade+ / Arg+ spores as S(exp) = 2 L C, where C

is collision frequency (Ade+ / CFU in the collision tester strains) and L is the coupling constant

between the GPD1:loxP:ade2 construct and the coupled heteroallele upon selection for Arg+

spores (Ade+ / Arg+ in the linkage control strains) . If L = 0.5, then there is no effect of Arg+

selection on the recovery of the GPD1:loxP construct. If L = 1, then all Arg+ spores are coupled

to GPD1. If L = 0, then no Arg+ spores contain GPD1.

If gene conversion and collision events occur independently and are unlinked, we expect

the collision frequency to be the same between unselected and gene conversion-selected spores,

i.e. S(exp) = C (where L = 0.5). But if the collision and conversion events occur independently

and are linked, the effects of coupling and repulsion between the two assays can become evident

if there are differential recombination parameters between the two heteroalleles. For example, if

all Arg+ spores were the result of arg4-bgl --> ARG4 gene conversion with no associated

crossing over, then Lbgl = 1 and Lnsp = 0, thus Sbgl(exp) = 2 C and Snsp(exp) = 0. Restated, if both

a collision and a gene conversion happen in the same meiotic cell, the Mendelian expectation is

that GPD1:loxP:ade2 and URA3:ARG4 would occupy the same spore 1/4 of the time, i.e. S = C.

But if the Arg+ gene conversion recipient in one linkage phase always ended up on a GPD1-

containing chromatid, then GPD1:loxP:ade2 and URA3:ARG4 are expected to occupy the same

spore 1/2 of the time, so S(exp) = 2 C, whereas in the other linkage phase S(exp) = 0 (FIGURE 2a

and 2b).

We estimated the values of Lbgl and Lnsp for heteroallele insertions at DED81 and THR1,

using derivative linkage control strains in which all loxP inserts possessed the loxP-proximal

ade2 ORF, while only one homolog carried the loxP-distal GPD1 promoter (FIGURE 2c and 2d).

In this way, the GPD1-containing chromatid could be followed independently of collisions. L

was measured as the frequency of Ade+ prototrophs amongst Arg+-selected spores (Ade+ / Arg+).

To evaluate potential cis effects of the GPD1 promoter on biases between the heteroalleles in

generating Arg+ spores and/or associated crossover frequencies, we measured the sum Lbgl + Lnsp,

which indicates the effect of the arg4∆::loxP heterozygosity on heteroallelic gene conversion

parameters. If Lbgl + Lnsp = 100%, then we would conclude that there is no cis effect of loxP

heterozygosity on the recombination parameters of the arg4 heteroalleles.

Calculation of collision frequency among crossover and non-crossover

recombinants: For experiments with DED81::arg4 heteroalleles that also contained a

segregating yhl022c∆::KanMx allele in repulsion to the GPD1:loxP (or GPD1:loxP:ade2)

construct (TABLE 1), we measured the observed and expected frequency of collisions associated

with crossover and non-crossover recombination. For collisions among crossovers, X(exp) = 2 M

C, where M is the frequency of Ade+ GenR / Arg+ spores in linkage control strains (SBY3155 and

SBY3156), whereas X(obs) is Ade+ GenR / Arg+ and C is Ade+ / CFU in collision tester strains

(SBY3153 and SBY3154). For collisions among non-crossovers, O(exp) = 2 N C, where N is the

frequency of Ade+ GenS / Arg+ spores in linkage control strains and O(obs) is this value in

collision tester strains.

For the collision tester strains (SBY3153 and SBY3154), only Arg+ Ade+ spores are

informative of a collision and gene conversion occurring in the same meiosis, where a GenS

genotype indicates a collision and NCR-associated gene conversion and GenR indicates a

collision plus CR-associated gene conversion. The two linkage phases are informative for

different classes of recombination events occurring with a collision (Arg+ Ade+ spores).

For the linkage control strains (SBY3155 and SBY3156), we could evaluate all four

detectable classes of recombination event among Arg+ gene conversions independent of

collision: Ade+ GenS and Ade- GenR indicate NCR-associated gene conversion, while Ade+ GenR

and Ade- GenS indicate CR-associated gene conversion. When GPD1 is coupled to arg4-bgl, the

Arg+ Ade+ GenS genotype indicates a bgl recipient with no crossover (Nbgl), while Arg+ Ade+

GenR indicates either a nsp recipient with a proximal CR or a bgl recipient with a distal crossover

(Mbgl). In the opposite linkage phase (when GPD1 is coupled to arg4-nsp), a reciprocal set of

events are reported upon; Arg+ Ade+ GenS indicates a nsp recipient and no crossover (Nnsp), but

Arg+ Ade+ GenR indicates either a nsp recipient and a distal CR or a bgl recipient and a proximal

crossover (Mnsp). Furthermore, if the presence of the loxP heterozygosity has no effect on

heteroallele recombination parameters, we expect that (Mbgl + Nbgl) + (Mnsp + Nnsp) = 1.

RESULTS

A two locus genetic assay to measure allelic pairing interactions and meiotic

recombination during budding yeast meiosis: To address whether or not allelic interactions

occur at higher levels for allelic sites adjacent to meiotic recombination events, we used Cre /

loxP site-specific recombination to analyze the relative magnitude of allelic interactions at the

ARG4 locus in relation to gene conversion events occurring at sites on the same or different

chromosomes (FIGURE 1).

First, heterozygous loxP constructs were used to replace the endogenous ARG4 locus on

chromosome VIII (FIGURE 1b and 1d). One loxP insert bears a centromere-distal constitutive

promoter (GPD1:loxP), while the other bears a centromere-proximal promoterless reporter

(loxP:ade2). Cre is supplied under the control of the galactose-inducible promoter (GAL1), and

Cre-mediated crossovers between loxP sites (loxP “collisions”) are detected by Ade+ prototroph

formation (FIGURE 1a and 1b). The collision frequency reports the relative spatial proximity of

pairs of loxP sites engineered into the yeast genome (BURGESS and KLECKNER 1999, PEOPLES et

al. 2002, PEOPLES-HOLST and BURGESS 2005, LUI et al. 2006). Cre-mediated loxP collisions are

only detected between homologous chromatids; sister-sister loxP recombinants are undetectable,

but their occurrence may influence the frequency of events between the homologs.

Second, arg4-bgl / arg4-nsp heteroallelic gene conversion reporters (WU and LICHTEN

1994; GOLDMAN and LICHTEN 1996) were added at one of three locations: a site immediately

adjacent to the loxP site at DED81 (3 kb, ~2 cM), a more distal linked site at THR1 (25 kb, ~20

cM), and an unlinked site at HIS4 (chromosome III, 50 cM) (FIGURE 1c and 1d). Strains

heterozygous for arg4-bgl and arg4-nsp can generate wild-type ARG4 information by gene

conversion, and Arg+ spores produced during meiosis represent recipients of meiotic DSB repair

initiated near one of the heteroallele sites (FIGURE 1c, NICOLAS and PETES 1994).

For each gene conversion reporter located at sites linked to loxP (i.e. at DED81 and

THR1), four diploid strains were constructed to control for the effects of linkage phase between

the heterozygous loxP constructs and the heterozygous arg4 constructs (FIGURE 2). If Arg+ gene

conversions and / or crossing over are biased between the arg4-bgl and arg4-nsp heteroalleles,

then the effects of coupling and repulsion between the heteroallele pair and the loxP constructs

will become evident. The heteroallele pair, arg4-bgl / arg4-nsp, was inserted in both linkage

phases relative to the collision tester constructs, GPD1:loxP / loxP:ade2 (FIGURE 2a and 2b), and

also derivative linkage control constructs, GPD1:loxP:ade2 / loxP:ade2 (FIGURE 2c and 2d). In

the former strains Ade+ spores are produced by Cre-mediated loxP collisions, whereas in the

latter strains the Ade+ phenotype segregates with GPD1-containing chromatids, independent of

collisions. For convenience, we refer to the two linkage phases as GPD1-bgl (FIGURE 2a and 2c)

and ade2-bgl (FIGURE 2b and 2d). For variables, we use subscripts bgl and nsp, respectively, to

refer to the two different linkage phases.

We performed random spore analysis for this set of nine strains (TABLE 1) to determine

the gene conversion frequency R (Arg+/CFU), the unselected collision frequency C (Ade+/CFU),

the relative effect of selecting for Arg+ on the recovery of GPD1-containing chromatids L, and

the conversion-selected collision frequency S(obs) (Ade+/Arg+) (TABLE 2).

Heteroallelic gene conversions at three loci are not affected by the presence of loxP

constructs or by Cre-induction: We first considered the frequency of Arg+ gene conversion (R)

at the three heteroallele inserts. The rate of gene conversion has been previously shown to

depend on chromosomal location of the heteroallele reporters, indicating a recombination

position effect (LICHTEN and HABER 1989). Arg+ spore frequencies differed between the

URA3:arg4 inserts: the lowest for heteroalleles at DED81, intermediate at THR1, and highest at

HIS4 (FIGURE 1d, TABLE 2 column C). Gene conversion frequencies at THR1 and HIS4 were

consistent with previously published values (WU and LICHTEN 1995; GOLDMAN and LICHTEN

1996). There was no significant difference in gene conversion for Cre-induced and un-induced

cultures, between the two linkage phases for DED81 and THR1 inserts, or between the collision

tester and linkage control strains (data not shown, p > 0.30 for all comparisons). We concluded

that the loxP constructs and Cre-induction have little or no cis effect on gene conversion

frequency.

Allelic loxP collisions during meiosis are not affected by the presence of heteroallele

constructs: We next considered the frequency of allelic loxP collisions for strains containing

each combination of heteroallele insert location and linkage phase. For cultures in which Cre

was induced, the allelic loxP collision frequency per chromatid (C, Ade+/CFU) was ~4.6% and

did not significantly differ with heteroallele insert location (TABLE 2 column D, all pair-wise p-

values > 0.50). This value per spore is comparable to the 16-24% collision frequency per

meiotic cell observed in return-to-growth experiments (J.C.M. and S.M.B. unpublished results;

PEOPLES et al. 2002). This number is also consistent with the 1:3 segregation of Ade+ observed

in 16 - 24% of tetrads; 2:2 segregants occur in ~1% of tetrads, indicating that the majority of

loxP collisions occur after DNA replication (data not shown; PEOPLES et al. 2002). In cultures

with no galactose induction, < 0.01% of spores were Ade+, indicating negligible crossing over

within the loxP sites in the absence of the Cre recombinase. These results indicate that the

presence of the heteroallele constructs does not appreciably affect the level of allelic interactions

in cis.

Linkage phase effects are exclusively due to differential repair of arg4 heteroalleles: We

next evaluated the effect of differences between Arg+ gene conversion of the arg4-bgl and arg4-

nsp heteroalleles on the recovery of the GPD1:loxP collision reporter construct for inserts at

DED81 and THR1 among Arg+-selected spores. In the linkage control strains, the homolog

bearing the GPD1 promoter always expresses the ADE2 open reading frame, independent of

allelic loxP collisions (TABLE 1, FIGURE 2c and 2d). In these strains, Ade+ spores accounted for

50% of the total with or without Cre-induction, consistent with Mendelian expectations. In

contrast, Ade+ / Arg+ spores (L) deviated from 50% in opposite directions of equal magnitude in

the two linkage phases (+30% and -30% for GDP1-bgl and ade2-bgl, respectively). This result

was consistent at both insert locations (TABLE 2 column E). Thus the loxP heterozygosity did not

exert a cis effect on repair biases between arg4-bgl and arg4-nsp, for example from a DSB

hotspot induced by the GPD1 promoter. These results indicate that Arg+ recombinants are

skewed toward either arg4-bgl gene conversion recipients associated with no proximal crossover

and / or arg4-nsp recipients associated with a proximal crossover, independent of linkage phase

and location relative to the loxP constructs (FIGURE 2c, TABLE 2, see below).

Non-random coincidence of allelic loxP collisions and gene conversion: We determined

the coincidence of Arg+ and Ade+ prototrophy in spores by measuring the apparent effect of Arg+

gene conversion at the three assayed sites on the Ade+ collision frequency (TABLE 2 columns E

to G). We compared the observed frequency of Ade+ / Arg+ spores in collision tester strains,

S(obs), to the expected frequency, S(exp), which was calculated as 2 L C (see MATERIALS AND

METHODS). For heteroallele inserts at DED81 and THR1, both linkage phases were evaluated.

When GPD1:loxP was coupled to arg4-bgl (FIGURE 2a, TABLE 2), spores with Arg+ gene

conversions at DED81 showed a significant increase in the frequency of Ade+ collisions

compared to expected: Sbgl(obs) = 13.3 ± 0.4% versus Sbgl(exp) = 7.0 ± 0.1% (p = 0.012). Indeed

S(obs) exceeded the maximum expected effect for complete coupling between Arg+ and Ade+: If

L = 1, then S(exp) = 8.7 ± 1.1% in one linkage phase and 0.0% in the other. These data indicate

a contribution of meiotic recombination in juxtaposing nearby allelic sites.

In contrast, Arg+ spores from conversions at THR1 had a significant decrease in the

frequency of Ade+ collisions: Sbgl(obs) = 4.8 ± 0.5% versus Sbgl(exp) = 7.5 ± 1.0% (p = 0.011).

Notably, the elevated coincidence of gene conversion and allelic loxP collision occurred over a

short physical and genetic distance. Within a distance of 25 kb (~20 cM), gene conversions and

allelic loxP collisions occur together less often than expected, indicating that recombination can

repress allelic interactions at more distant sites.

In the opposite linkage phase, when GPD1:loxP was coupled to arg4-nsp (FIGURE 2b), a

similar trend was observed, however the differences were not statistically significant (TABLE 2) .

Thus it appears that only some classes of gene conversions are significantly coincident with

allelic loxP collisions: Either bgl recipients with no or distal CR and / or nsp recipients with

proximal CR events (FIGURE 2a)—but not nsp recipients with no CR or distal CR and / or bgl

recipients associated with proximal CR events (FIGURE 2b)—are correlated with elevated

collisions at DED81 and decreased collisions at THR1. These data indicate that not all

recombination events substantively influence allelic interactions in cis (see below).

When the heteroallele pair was located at HIS4 on chromosome III, unlinked from the

loxP sites, we unexpectedly observed a marginal decrease in allelic collisions compared to

expected (TABLE 2; p = 0.048). However in repeated experiments this difference reproducibly

bordered on statistical significance, with p = 0.050 ± 0.005. While unknown biases in the

experimental set-up could create this marginal trend, biologically relevant causes cannot be ruled

out. Selection for gene conversions at HIS4 may effectively enrich for a subset of cells, in which

a component of the recombination machinery (perhaps a crossover-specific factor) was titrated

away from other sites in the nucleus (i.e. adjacent to the loxP site). This effect may be similar to

the decrease in allelic loxP collisions observed when meiotic cells are exposed to γ-irradiation to

induce exogenous DSBs (unpublished data). Regardless, the marginal effect of gene conversion

at HIS4 on loxP collisions observed is insufficient to explain the magnitude and significance of

coincidence between gene conversion and collisions when the assays were at linked locations in

the GPD:loxP arg4-bgl linkage phase.

Endogenous meiotic recombination aids Cre-mediated recombination at nearby

loxP sites: We sought to distinguish whether recombination locally aids loxP collisions or the

converse by inducing the expression of Cre at different time points in synchronized meiotic

cultures. If loxP collisions facilitate gene conversion at nearby sites, then induction of the Cre

recombinase at later time points in early prophase I should cause a decrease in Sbgl(obs) towards

expected, since cells at later time points would have already initiated meiotic recombination.

Alternatively, if recombination aids collision, we would not observe a decrease in coincident

spores at late Cre induction times.

We added galactose to synchronized meiotic cultures of SBY2186 and SBY2709

(DED81 heteroallele inserts in the GPD1-bgl linkage phase) to induce Cre expression at 1, 2, 3

or 4 hours after transfer to sporulation media and evaluated the frequency of Ade+, Arg+, and

Ade+ Arg+ spores after sporulation was complete (FIGURE 3). Heteroallelic gene conversion at

DED81::arg4 was unchanged for different induction times in both the tester and control strains

(data not shown). Varying induction time also had no effect on Ade+ segregation in the linkage

control SBY2709; for all four induction times, control Ade+ spores arose at a 50% frequency,

and Ade+ constituted 80% of Arg+ spore clones (L), as expected. These results indicate that

inducing Cre at later time points does not affect gene conversion frequency or the relative bias in

heteroallele repair between arg4-bgl and arg4-nsp.

Allelic loxP collisions were less frequent when galactose was added at later points in

meiosis. This result was expected, since the length of time Cre had to act on the loxP sites

before the meiosis I division was decreased. In the collision tester SBY2186, the frequency of

Ade+ collision was 3.9 ± 0.3% of spores for induction at t = 1 hour, decreasing to 1.9 ± 0.5% and

2.0 ± 0.6% for t = 2 and t = 3 hour induction respectively, and was further reduced to 1.0 ± 0.1%

with a t = 4 hour induction time. However, the coincidence of allelic collisions and nearby gene

conversions showed a much less appreciable change: Sbgl(obs) was 17.9 ± 3.2% for induction at

t = 1 hour and 11.7 ± 1.7% for the t = 4 hour induction time. Thus, Sbgl(obs) was 3-fold greater

than S(exp) for early Cre induction, increasing to 7-fold for late Cre induction (FIGURE 3). These

data strongly argue that Cre-mediated loxP crossovers do not affect meiotic recombination in cis.

Instead ongoing meiotic recombination adjacent to the loxP sites increases the probability of a

Cre-mediated event.

A role for crossover-bound recombination intermediates in juxtaposing nearby

allelic sites: Taken together, the results of the gene conversion experiments described above

demonstrate a cis effect of a subset of recombination intermediates on the frequency of allelic

loxP collisions. We hypothesized that CR recombination intermediates play a specific cis role in

affecting loxP collisions, whereas NCR recombination intermediates do not: If CR

recombination intermediates and / or products aid loxP collisions locally, then collisions may be

more likely when crossovers occur nearby, yet less likely when crossovers occur at a more

distant linked site, due to genetic interference. Selection for gene conversion at a particular site

effectively enriches for spores with associated crossover events, while chromosomal interference

simultaneously depletes the occurrence of incidental crossovers at linked sites. NCR

recombination intermediates are unlikely to repress loxP collisions at a distance, since NCR

products do not show chromosomal interference (HURST et al. 1972, MORTIMER and FOGEL

1974; MALKOVA et al. 2004).

To distinguish the contributions of CR and NCR recombination to the allelic loxP

collision frequency, we added a heterozygous KanMx antibiotic marker (which confers a GenR

phenotype) at the YHL022c locus, immediately distal to DED81, to produce strains SBY3153

through SBY3156, representing both collision tester and linkage control strains for

recombination at DED81 in both linkage phases (TABLE 1, FIGURE 4). We conducted random

spore analysis with these new strains using galactose induction at t = 1 hr in SPM (TABLES 3 and

4). The genotypic frequencies of Ade+, Arg+, and Ade+ Arg+ spores were comparable to those

previously observed in the absence of KanMx for all four DED81::arg4 diploids (data not

shown), and 50% of random spores were GenR, as expected. In the linkage control strains, 9.0 ±

1.9% and 9.1 ± 1.0% (for GPD1-bgl and ade2-bgl, respectively) of GenR spores were Ade+,

indicating a 9 cM map distance between YHL022c and arg4∆::loxP. Galactose induction had no

effect on spore genotypic frequencies, except that in the collision testers all spores were Ade-

(data not shown).

Linkage control strains show the predicted distribution of crossovers and non-crossovers

associated with gene conversion: In the linkage control strains (SBY3155 and SBY3156), we

could measure the frequency of CR-associated Arg+ GenR Ade+ (M) and Arg+ GenS Ade- spores,

as well as NCR-associated Arg+ GenS Ade+ (N) and Arg+ GenR Ade- spores. In both linkage

phases, the frequency of Arg+ spores associated with CR was ~60%, but the two linkage phases

differed substantially in the values of M and N (FIGURE 4, TABLE 3). That is, in the GPD1-bgl

linkage phase, informative Arg+ Ade+ spores were mostly CR-associated (~2-fold greater than

NCR). By contrast in the ade2-bgl linkage phase, the informative Arg+ Ade+ spores were

predominantly NCR-associated (~6-fold greater than CR). These data are consistent with our

inference that CR, but not NCR, recombination contributes to loxP recombination in cis.

Notably the ~20-fold difference between Mbgl and Mnsp suggests that nsp and bgl

conversion recipients have dramatically higher probabilities of being resolved as proximal and

distal CRs, respectively. This strong bias might be related to the relative location of nsp towards

the arg4 proximal DSB site and bgl towards the arg4 distal DSB site (BORDE et al. 1999).

Collision tester strains show the predicted excess of loxP collisions among crossover-

associated Arg+ spores in both linkage phases: We used the linkage control strains to calculate

X(exp) and O(exp), the expected Ade+ collision frequency with CR-associated and NCR-

associated Arg+ gene conversion, respectively ( TABLE 4). Both linkage phases showed a

significant excess of observed CR-associated loxP collisions above expected (~2-fold, p = 0.029

and p = 0.029), while neither linkage phase significantly differed in the level of NCR-associated

collisions (p = 0.311 and p = 0.476). These results conclusively show that CR-associated gene

conversions play a special role in increasing nearby loxP collisions, while NCR-associated gene

conversions neither elevate nor inhibit nearby loxP collisions.

DISCUSSION

Meiotic recombination facilitates homologous chromosome interactions in cis: The

importance of genes required for early steps of meiotic recombination in the co-alignment and

juxtaposition of homologous chromosomes in many eukaryotes led us to wonder whether

homolog interactions occurred at higher levels for sites in cis to meiotic recombination events.

We used a two-locus assay in budding yeast to measure the coincidence of meiotic

recombination (gene conversion of arg4 heteroalleles yielding Arg+ spores) and Cre-mediated

recombination between allelic loxP sites (allelic loxP “collisions” yielding Ade+ spores). We

found that the frequency of coincident Ade+ Arg+ meioses exceeded random expectations when

the loxP and heteroallele constructs were at tightly linked positions, suggesting that meiotic

recombination can bring nearby loxP sites into close proximity for Cre to act.

An alternate possibility is that Cre-mediated loxP crossovers increase the chance of

nearby heteroallelic gene conversion, but several lines of evidence argue against this. First, the

frequency of heteroallelic gene conversions was neither affected by the presence of nearby loxP

constructs nor by expression of Cre. Second, the coincidence of gene conversions and allelic

loxP collisions did not decrease –but rather increased—when Cre was induced at progressively

later time points in synchronized sporulations. These data suggest that ongoing recombination

serves to locally juxtapose nearby chromosomal regions, rather than Cre-mediated loxP

crossovers influencing nearby recombination.

The extent to which meiotic recombination elevates nearby allelic interactions is likely

underestimated by our measurements, since the total number of meiotic recombination events

that initiate near the assayed site exceeds the levels of Arg+ gene conversions. Many

recombination events initiated within or near the heteroalleles may not include either arg4

mutation in heteroduplex DNA. Furthermore, the outcome of mismatch repair can lead to either

restoration or conversion of ARG4 / arg4 heteroduplex. Indeed, there are about ten times more

DSBs per chromatid around the arg4 heteroallele constructs than Arg+ spores, independent of the

recombination position effect (BORDE et al. 1999).

Crossover recombination plays a special role in juxtaposing nearby homologous

sites: Only some recombination-mediated interactions exerted a cis effect on allelic loxP

collisions, whereas others had no detectable effect. In one linkage phase, gene conversion

nearby the loxP site (at DED81) significantly increased the probability of a collision. When gene

conversion occurred further away from loxP (at THR1), there was significantly less probability

of collision. In the other linkage phase, we observed no significant deviation from random

expectations at either site. From these data, we inferred a unique role for CR recombination

intermediates in tethering nearby allelic sites, which predicted a strong difference in the

probability of informative Arg+ spores associated with CRs between the two linkage phases. We

verified our prediction for gene conversions at DED81, and further found that CR-associated

gene conversion significantly elevated nearby allelic loxP collisions in both linkage phases,

while NCR-associated gene conversion did not.

While the formation of recombinant products may occur synchronously for CRs and

NCRs, their respective joint molecule intermediates likely have different stabilities and / or

persistence times (MALOISEL et al. 2004, MERKER et al. 2003). Recent evidence suggests that

strand invasion recombination intermediates bound for an NCR fate are indeed considerably less

stable than those bound for a CR fate (MACMAHILL et al. 2007). Our data suggest that the

greater stability of CR-bound joint molecules increases the time available for Cre to act on close

by loxP sites located at allelic sites on homologous chromatids. The phenomenon of genetic

interference between CRs then explains the decreased collisions for conversions 25 kb away at

THR1, as well as the lack of effect of NCR recombinants. We suspect that NCR-bound SDSA

recombination intermediates still make contributions to the end-to-end co-alignment of

homologous chromosomes, but individual events have short-lived joint molecules that make

minor cis contributions to stable interactions between allelic sites.

Crossover interference and homologous chromosome interactions: Future sites of

CRs are marked by “synapsis initiation complexes” (SICs) containing Zip3p, formed at discrete

sites along chromosomes during prophase I, and these SIC foci show chromosomal interference

(FUNG et al. 2004). Zip3p interacts with a number of other proteins including Zip2p and Mer3p,

all of which play important structural or biochemical roles in the formation of CR recombination

products, perhaps by stabilizing joint molecule recombination intermediates (BORNER et al.

2004).

The zip2∆, zip3∆, and mer3∆ mutants have all been shown to have intermediate defects

in allelic loxP collisions during meiosis, presumably due to a decrease in recombination

intermediate stability in the absence of SICs (PEOPLES-HOLST and BURGESS 2005). In this study

using wild-type yeast, selecting for gene conversions nearby the loxP contructs (at DED81) may

effectively enrich for the local presence of SICs. Since SICs display chromosomal interference,

selection for recombinants at a more distant site (THR1) may deplete the presence of SICs

immediately adjacent to the loxP sites. In effect, this latter case locally mimics a zip2∆, zip3∆,

or mer3∆ mutant around the loxP sites.

Crossover product formation is not required for SIC interference or wild-type allelic

interactions. Null mutants of ZIP1 and MSH4 cause defects in crossover formation and genetic

interference, yet still display interfering Zip3p foci (FUNG et al. 2004). The zip1∆ and msh4∆

mutants also have wild-type or nearly wild-type levels of allelic interactions as measured by the

Cre / loxP collision assay (PEOPLES-HOLST and BURGESS 2005). These data indicate that

stabilized joint molecule recombination intermediates conducive to CR formation, rather than

CR products per se, are responsible for increasing local allelic interactions during meiosis and

imposing interference. Furthermore, Cre-mediated loxP crossovers are insufficient to impose

crossover interference on the immediately adjacent interval, consistent with other evidence that

the “substrate” for interference signaling depends on meiotic recombination intermediates

present prior to crossover formation (BISHOP and ZICKLER 2004).

Acknowledgements: Special thanks to David Liang for technical assistance, to John

McKay for statistical consulting, and to Michael Lichten and Alistair Goldman for plasmids. We

thank Joanne Engebrecht, Wolf-Dietrich Heyer, and Michael Catlett for helpful discussions. We

also thank Dan Ohde, Kelly Komachi, Ira Hall, and helpful reviewers for critical evaluation of

the manuscript. This work was supported by a grant from the American Cancer Society (RSG-

01-053-01-CCG) and the National Institutes of Health (5RO1GM75119-2) to S.M.B. and a

University of California Dissertation Year Fellowship to J.C.M.

TABLE 1

Yeast strains

Diploid Haploid Genotype

Collision testers:

MATa his4::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP

SBY2074 MATα his4::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa THR1::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP

SBY2257 MATα THR1::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa THR1::URA3:arg4-nsp arg4∆::LEU2:GPD1:loxP

SBY2258 MATα THR1::URA3:arg4-bgl arg4∆::LEU2:loxP:ade2

MATa DED81::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP

SBY2186 MATα DED81::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa DED81::URA3:arg4-nsp arg4∆::LEU2:GPD1:loxP

SBY2185 MATα DED81::URA3:arg4-bgl arg4∆::LEU2:loxP:ade2

MATa YHL022c DED81::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP

SBY3153 MATα yhl022c∆::KanMx DED81::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa YHL022c DED81::URA3:arg4-nsp arg4∆::LEU2:GPD1:loxP

SBY3154 MATα yhl022c∆::KanMx DED81::URA3:arg4-bgl arg4∆::LEU2:loxP:ade2

Linkage Controls:

MATa THR1::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP:ade2

SBY2712 MATα THR1::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa THR1::URA3:arg4-nsp arg4∆::LEU2:GPD1:loxP:ade2

SBY2711 MATα THR1::URA3:arg4-bgl arg4∆::LEU2:loxP:ade2

MATa DED81::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP:ade2

SBY2710 MATα DED81::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa DED81::URA3:arg4-nsp arg4∆::LEU2:GPD1:loxP:ade2

SBY2709 MATα DED81::URA3:arg4-bgl arg4∆::LEU2:loxP:ade2

MATa YHL022c DED81::URA3:arg4-bgl arg4∆::LEU2:GPD1:loxP:ade2

SBY3155 MATα yhl022c∆::KanMx DED81::URA3:arg4-nsp arg4∆::LEU2:loxP:ade2

MATa YHL022c DED81::URA3:arg4-nsp arg4∆::LEU2:GPD1:loxP:ade2

SBY3156 MATα yhl022c∆::KanMx DED81::URA3:arg4-bgl arg4∆::LEU2:loxP:ade2

All strains used in this study were derived from SK1 haploids SBY1756 (MATa ho::hisG

leu2::hisG ura3∆::hisG trp1::hisG ade2∆::hisG GAL3 lys2::GAL1:Cre:LYS2) and SBY1074

(MATα ho::hisG leu2::hisG ura3∆::hisG trp1::hisG ade2∆::hisG GAL3 lys2).

TABLE 2

Coincidence of Collisions and Conversions at Three Distances

A B C D E F G H

Inserta Phaseb Arg+/CFU c Ade+/CFU d Le S(exp)f S(obs)g p-valueh

GPD1-bgl 0.24 ± 0.01% 4.4 ± 0.5% 81 ± 5% 7.0 ± 0.9% 13.3 ± 0.4% 0.012* DED81

ade2-bgl 0.24 ± 0.03% 4.5 ± 0.8% 23 ± 2% 2.1 ± 0.6% 2.7 ± 0.4% 0.214

GPD1-bgl 0.98 ± 0.13% 4.8 ± 0.8% 78 ± 3% 7.5 ± 1.0% 4.8 ± 0.5% 0.011* THR1

ade2-bgl 0.80 ± 0.14% 4.5 ± 0.5% 20 ± 5% 1.8 ± 0.6% 1.5 ± 0.1% 0.400

HIS4 n/a 1.96 ± 0.24% 4.7 ± 0.5% 50% 4.7 ± 0.5% 3.2 ± 0.7% 0.048

Arg+, Ade+, and Ade+ / Arg+ at varying heteroallele sites relative to loxP constructs.

Percentage values indicate average ± standard deviation from triplicate experiments. Error was

propagated to calculate S(exp) = 2 L C.

a Location of URA3:arg4-bgl and URA3:arg4-nsp heteroallele constructs

b Coupling of URA3:arg4-bgl to either GPD1:loxP or loxP:ade2 (TABLE 1, FIGURE 2)

c R, Arg+ gene conversion frequency per chromatid at arg4 heteroalleles

d C, Ade+ allelic loxP collision frequency per chromatid

e L, Linkage phase deviation: Ade+ / Arg+ chromatid in linkage control strains, containing

GPD1:loxP:ADE2

f S(exp), Expected Ade+ / Arg+ chromatid ( 2 L C )

g S(obs), Observed Ade+ / Arg+ chromatid in collision tester strains

h Student's t-tests (2-tails) compare S(obs) to S(exp). * indicates a significant difference (p <

0.05). For HIS4, repeated experiments showed a trend towards a decrease, but it was always

borderline significant.

TABLE 3

Frequencies of CR and NCR genotypes among Arg+ gene converted spores

CR1 NCR1 NCR2 CR2

Phase GenR Ade+ (M) GenS Ade+ (N) GenR Ade- GenS Ade-

GPD1-bgl 55.4 ± 1.9% 26.9 ± 1.3% 15.1 ± 2.0% 2.6 ± 1.3%

ade2-bgl 2.5 ± 1.0% 15.1 ± 3.0% 28.0 ± 3.3% 54.3 ± 5.2%

Frequencies of the four possible Gen and Ade genotypes among Arg+ gene converted

spores for the linkage control strains SBY3155 and SBY3156, as shown schematically in FIGURE

4. Phase indicates which loxP construct is coupled to the arg4-bgl heteroallele; GPD1-bgl

indicates data obtained from SBY3155, and ade2-bgl indicates data from SBY3156. The first

two columns indicate CR-associated and NCR-associated Arg+ Ade+ spores, respectively, which

are used as M and N. The second two columns indicate NCR-associated and CR-associated Arg+

Ade- spores, respectively.

TABLE 4

Frequencies of Ade+ collisions amongst CR and NCR associated Arg+ spores

Ade+ GenR / Arg+ Ade+ GenS / Arg+

Xbgl Xnsp Obgl Onsp

Obs 13.2 ± 3.2% 0.78 ± 0.17% 2.9 ± 1.0% 2.5 ± 1.0%

Exp 7.4 ± 1.0% 0.35 ± 0.15% 3.6 ± 0.3% 2.0 ± 0.5%

p-value 0.029* 0.029* 0.311 0.525

Observed and expected values of Ade+ collisions amongst CR-associated Arg+ gene

convervants (X) and NCR-associated Arg+ gene convertants (O) were measured as the frequency

of Ade+ GenR and Ade+ GenS genotypes among Arg+ spores, respectively. X(exp) and O(exp)

were calculated as 2 M C and 2 N C, respectively. X(obs) and O(obs) were determined using

SBY3133 and SBY3134 for GPD1-bgl and ade2-bgl linkage phases respectively. P-values were

calculated using 2-tailed t-tests.

FIGURE LEGENDS

FIGURE 1: Two-locus assay system for measuring collisions and gene conversions: (a) The

frequency of Cre-mediated loxP recombination (or collisions) indicates the relative spatial

proximity of two loxP sites engineered into the genome. (b) Structure of the heterozygous loxP

constructs inserted at the arg4∆ locus. Collisions are measured as the frequency of Ade+

prototroph formation upon Cre induction from a GAL1 promoter in cells synchronized to enter

meiosis. Only one of the two interacting homologous chromatids expresses ADE2 following

Cre-mediated recombination. (c) Structure of the arg4-bgl/arg4-nsp heteroallele pair. Either the

proximal nsp mutation or the distal bgl mutation can be gene converted using wild type

information from the other allele to generate an ARG4 chromatid. Double-strand DNA break

(DSB) hotspots are located immediately proximal and distal to the arg4 gene. (d) Schematic

map (not to scale) indicating the relative positions of the loxP collision reporter at arg4∆ (black

vertical bar) and the URA3:arg4 heteroallele insertion sites (black triangles). DED81, THR1,

and HIS4 are 3kb, 25kb, and unlinked to the loxP constructs respectively.

FIGURE 2: The linkage phase of the arg4 heteroalleles and the loxP constructs affects the

coincidence of Arg+ gene conversion and Ade+ collision in spores: In (a) and (b) collision tester

strains are illustrated (SBY2186 and SBY2185 respectively for DED81 inserts; SBY2257 and

SBY2258 respectively for THR1 inserts; TABLE 1). In (c) and (d), control strains are illustrated

(SBY2709 and SBY2710 respectively for DED81 inserts; SBY2711 and SBY2712 respectively

for THR1 inserts). In (a), where arg4-bgl is coupled to the GPD1:loxP allele, coincident Ade+

Arg+ spores are generated by a collision and a gene conversion of arg4-bgl associated with no

crossover (NCR) or a distal crossover (dCR) between arg4 and loxP, or alternatively by

conversion of arg4-nsp with an associated proximal crossover (pCR). In (b), where arg4-bgl is

coupled to the loxP:ade2 allele, the situation is reversed, with collisions accompanied by arg4-

nsp conversions associated with NCR or dCR and arg4-bgl pCR conversions generating Ade+

Arg+ spores. In (c) where arg4-bgl is coupled to the GPD1:loxP:ade2 allele, coincident Ade+

Arg+ spores are generated by gene conversion of arg4-bgl with NCR or dCR between arg4 and

loxP, or alternatively by conversion of arg4-nsp with an associated pCR. In (d), where arg4-bgl

is coupled to the loxP:ade2 allele, the situation is reversed, with arg4-nsp NCR/dCR conversions

and arg4-bgl pCR conversions generating Ade+ Arg+ spores. Note that only 2-strand double

events are illustrated; one of the two classes of 3-strand doubles can also generate coincident

spores, whereas the 4-strand double and other class of 3-strand double can never generate

coincident spores.

FIGURE 3: Gal-induction time course shows increased coincidence of gene conversions and

allelic loxP collisions for tightly linked sites in late prophase I. Cre was induced with galactose

at t=1, 2, 3, or 4 hours after transfer to sporulation medium for SBY2186 and SBY2709, as

indicated on the x-axis. On the primary y-axis, yellow bars indicate the expected frequency of

Ade+ among Arg+ spore clones, Sbgl(exp) = 2 L C, while dark blue bars indicate the observed

frequency of Ade+ among Arg+ spore clones, Sbgl(obs). On the secondary y-axis, the red line

shows the ratio Sbgl(obs) / Sbgl(exp).

FIGURE 4: Schematic of linkage control strains for measuring relative CR and NCRs per Arg+

gene converted spore: (a) SBY3155, and (b) SBY3156. Data are summarized in TABLE 3.

Annotations are as in FIGURE 2, except that blue boxes indicate a KanMx marker (conferring a

GenR phenotype) inserted immediately distal to DED81 at YHL022c. Below each schematic are

shown the recombinant classes sorted by genotype. For the collision tester strains, the situation

is the same, except only Ade+ spores generated by loxP collision are informative regarding the

class of associated Arg+ gene conversion.

FIGURE 1

FIGURE 2

COLLISION TESTERS:

LINKAGE CONTROLS:

FIGURE 3

FIGURE 4

REFERENCES

ALLERS, T., and M. LICHTEN, 2001 Differential timing and control of noncrossover and

crossover recombination during meiosis. Cell 106: 47-57.

BISHOP, D. K., and D. ZICKLER, 2004 Early decision; meiotic crossover interference prior to

stable strand exchange and synapsis. Cell 117: 9-15.

BORDE, V., T. C. WU and M. LICHTEN, 1999 Use of a recombination reporter insert to define

meiotic recombination domains on chromosome III of Saccharomyces cerevisiae. Mol

Cell Biol 19: 4832-4842.

BORNER, G. V., N. KLECKNER and N. HUNTER, 2004 Crossover/noncrossover differentiation,

synaptonemal complex formation, and regulatory surveillance at the leptotene/zygotene

transition of meiosis. Cell 117: 29-45.

BURGESS, S. M., and N. KLECKNER, 1999 Collisions between yeast chromosomal loci in vivo are

governed by three layers of organization. Genes & Dev. 13: 1871-1883.

CARPENTER, A. T., 1987 Gene conversion, recombination nodules, and the initiation of meiotic

synapsis. Bioessays 6: 232-236.

DE LOS SANTOS, T., N. HUNTER, C. LEE, B. LARKIN, J. LOIDL et al., 2003 The Mus81/Mms4

endonuclease acts independently of double-Holliday junction resolution to promote a

distinct subset of crossovers during meiosis in budding yeast. Genetics 164: 81-94.

DERNBURG, A. F., K. MCDONALD, G. MOULDER, R. BARSTEAD, M. DRESSER et al., 1998

Meiotic recombination in C. elegans initiates by a conserved mechanism and is

dispensable for homologous chromosome synapsis. Cell 94: 387-398.

FRANCIS, K. E., S. Y. LAM, B. D. HARRISON, A. L. BEY, L. E. BERCHOWITZ et al., 2007 Pollen

tetrad-based visual assay for meiotic recombination in Arabidopsis. Proc Natl Acad Sci U

S A 104: 3913-3918.

FUNG, J. C., B. ROCKMILL, M. ODELL and G. S. ROEDER, 2004 Imposition of crossover

interference through the nonrandom distribution of synapsis initiation complexes. Cell

116: 795-802.

GASIOR, S. L., H. OLIVARES, U. EAR, D. M. HARI, R. WEICHSELBAUM et al., 2001 Assembly of

RecA-like recombinases: distinct roles for mediator proteins in mitosis and meiosis. Proc

Natl Acad Sci U S A 98: 8411-8418.

GOLDMAN, A. S., and M. LICHTEN, 1996 The efficiency of meiotic recombination between

dispersed sequences in Saccharomyces cerevisiae depends upon their chromosomal

location. Genetics 144: 43-55.

HILDEBRANDT, E. R., and N. R. COZZARELLI, 1995 Comparison of recombination in vitro and in

E. coli cells: measure of the effective concentration of DNA in vivo. Cell 81: 331-340.

HUNTER, N., and N. KLECKNER, 2001 The single-end invasion: an asymmetric intermediate at

the double-strand break to double-holliday junction transition of meiotic recombination.

Cell 106: 59-70.

HURST, D. D., S. FOGEL and R. K. MORTIMER, 1972 Conversion-associated recombination in

yeast (hybrids-meiosis-tetrads-marker loci-models). Proc Natl Acad Sci U S A 69: 101-

105.

KANE, S. M., and R. ROTH, 1974 Carbohydrate metabolism during ascospore development in

yeast. J Bacteriol 118: 8-14.

KEENEY, S., C. N. GIROUX and N. KLECKNER, 1997 Meiosis-specific DNA double-strand breaks

are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88: 375-

384.

KLAPHOLZ, S., and R. E. ESPOSITO, 1982 A new mapping method employing a meiotic rec-

mutant of yeast. Genetics 100: 387-412.

LESSER, C. F., and C. GUTHRIE, 1993 Mutational analysis of pre-mRNA splicing in

Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics 133: 851-

863.

LOIDL, J., F. KLEIN and H. SCHERTHAN, 1994a Homologous pairing is reduced but not abolished

in asynaptic mutants of yeast. J Cell Biol 125: 1191-1200.

LOIDL, J., H. SCHERTHAN and D. B. KABACK, 1994b Physical association between

nonhomologous chromosomes precedes distributive disjunction in yeast. Proc Natl Acad

Sci U S A 91: 331-334.

LUI, D. Y., T. L. PEOPLES-HOLST, J. C. MELL, H. Y. WU, E. DEAN et al., 2006 Analysis of close

stable homolog juxtaposition during meiosis in mutants of Saccharomyces cerevisiae.

Genetics.

MCMAHILL, M. S., C. W. SHAM and D. K. BISHOP, 2007 Synthesis-dependent strand annealing in

meiosis. PLoS Biol 5: e299.

MAHADEVAIAH, S. K., J. M. TURNER, F. BAUDAT, E. P. ROGAKOU, P. DE BOER et al., 2001

Recombinational DNA double-strand breaks in mice precede synapsis. Nat Genet 27:

271-276.

MALKOVA, A., J. SWANSON, M. GERMAN, J. H. MCCUSKER, E. A. HOUSWORTH et al., 2004

Gene conversion and crossing over along the 405-kb left arm of Saccharomyces

cerevisiae chromosome VII. Genetics 168: 49-63.

MALOISEL, L., J. BHARGAVA and G. S. ROEDER, 2004 A role for DNA polymerase delta in gene

conversion and crossing over during meiosis in Saccharomyces cerevisiae. Genetics 167:

1133-1142.

MARTIN, S. S., V. C. CHU and E. BALDWIN, 2003 Modulation of the active complex assembly

and turnover rate by protein-DNA interactions in Cre-LoxP recombination. Biochemistry

42: 6814-6826.

MCKEE, A. H., and N. KLECKNER, 1997 Mutations in Saccharomyces cerevisiae that block

meiotic prophase chromosome metabolism and confer cell cycle arrest at pachytene

identify two new meiosis-specific genes SAE1 and SAE3. Genetics 146: 817-834.

MERKER, J. D., M. DOMINSKA and T. D. PETES, 2003 Patterns of heteroduplex formation

associated with the initiation of meiotic recombination in the yeast Saccharomyces

cerevisiae. Genetics 165: 47-63.

MOENS, P. B., D. J. CHEN, Z. SHEN, N. KOLAS, M. TARSOUNAS et al., 1997 Rad51

immunocytology in rat and mouse spermatocytes and oocytes. Chromosoma 106: 207-

215.

MORTIMER, R., and S. FOGEL, 1974 Genetical interference and gene conversion. Plenum Press,

New York/London.

NAG, D. K., H. SCHERTHAN, B. ROCKMILL, J. BHARGAVA and G. S. ROEDER, 1995 Heteroduplex

DNA formation and homolog pairing in yeast meiotic mutants. Genetics 141: 75-86.

NICOLAS, A., and T. D. PETES, 1994 Polarity of meiotic gene conversion in fungi: contrasting

views. Experientia 50: 242-252.

NICOLAS, A., D. TRECO, N. P. SCHULTES and J. W. SZOSTAK, 1989 An initiation site for meiotic

gene conversion in the yeast Saccharomyces cerevisiae. Nature 338: 35-39.

PADMORE, R., L. CAO and N. KLECKNER, 1991 Temporal comparison of recombination and

synaptonemal complex formation during meiosis in S. cerevisiae. Cell 66: 1239-1256.

PAGE, S. L., and R. S. HAWLEY, 2003 Chromosome choreography: the meiotic ballet. Science

301: 785-789.

PAPAZIAN, H. P., 1952 The Analysis of Tetrad Data. Genetics 37: 175-188.

PAQUES, F., and J. E. HABER, 1999 Multiple pathways of recombination induced by double-

strand breaks in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 63: 349-404.

PAWLOWSKI, W. P., and W. Z. CANDE, 2005 Coordinating the events of the meiotic prophase.

Trends Cell Biol 15: 674-681.

PEOPLES, T. L., E. DEAN, O. GONZALEZ, L. LAMBOURNE and S. M. BURGESS, 2002 Close, stable

homolog juxtaposition during meiosis in budding yeast is dependent on meiotic

recombination, occurs independently of synapsis, and is distinct from DSB-independent

pairing contacts. Genes Dev 16: 1682-1695.

PEOPLES-HOLST, T. L., and S. M. BURGESS, 2005 Multiple branches of the meiotic

recombination pathway contribute independently to homolog pairing and stable

juxtaposition during meiosis in budding yeast. Genes Dev 19: 863-874.

PETRONCZKI, M., M. F. SIOMOS and K. NASMYTH, 2003 Un menage a quatre: the molecular

biology of chromosome segregation in meiosis. Cell 112: 423-440.

ROCKMILL, B., J. A. ENGEBRECHT, H. SCHERTHAN, J. LOIDL and G. S. ROEDER, 1995 The yeast

MER2 gene is required for chromosome synapsis and the initiation of meiotic

recombination. Genetics 141: 49-59.

SCHENA, M., D. PICARD and K. R. YAMAMOTO, 1991 Vectors for constitutive and inducible gene

expression in yeast. Methods Enzymol 194: 389-398.

SMITHIES, O., and P. A. POWERS, 1986 Gene conversions and their relation to homologous

chromosome pairing. Philos Trans R Soc Lond B Biol Sci 312: 291-302.

TARSOUNAS, M., T. MORITA, R. E. PEARLMAN and P. B. MOENS, 1999 RAD51 and DMC1 form

mixed complexes associated with mouse meiotic chromosome cores and synaptonemal

complexes. J Cell Biol 147: 207-220.

TESSE, S., A. STORLAZZI, N. KLECKNER, S. GARGANO and D. ZICKLER, 2003 Localization and

roles of Ski8p protein in Sordaria meiosis and delineation of three mechanistically

distinct steps of meiotic homolog juxtaposition. Proc Natl Acad Sci U S A 100: 12865-

12870.

THORNE, L. W., and B. BYERS, 1993 Stage-specific effects of X-irradiation on yeast meiosis.

Genetics 134: 29-42.

WACH, A., A. BRACHAT, R. POHLMANN and P. PHILIPPSEN, 1994 New heterologous modules for

classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-

1808.

WEINER, B. M., and N. KLECKNER, 1994 Chromosome pairing via multiple interstitial

interactions before and during meiosis in yeast. Cell 77: 977-991.

WU, H. Y., and S. M. BURGESS, 2006 Ndj1, a telomere-associated protein, promotes meiotic

recombination in budding yeast. Mol Cell Biol 26: 3683-3694.

WU, T. C., and M. LICHTEN, 1994 Meiosis-induced double-strand break sites determined by

yeast chromatin structure. Science 263: 515-518.

WU, T. C., and M. LICHTEN, 1995 Factors that affect the location and frequency of meiosis-

induced double-strand breaks in Saccharomyces cerevisiae. Genetics 140: 55-66.

XU, L., M. AJIMURA, R. PADMORE, C. KLEIN and N. KLECKNER, 1995 NDT80, a meiosis-

specific gene required for exit from pachytene in Saccharomyces cerevisiae. Mol. Cell

Biol. 15: 6572-6581.