Copyright 8 1995 by the Genetics Society of America
Factors That Affect the Location and Frequency of Meiosis-Induced
DoubleStrand Breaks in Saccharomyces c e r e e
Tzu-Chen Wu and Michael Lichten
Laboratmy of Biochemistry, Division of Cancer Biology, Diagnosis and Centers, National Cancer Institute, Bethesda, Maryland 20#2
Manuscript received November 11, 1994
Accepted for publication January 20, 1995
ABSTRACT
Double-strand DNA breaks (DSBs) initiate meiotic recombination in Saccharomyces cerevisiae. DSBs
occur at sites that are hypersensitive in nuclease digests of chromatin, suggesting a role for chromatin
structure in determining DSB location. Weshow here that the frequency ofDSBs at a site is not
determined simply by DNA sequence or by features of chromatin structure. An argkontaining plasmid
was inserted at several different locations in the yeast genome. Meiosis-induced DSBs occurred at similar
sites in pBR322derived portions of the construct at all insert loci, and the frequency of these breaks
varied in a manner that mirrored the frequency of meiotic recombination in the arg4 pcrtion of the
insert. However, DSBs did not occur in the insert-borne a@ gene at a site that is frequently broken at
the normal ARM locus, even though the insert-borne arg4 gene and the normal ARM locus displayed
similar DNase I hypersensitivity patterns. Deletions that removed active DSB sites from an insert at HIS4
restored breaks to the insert-borne arg4 gene and to a DSB site in flanking chromosomal sequences.
We conclude that the frequency of DSB at a site can be affected
by sequencesseveral thousand nucleotides
away and suggest that this is because of competition between DSB sites for locally limited factors.
M
EIOTIC recombination plays important roles
both in potentiating homologue pairing and in
ensuring the properdisjunction of homologues during
the first meiotic division (ROEDER 1990; KLECKNER et
al. 1991). In the yeast Saccharomyces cerevisiae, much,
and possibly all, meiotic recombination is initiated via
the formation and subsequent repair of double-strand
DNA breaks ( DSBs) . Sequences necessary for theactivity ofmeiotic recombination hot spots have been shown
to be the site of transient meiosis-induced DSBs (SUN
et al. 1989; CAO et al. 1990; GOLDWAY et al. 1993; NAG
and PETES1993). Thetime of formation and repair of
these breaks is consistent with a role in initiating meiotic recombination (PADMORE
et al. 1991; GOYON and
LICHTEN1993), and deletions and other rearrangements that alter the
frequency of meiotic exchange
events display parallel alterations in the DSB frequencies (ROCCO et al. 1992; DE MASSY and NICOLAS
1993;
FAN et al. 1995). DSBs also occur in regions that do
not contain obvious recombination hotspots, and the
distribution of these breaks closely parallels that of meiotic crossovers, both over entire chromosomes (GAME
1992; ZENVIRTHet al. 1992) and over smaller regions
( WU and LICHTEN1994).
Factors other than primary DNA sequence, most notably chromatinstructure,helptodetermine
where
DSBs occur. DSB sites are hypersensitive in both DNase
I and micrococcal nuclease digests of chromatin, and
changes in chromatin structure that alter a site’s sensiCorresponding author: Michael Lichten, Bldg. 37, Room 4D14,
N.I.H., Bethesda, MD 20892. E-mail: lichten@helix.nih.gov
Genetics 140: 55-66 (May, 1995)
tivity to exogenous nucleases are accompanied by parallel changes in DSB patterns ( OHTAet al. 1994; WU and
LICHTEN1994). Studies of DSBs at theARG4 locus also
provide evidence for the importance of factors other
than primary DNA sequence. DSB occur at this locus
“200 bp upstream of coding sequences, coincident
with a region that contains a hot spot for the initiation
of meiotic recombination (NICOLAS
et al. 1989; SUN et
al. 1989). Rearrangements that change the chromosomal environment of the ARM gene can significantly
alter DSB frequencies at this site (ROCCO et al. 1992;
DE MASSY and NICOLAS1993; GOYON and LICHTEN
1993). In one case, a rearrangement that significantly
reduced thefrequency of DSBs contained normalARG4
locus sequences for more than 1 kb on either side of
the ARG4 DSB site ( GOYON and LICHTEN1993) . It is
therefore likely that entities that influence DSB frequencies act over at least this distance.
Evidence for action at a distance is also provided by
studies of meiotic recombination between a pair of leu2
mutant alleles inserted at various locations in the yeast
genome ( LICHTENet al. 1987). The set of insert loci
examined displayed a 30-fold range in frequencies of
LEU2 meiotic recombinants. Because these inserts
shared sequence identity for more than 2 kbp on each
side of the leu2 alleles used, it was concluded that entities present in flanking chromosomal sequences could
act over at least this distance to modulate the frequency
of meiotic recombination within an interval.
To further examine theidentity of factors capable
of influencing the frequencyof DSBs at a given locus,
we determined thelocation and frequency of meiosis-
T.-C. Wu and M.Lichten
56
TABLE
1
Yeast strains
Name
Relevant genotype"
Strains used for random spore analysis
arg4-bgl h 2 - R
~-
MJL1059
MJL1496
MJL1077,
MJLlO78
MJL1206
MJL1211
MJLI501
MJL1094
MJL1068
MJL1124
MJL1069
MJL1096,
MJL1097
MJL1016,
MJL1017
MJL1018,
MJLlO19
MJL1014, MJL1015
MJLlOSO
arg4-nsp h 2 - K
arg4-nsp his4-X h 2 - K
arg4-bgl his4-B h 2 - R
arg4-nsp, bgl h 2 - R his4::URA3. arg4nsp
arg4-nsp, bgl leu2-X his4::URA3- are-bgl
arg4-nsp, bgl h 2 - R his4::URA3. arg4-nsp
arg4-bgl
h 2 - K h154
arg4-nsp, bgl h 2 - K his4::URA3. arg4-bgl
arg4-nsp
leu2-R HIS4
arg4-nsp, bgl h.%R his4A(sal-Cla)::UR43A (Sm-Barn) mg4-nsp.A(Sal-Eo)
arg4-nsp, bgl h 2 - K his4A(Sal-Cla)::URA3A(Sma-Barn).arg4-bgl. A(Sa1-Eco)
arg4-nsp, bgl h2::URA3. arg4-nsp
a@-nsp, bgl h2::URA3- arg4-bgl
a@-nsp, bgl h 2 - R MTa::URA3. a@-nsp
arg4-nsp, bgl h 2 - K MATcu::URA3*arg4bgl
arg4A(Hpa)::URA3 leu2-R MATa::URA3* arg4-nsp
arg4A(Hpa)::URA3 leu2-K MATcu::URA3. arg4-bgl
wg4-nsp, bgl h 2 - R u~a3::UR43. arghsp
ar@-nsp, bgl leu2-K ura3::URA3- arg4-bgl
arg4-nsp, bgl h 2 - R CHAl::URA3*arg4-nsp
arg4-nsp, bgl h 2 - K WAI::URA3. arg4bgl
leUZA(K-R) his4::URA3.h2-R
h 2 A ( K - R ) his4::URA3*h 2 - K
h 2 A ( K - R ) MATa::URA3- h 2 - R
le~2n(K-R) MATc~::URA~*
h2-X
h 2 A ( K - R ) ura3::URA3- h 2 - R
leuPA(K-R) ura3::URA3. h 2 - K
h 2 A 1 K - R ) CHAl::URA3*h 2 - R
h Z A ( K - R ) CHAI::UR43- h 2 - K
rad5OS strainsb
NKY1002
MJLll74
MJLl170
MJLll7S
MJL1373
MJLl248
MJL1382
MJLl384
rad5@KI81- U ! 3
rad50-Kl81- URA3
rad5@Kl8I::URA3 h 2 A ( K - R ) his4::URA3- leu2-R
rad5O-ZU81::URA3 1eu2
h154
rad50-U81* URA3 arg4+~p,
bgl h 2 - R his4::UL43* arg4nsp
rad5@IU81*cTRA3 ARG4
h 2 - R his4::UR43* arg4-nsp
rd50-KI81 URA3 arg4-nsp, bgl h 2 - R his4::URA3. a e - n s p
rad5@Kl8I*URA3 ARG4
LEU2 HIS4
rad50-KI81. URA3 his4::URA3. A(Sma-Barn) * arg4-nsp
rad5@KI81*URA3 his4::URA3. A (Sma-Bam)* arg4-nsp
rad5@KI81 URA3 h Z - R his4::URA3. A(Sma-Barn) arg4-nsp
rad5@Klt?l*URA3 h 2 - R HIS4
rad50"81* ura3::LEU2 h 2 A ( K - R ) his4A(Sal-Clu)::URA3. arg4-nsp-A(Sa1-Eco)
rad50-Kl81- ura3::LEU2 h 2 A ( K - R ) his4A(Sal-Cla)::URA3-arg4-bgl- A(Sa1-Eco)
rad5@KI81*ura3::LEUZ h 2 A ( K - R ) HIS4
rad5lkKl81- ura3::LEUZ k 2 A ( K - R ) his4A(Sal-CLa)::URA3-a ~ 4 - b g lA(Sal-Eco)
.
57PositionMeiosis
Effects in Yeast
TABLE 1
Continued
Name
genotype”
rad5@KI81*URA3 ARG4
h 2 - R his4A(SaGClu)::URA3*A(Sma-Bam) arg4-nsp. A(Sa1-Eco)
rad5@KI81- URA3 arg4-nsp,bgl LEU2 his4A(Sal-Cla)::URA3*A(Sma-Bam)* arg4-bgl- A(Sa1-Eco)
HIS4
rad5@KZ81*URA3 ARG4
rad5CLKI81- URA3 a@nsp,bgl his4A(Sal-Cla)::URA3*A(Sma-Bam) arg4-bgl. A(Sa1-Eco)
rad5@H81-URA3 arg4-nsp, bgl h2::URA3- arg4-nsp
rad5@KI81- URA3 ARG4
LEU2
rad5@KI81*URA3 arg4-nsp, bgl h 2 - R MATa::URA3- a@-nsp
LEU2 MATa
rad5@KI81*URA3 ARG4
rad5@KI81*URA3 arg4nsp,bgl h 2 - K MATa::URA3. arg4-nsp
LEU2 MATa
rad5@KI81- URA3 ARG4
radS@KI81*URA3 ar@nsp,bgl h 2 - R ura3::URA3. arg4-nsp
LEU2 ura3
rad5@H81URA3 ARG4
rad5@KI81*URA3 h 2 - R CHAl::URA3. arg4-nsp
rad5@KI81*URA3 LEU2 CHAl
MJL1515
-
MJL1517
MJL1184
MJLl176
MJL1481
MJL1177
MJLll85
Strains MJL1059, MJLlO68 and MJL1124 are from GOYONand LIGHTEN1993; strain NKY1002 is from CAO et al. 1990.
a The h4ATa parent of each diploid is on top. All strains are homozygous for the following: ura3, 4s2, ho::LYS2. Mutant alleles
are described in Table 2. Insert structures are illustrated in Figure1. Unless indicated, all strains wereconstructed for this work.
*All rad50S strains contain a URA3 insert adjacent to the RAD50 locus, denoted here as rad5@Kl81-URA3. In MJL1382 and
MJL1384, this URA3 insert is disrupted by a LEU2 gene insert.
induced DSB within argkontaining
an
pBR322 construct. We examinedtheeffect
of locationin the
genome On both the frequency Of DSB and the fiequency of meioticrecombination within the construct. We also determined theeffect of deleting portions of this construct on DSBs within the construct
and at a nearby site’ we
that entities
thousand
away can affect the frequency
of breaks at a site and discuss possible mechanisms
responsible for these effects.
MATERIALS
AND
METHODS
Yeast strains: Allyeast strains are isogenic derivatives of
SK1 (WE
and ROTH1974). Genotypes are listed in Table
1; mutant alleles are described in Table 2. The h 2 A ( K - R )
allele was made by digesting a LEU2 derivative ofpMJ55
(LIGHTEN et al. 1987) with Asp718 and EcoH, conversion to
flush ends with T4 DNA polymerase, and ligation with T4
DNA ligase to form pMJ75. The h 2 A ( K - R )allele was inserted
at LEU2 by two-step replacement ( SCHERER
and DAVIS1979),
with plasmid excision being selected with 5-fluorwrotic acid
(BOEKEet al. 1984).
TABLE 2
Mutant alleles
Allele
arg4-nsp
arg4-bgl
a@-mp, bgl
a~g4A(Hpal)::UR43
his4-X
his4-B
his4A(Sal-Cla)
h2-K
h2-R
h2A(K-R)
rad5@M81
A(Sma-Bam)
A(Sa1-Eco)
Description
G -+ C transversion in ARG4 initiation
codon
4bp fill-in of BglII site at in ARM
Double mutation
Deletes entire ARM gene
4bp fill-in of XhoZsite in HIS4
4bp fill-in of BglII site in HZS4
Deletes between SalI and ClaI in HZS4
4bp deletion of the LEU2 QnI site
4bp fill-in of the LEU2 EcoRI site
Deletes between QnI and EcoRI in LEU2
Separation of function mutation; referred
to as rad50S in text
Deletes plasmid sequences from a SmaI
site in the URA3 Hind111 fragment and
the BamHI site in pBR322; referred to
as A L
Deletes pBR322 sequences from SalI to
EcoRI; referred to as AR
Refer
NICOLASet al. (1989)
NICOLASet al. (1989)
GOYON and LICHTEN
(1993)
GOYON and LIGHTEN
(1993)
CAO et al. (1990)
CAO et al. (1990)
This work
LIGHTEN
et al. (1987)
et al. (1987)
LIGHTEN
This work
ALANI et al. (1990)
This work
This work
C.
58
Wu and M. Lichten
Yeast strains with a@ or leu2 alleles inserted at loci other
than the normal locus (designated as X ::arg4 or X ::leu2)
contain the pBR322derived plasmid inserts illustrated
in Figure 1. Plasmids for leu2 inserts at HIS4 (pLED2, pLED3),
MAT (pMJ24, pMJ25), and at URA3 (pMJ54, pMJ55) have
been described ( LICHTEN
et al. 1987). Plasmids for inserts at
CHAl were made by inserting a 1.4kb HindIII-BamHI fraget al.
ment [nt 14838-16263 on chromosome ZII (OLIVER
1992) ] at the EcoRI site of pMJ54 or pMJ55 to create pMJ147
and pMJ140. Plasmids for arg4-nsp and urg4-bgi inserts at MAT
(pMJ109 and pMJl11) have been described ( GOYON and
LICHTEN1993); plasmids for full-lengthinserts at HIS4
(pMJl2l and pMJ123), at URA3 (pMJll3 and pMJ115) and
at CHAl (pMJ173 and pMJ174) are identical to those used
for leu2 inserts at these loci, except that 3.3-kb arg4 fragments
from pMJ109 or pMJlllinserted between the pBR322 B u d 1
and Sal1 sites replace the leu2 gene. leu2::arg4 inserts were
constructed by inserting a BstEII-EcoRV fragment internal to
LEU2 (ANDREADIS et al. 1984) at the EcoRI site of pMJll3 and
pMJ115. Plasmids for his4:: URA3. A(Sma-Bum). arg4 inserts
(pMJ250 and pMJ251) were created by digesting pMJ121 or
pMJ123 with SmaI, ligation to BamHI linkers, digestion with
BumHI, and subsequent ligation. Plasmidsforhis4A(SalCla)::URA3*arg4-A(Sal-Eco)inserts (pMJ320 and pMJ321)
were created by replacing the SalI-PmII segment of pMJl21
and pMJ123 with a 1.1-kb SalI-AruIIfragment from HIS4 (nt
67826-68951 on chromosome ZZI). Plasmids for his4A(SalCla) ::URA3A(Sma-Bam) arg4- A(Sal-Eco) inserts werecreated by combining the appropriate portions ofpMJ250,
pMJ251, pMJ320 and pMJ321.
Insert-containing rd5OS haploid strains wereusually made
by crossing the appropriate RAD50 strain with a haploid parent ofNKYl002 (ALANI et al. 1990). Haploid parents of
MJLl382 and MJLl384 wereconstructed by transforming the
two haploid parents of NKYl002 with a LEU2 disruption of
the URA3 gene (kindly given by J. HABER) , to yield radS0KI81. ura3::LEUZ. These strains were then transformed directly with pMJ320 or pMJ321.
Genetic analysis of meioticsegregants: Random spore
analysis was performed as previously described ( LICHTEN et
al. 1987).
Analysii of DNA and chromatin from premeiotic and meioticcells. Sporulation and DNA preparation were as described (GOYONand LICHTEN 1993). Meiotic DNA from
rad5OSstrains was prepared 5-6.5 hr after initiation of sporulation. Chromatin was prepared and digested with DNase I
as described (Wu and LICHTEN1994). DNAwas digested
with restriction endonucleases, displayed on agarose gelsand
transferred to membranes using establishedtechniques (SA"
BROOK et al. 1989) . Radioactive probes were prepared by random hexamer priming of DNA fragments that were agarose
gel purified, either from restriction endonuclease digests or
from polymerase chain reactions using appropriate plasmids
and oligonucleotide primers. A small amount of bactericphage A DNA (usually 0.2% of the mass of DNA) wasincluded
in labeling reactions to allow detection of Hind111 or BstEII
digests of A DNA included in gels as size standards. Membranes were hybridized with probe as described ( GOYON and
LICHTEN
1993) except that 2 X SSPEwas used for hybridization and filters were washed once at room temperature in 2X
SSPE, 0.1% SDS for 15 min and twice in 55" 0.1X SSPE, 0.1%
SDS. Radioactivity was visualized by autoradiography or with
a Fuji BAS2000 phosphorimager.
Determination of DSB location and frequencies: DSB locations were determined by comparing DSB band positions with
those of bands in size standards. Hind111 or BstEII digests of
bacteriophage A DNA and digests of mitotic yeast DNA with
appropriate restriction endonucleases were used as size stan-
dards. Restriction enzyme combinationsand dilutions of DNA
were chosen that yielded bands in standard lanes of about
the same size and intensity as the DSB bands to be examined.
DSB frequencies were determined by a combination of video
densitometry of autoradiograms ( GOYON and L~CHTEN1993)
and phosphorimage analysis.DSB band intensity was compared with the intensity of bands in standard lanes by video
densitometry; the amount of radioactivity at standard band
positions was in turn compared to total lane radioactivity or
to radioactivityat full-length fragment band positions. In early
experiments, this was accomplished by including dilutions of
standards sufficient to span the range of intensities between
DSB bands and uncut full-length fragments, thus bypassing
the limitations on linearity imposed by film. In later experiments, phosphorimage analysis was used the extended range
of linear measurement offered by this approach allowed a
reduction in the number of dilutions used.
RESULTS
Meioticrecombinationat leu2 and arg4 insert loci
displayspositioneffects:
We wished todetermine
whether or not theposition effects observed in studies
of meiotic recombination within leu2 inserts (LIGHTEN
et al. 1987) were unique to the gene used, and to ask
if meiosis-induced DSBs were also subject to position
effects. We therefore created, in the SK1 background,
a series of diploid strains containing leu2 inserts similar
to those previously used and a series of diploids in which
the leu2 segment of the insert was replaced by a 3.3-kb
ARM fragment marked with either arg4-nsp or arg4-bgl
( GOYONand LICHTEN1993), The structures of these
inserts are shown in Figure 1. Meiotic recombination
at each locus was monitored by measuring the frequency of Leu' or k g + spores produced by heteroallelic diploids.
Frequencies of meiotic recombination at leu2 and
arg4 insert loci displayedsimilar position effects (Table
3 ) . The set of diploids with leu2 inserts displayed about
a 10-fold range in the frequency of Leu+ recombinants,
consistent with that previously observed (LIGHTEN
et al.
1987). The range in frequencies of Arg' recombinants
was less (about fourfold), butthe order of loci was the
same for both arg4 and leu2 inserts (HIS4 > MAT >
URA3 2 CHAl) . All insert loci share 3.7 kb of PBR322
sequences to the rightof the arg4- and leu2-specific fragments and, with the exception of the inserts at URA3,
share 1.5 kb of pBR322 and URA3 sequences to the left
(Figure 1) . These locus-dependent effects on recombination must therefore involve action across these adjacent constant regions.
DSBs aresubject to position effects: The position
effects described above might be because of the action
of entities in flanking sequences that modulated the
frequency of DSB within the insert, which in turn initiated events producing Arg+or Leu+recombinants. To
examine this possibility, we determined the location
and frequency of DSBs in diploids that were homozygous for rad50-Kl81 (hereafter referred to as rad5OS;
ALANI et al. 1990). In these strains, DSBs persist through
59
Position Effectsin Yeast Meiosis
A
B
URA3
ARG4
URA3 LEU2
0 Q
i
i
E
ii
H
MAT
Pv
ii
c
his&
Pv C h l B
iii
ura3
LEU2
URA3
IV
iii
his%A3
-
? P
ARG4
P
FfEkEv
I I
E/%
C S Pv
his4 5'
Ekcs
Pv
A"
RG4 his4
5'
iv
Pv C W B
V
V
FIGURE 1.-Structure of leu2 and arg4 inserts. Thick lines are pBR322 sequences; unless otherwise indicated, thin lines are
chromosomal sequences that are duplicated as a result of plasmid integration. Hatched boxes are the URA3 HindIII fragment.
Open boxes are either the LEU2 XhoI-Sal1fragment or the ARG4 PstI fragment; horizontal arrows indicate the coding sequences
of these two genes. Restriction sites are as follows: B, BamHI; Bg, BglII; Bs, BstEII; C,
C M ; E, EcoRI; Ev,EcoRV; H, HindIII; P,
PstI; F'v, PVUII; S, SalI; Sm, SmaI; X, XhoI. For the sake of clarity, only relevant sites are shown. Strikeouts indicate restriction
sites that were destroyed during plasmid construction. 63 and Ei indicate the location of leu2-K and h2-R, respectively; 0 and
indicate the location ofarg4-nsp and a@-bgl, respectively.For further details of insert structures and construction, see
MATERIALSAND METHODS. Plasmid inserts at HIS4, LEU2, and ARG4 lead to loss of gene function; inserts at all other loci retain
at least one functional gene copy. ( A ) leu2 inserts; top: pMJ55 and pMJ56 linearized at the EcoRI site of pBR322. ( i ) : his4::leu2;
(ii) : MAT::leu2; (iii) : URA3::leu2. The normal URA3 locus in the SKI strains used here contains a large ( ca. 6 kb) insertion
-470 bp from the left hand HindIII siteas drawn here (T.-C. WU and M. LICHTEN, unpublished results) . The point of integration
of pMJ55 and pMJ56 at URA3 is to the right of this insertion mutation. (iv) : a@::leu2; (v) : CHAl ::h2. ( B ) a@ inserts; top:
pMJll3 and pMJll5 linearized at the EcoRI site of pBR322. The structures of flanking sequences at MAT::arg4, URA3::arg4,
and CHAl ::a@ are identical to their leu2 counterparts. ( i ) : leu2::arg4; (ii): his4::arg4. Integration of the plasmids at HIS4
duplicates a PVUII-ClaI fragment internal to HIS4. The additional CluI-huII fragment is shown for comparison with deletion
(AL);(iv):
his4A(Sal-Cla)::URA3*arg4*A(Sal-Eco)(AR);(v): his4A
derivatives. (iii): his.l::URA3*A(Sma-Bam)-arg4
(Sal-Cla):: URA3A(Sm-Bam)- a@. A(Sa1Eco) (ALAR).
the endof meiosisand do not
suffer the 5 ' to 3' recision
that occurs in wild-type cells (ALANI et al. 1990; SUN et
al. 1991) . This allows accurate mapping of break sites
and measurement of DSB frequencies.
Strains with inserts at HIS#, MAT, and CHAl segregate k g + meiotic recombinants at high, intermediate,
and low frequencies, respectively (Table 3 ) . DSB occurred at similar sites within the plasmid insert at all
three loci (Figure 2 ) . Breaks occurred at two sites between URA3 and a@-near
the junction of pBR322
and ARM sequences and near the junctionof pBR322
and URA3 sequences. The close proximity of these two
break sites precludedthe separate measurement of
break frequencies ateach site; we will refer to bothsites
collectively as DSB-left. Breaks also
occurred at all three
insert loci in a region between the pBR322 Sal1 and
EcuRI sites. These break sites were broadly distributed
and were not readily resolved; wewill refer to these
sites collectively as DSB-right.
A lack of appropriate restriction enzyme sites and
probes prevented us fkom determining the distribution
of DSB across the entire length of inserts at LEU2 and
URA3. However, usingrestriction sites and probes within
insert sequences, we were able to determine thelocation
60
T.-C. Wu and M.Lichten
TABLE 3
Position effects in meiotic recombination
Strain
ARG4 spores
X 103a
total spores
location Insert
MJL1059
none
48
11
5
0.4
LEU2 spores
total spores
X
lo4"
f 16b
leu2 inserts
MJL1017
MJL1016,
MJL1018, MJL1019
MJL1014, MJLlOl5
MJLlO80
arg4 inserts
MJLlO77,
MJL1078
MJL1094
MJLl124
MJL1068,
MJL1069
MJL1096,
MJL1097
(I
HIS4
MAT
m 3
CHAl
HIS4
LEU2
MAT
uRA3
CHAl
17(1.6)
f 1.7
19 5 0.7 (1.8)
9.4 5 2.7 (0.9)
5.7 ?
(0.5)
410.9
4.7(0.4)
? 0.3
(1.3) 62 f 13
15
(0.3)
f 2.4
7.7 f 1.3 (0.16)
(0.15) 7.0 ? 0.8
?
2.1 (0.4)b
49 ? 7.0 (l.O)b
f
(0.8)b
3.1
44(o.9)b
+- 3.4
Mean ? SD (frequency);values in parentheses are relative to MJL1059.
Leu' recombinants produced by recombination between leu2-K and h 2 - R at the normal LEU2 locus.
and frequency of breaks in DSB-left at all five insert loci
(Figure 3 ) . Break locationsand frequencies were similar
to those found by examination of the entire insert.
Break frequencies (Table 4) displayed position effects similar to those seen with recombination between
arg4 heteroalleles. The ratio of frequencies of total DSB
at his4::a@,A442"::arg#,
and CHAI::arg4 (1:0.4:0.2)
was similar to the ratio of frequencies of meiotic recombinants (1:0.6:0.3). The magnitude of these position
effects was different for thetwo subregions of the insert.
DSB frequencies at DSB-left varied by a factor of 10,
while those in DSB-right varied by a factor of three.
DSBs occur at the normalARG4 locus at a site -200
nucleotides upstream of ARG4 coding sequences in 34% of chromosomes (SUN et al. 1989; DE MASSY and
NICOLAS1993; GOYON and LIGHTEN1993). This level
of breaks was seen at the normal ARG4 locus in all
strains used in this study (data not shown). However,
in no case did we observe breaks at this site within arg4
insert loci (Figures 2 and 3 ) . We estimate the lower
limit of detection in these experiments to be 0.3% of
chromosomes. Therefore, insertion of arg4 sequences
in a full-length plasmid construct results in at least a
10-fold reduction in the frequency of DSBs at this promoter site. The arg4fragment used in these constructs
included sequences from the normal ARG4 locus 1.2
kb to the left and 2.1 kb to therightofthe
ARG4
promoter DSB site. Therefore, entities responsible for
the loss of DSB from the ARG4 promoter in plasmid
inserts must act over 21.2 kb.
We also examined thelocation and frequency of DSB
within leuhontaining inserts. At his4 ::leu2, the distribution and frequency of DSB in DSB-right weresimilar
to those observed at his4::arg4 (compare Figure 2, D
with A). DSB in the U R A 3 h 2 portion of the insert
were broadly distributed over an 800-bp region that
extended from the URA3pBR322 junction to a point
-200 bp into LEUMerived sequences. DSB occurred
in the same region at other leu2containing insert loci,
but atsignificantly lowerfrequencies (data notshown) .
The broad distribution and low frequency of these
breaks precluded accurate mapping and quantitation.
Deleting pBR322 sequences from his4::urgl restores
DSB to normal levels in the ARG4 promoter and in the
nearby HIS4 promoter: We considered the possibility
that the strong DSB sites in pBR322 sequences were
responsible for the loss of breaks in the arg4 promoter
at insert loci. We therefore deleted these portions of
the his4 ::arg4 locus, and examined the effect of these
deletions on the location (Figure 4 ) and frequency
(Table 5 ) ofDSBs. Deleting sequences between the
URA3 and arg4 fragments ( A L ) restored DSBs in the
ARG4 promoter site to a detectable level ( 1% of chromosomes) . DSB occurred at thenewly formed junction
of URA3 and ARM sequences, although at a lower frequency than was seen in the full-length insert. Deleting
pBR322 sequences to the right of ARG4 ( A R ) also restored DSBs to the ARG4 promoter region and significantly increased the frequency of breaks at DSB-left.
Deleting plasmid sequences on both sides of the ARG4
fragment ( A L A R ) resulted in a further increase in the
frequency of DSBs at the ARG4 promoter (to frequencies approaching that observed at the normal ARG4
locus) and at the new URA3ARG4junction (from 3%
in A L to 9% in A L A R ) . In all cases, similar patterns
and frequencies ofDSB
occurred within insert sequences in strains with hemizygous his4::arg4 inserts
and in strains with homozygous his4::arg4 inserts.
his4 ::a@ inserts affect DSBs in the HIS4 promoter: In normal strains, DSBs occur in the HIS4 promoter in 2-3% of chromosomes (NAG and PETES
1993). The presence of a homozygous full-length his4
arg4 insert reduced the frequency ofDSB at this site
about eightfold (Table 6 ) . Deleting insert sequences
-
61 Position
Meiosis
Effects in Yeast
C
CHAl Xhol-BplX361 fragment
I
CHAl::arg4Xhd fragment
1
B
1
MATa Bgll-BsfXI fragment
MtTa Epul1021-BsrXIfragment
MATa::arg4 Bpul1021 fragment
1
Mitotic
Meiotic
Mitotlc
Meiotic
///A/
RCURE2.-DSB in
his4::arR4, MAT::a@, CHAl ::a@, and his4::leu2 insert loci. DNAwas prepared from mitotic or meiotic
cultures of rri50.T strains, displayed on Southern blots and hybridized with the radioactive probes described below. The identities
of major bands are indicated above each pair of lanes. Internal and external size standards (not shown, see MATERIALS AND
METHODS) were used to determine DSB locations, which are indicated on a map of the insert locus below each pair of lanes. Thick
black lines are pBR322 sequences; thin black lines are flanking chromosomal sequences; lower lines indicate probe sequences. In
A-C, a hollow vertical arrow indicates the ARC4 promoter DSB site. (A) MJL1178 (his4::arg4/HIS4). DNA was digested with
X6aI.X6aI sites are located at nt 65967 and at about nt 67800 on chromosome III; this latter site is present in strain SKI but not
in the published sequence. The probe was an X6uI-AruII fragment ( n t 65967-66522). The small major band is a 1.8kb X6aI
fragment from the copy of chromosome 111 lacking an insert; it partially obscures a band produced byDSB at the URA5his4 3'
junction. ( B ) MJL1481 ( MATa ::a@/ MATa) . DNA was digested with BplllO21 ( n t 199271) , BsKI ( n t 201208). and B,LII. The
BfLl~1102Isite is in MATa-specific sequences. The BglII site is located in MATa-specific sequences 1694 nt centromere-proximal to
the BslXI site. The hybridization probe was a 450 nt MAT centromeredistal fragment that extends from a MATaX141 XhoI-linker
insertion (TATC:HEI.L
~t al. 1981) to a Hind111 site at nt 200821. The band present in both lanes is due to contamination of probe
with unrelated sequences. ( C ) MJL118.5 ( CHAl ::a@/ C H A l ) . DNA was digested with XhoI and Bsu36I. XhoI sites are located at
nt 15395 and 15494; the Bsu36I site is at nt 16687. The hybridization probe is a XhoI-BamHI fragment (nt 15494-16263). (D)
MJLll74 ( h i d : : l m 2 / H I S 4 ) .Restriction digests and probe are identical to those used for his4::arffl.
either to the left or to the right of the A R M fragment
significantly increased the frequency of breaks in the
HIS4 promoter; deletion of both regions restored
breaks to frequencies close to that seen in strains lacking this insert.
Changes in DSB levels at the ARG4 promoter are not
accompanied by markedchangesinchromatinstructure: We reasoned that the transfer of the A R M gene
from its normal chromosomal context to the pBR322based insert at his4::arg4 might alter chromatin structure at the ARC4 DSB site in such a way as to render it
inaccessible to DSBforming activities and that such an
occlusion might be detected as an alterationin the local
pattern of DNaseI hypersensitivity in digests of chromatin. To test this hypothesis, we performed DNase I diges
tion on chromatin from MJLlO77,which contains AKG4
sequences both at the normal locus and in a his4::arg4
insert (Figure 5 ) . The normal ARM locus (Figure 5A)
was DNase I hypersensitive in the DFB8I-DFBB1, A R M ,
and YSC83YSC84 5' intergenic regions, each of which
contain DSB sites. The full-length his4::arg4 insert also
containedthree hypersensitive regions (Figure5B) ,
but only two of these ( DSB-left and DSBright)suffered
DSBs; a third hypersensitive site was located at the expected place in the ARG4 promoter. Similar patterns
of DNaseI hypersensitivity were also observed strains
in
with full-length inserts at MAT and at CHAI (data not
shown) . Thus, theloss of DSBs from the arg4 promoter
within these inserts is not accompanied by a loss of
DNase I hypersensitivity at this site.
We also examined chromatin structure in a strain
(MJLlSOl) where insert sequences surrounding the
T.-C. Wu and M. Lichten
62
AR
"
DSB-left
a
-3
&' , I
+uARG4
Pvul I
probe
FIGURE
3.-DSBs in the URA3arR.l portion of inserts. DNA
was digested with RmII andSlvI, displayed on Southernblots,
and hybridized with probe preparedfrom the Snll-IzvuIl fragment of pRR322. Samples are from meiotic culturesof (from
left to right):
MJLll78,
MJL1184,
MJL1176,
MJLl177,
MJLll85. Mitotic DNA is from MJLl178. Solid lines indicate
pBR322 sequences; the lower line indicates probe sequences.
The expectedposition of a DSB in the ARC4 promoter region
is indicated by a hollow arrow.
arg4 portion of his4::nrg4 were deleted; this deletion
(AZAR) restored DSB in the nrg4 promoter to near
wild-typelevels. Chromatin from this strain displayed
DNase I hypersensitivity at both the ARC# DSB site and
at the new DSB site at the URA3ARG4 junction.
TABLE 4
Frequencies of doublestrand breaks
within a@ plasmid inserts
Insert
Frequency of double-strand
breaks ( %) "
right'
DSEleft"
location Strain
MJLll70,
10MJLll78
MJLl184
MJLll76,
MJL1481
MJLll77
MJLll85
HIS4
LEU2
MAT
uRA3
CHAl
10 2
t
82
32
4t
42
1.1 t
0.8 2
3 (6)
1 (2)
0.4 (2)
1 (3)
1 (2)
0.3 (2)
0.2 (3)
0.2 (2)
6 t 2 (5)
ND"
ND
3 t 0.6 (3)
ND
ND
2 t 0.1 (2)
ND
" Fraction of chromosomes expressed as means 2 SD with
a doublestrand break (DSB) in the indicatedregion with
number of determinations in parentheses. When two sets of
frequencies aregiven, the first is from experiments thatexamined the entire insert; the second is from experiments that
examined only the URA3-urg4portion of the insert.
'Double-strand breaks between the pBR322 Hind111 and
BumHI sites.
' Doublestrand breaks between the pBR322 SaA and EmRIsites.
,INot done.
AL
URA3A ARG4
"
"
"
"
P
"
hemizygous
homozygous
hemizygous
homozygous
homozygous
hemizygous
hemizygous
homozygouz
71
1
1
I
1
Full length
AL
AR
ALAR
FIGURE4.-Deleting
plasmid scqwnces alters the frcquency of DSB in his4::nrg4. DNA from meiotic cdtures (.i6 hr after transfer to sporulation medium) was digested with
XhuI (full-length and AI. inserts) or with x11111
and X ~ I J (AI<
I
or AIARinserts) .Thelocation of XbuI sites and thefragment
used as probe aredescribed in Figure 2A. Arrows indicate the
expectedlocations of DSBs within inserts. Fraglueuts p I c ~
duced byDSBs in the ARCH promoter migrate to either of
two positions, depending on whether theinsert contains AI.
or not. For each lane, theinsert deletion genotypeis indicated
on the right; whether this insert was hemizygous oI hornozygous is indicated on the left. The dark band on the far left
in lanes with DNA from strains hemizygous for the insert is
the XbuI fragment from the copy of chromosome III lacking
an insert. The rightmost band inall lanes is the fragment
containing theinsert construct. Sourcesof DNA are, from top
to bottom: MJL1178; MJL1170; MJL1248;MJL1373;MJL1382;
MJLl384; MJL1517; MJL1.515.
Similar results were obtained when we examined
chromatin structure at the HIS4 promoter. A wild-type
strain (MJL1059) and a his4::nrg.l strain (MJL1178)
that displayed an eightfold difference in the frequency
of breaks at this site(Table 6 ) displayed similar patterns
of DNase I hypersensitivity (data not shown).
In summary, we conclude that the reduced frequencies of DSBs at the ARC4 promoter and at the HIS4
promoter seen in his4::nrgg strains are not due to
changes in chromatin structure detectable at the level
of resolution and sensitivity provided by DNase I digestion of chromatin.
A his4::a@ insert reducesmeiotic recombination at
the nearby LEU2 locus: In the diploid strains with nrg4
inserts used in this study, the normal IXU2 locus was
marked with leu2-Ron one chromosome IIIhomologue
and withleu2-K on the other. Mostof these diploids
produced Leu+ recombinants atthe frequency o h
served in insertless strains. MJL1077 and MJL1078,
which contain his4::nrg4 inserts on both copies of chromosome III, displayed a significantly lower frequency
of Leu+ recombinants (Table 3 ) . This was not a result
of histidine auxotrophy, as an insertless his4 diploid
(MJL1496) produced Leu+ recombinants at the normal frequency (Table 7 ) . This reduction was seen in
all diploids with a his4::arg4 insert present in cis to
inPosition
Meiosis
Effects
Yeast
63
TABLE 5
Effect of deleting pBR322 double-strand break sites on double-strand breaks at his4::arg4
Frequency of double-stranded breaks (%)'
DSB-left
ARC4 promoter
DSB-right
10 2 63 (3)
10 2 3 (2)
3 t 2 (4)
2 2 1 (2)
31 2 9 (8)
30 2 10 (6)
13 2 4 (7)
12 2 3 (6)
-
6 2 2 (4)
6 (1)
7 (1)
1 1 (1)
structure"Insert
Full length hemizygous
Full length homozygous
A L hemizygous
A L homozygous
A R hemizygous
A R homozygous
A L A R hemizygous
A L A R homozygous
1.1 2 0.7 (2)
0.4 (1)
1.6 2 0.8 (4)
1.3 2 0.5 (3)
4.0 2 1.7 (6)
3.6 t 1.6 (5)
-
" Deletions of plasmid insert sequences are A L - A(Sma-Bum); A R - A (Sal - Eco) as illustrated in Figure
1. Names of strains are given in the legend to Figure 4.
Fraction of chromosomes expressed as means t SD suffering a double-strand break (DSB). Numbers in
parentheses indicate the number of independent determinations. Regions are as illustrated in Figure 4. Values
for breaks in inserts from strains with hemizygous inserts and homozygous inserts are reported separately.
DSBs could not be measured in any experiments but one, where a value of 0.3% was obtained.
h 2 - K (MJL1077, MJL1078
and MJLl211). No such
reduction occurred in a diploid with a hemizygous insert present in cis to h 2 - R (MJL206).Because most
Leu+ recombinants from h B K / h 2 - R diploids are the
products of gene conversion of h 2 - K to LEU2 ( LICHTEN et al. 1987),these data are consistent with the suggestion that a h i s 4 : : a e insert acts in cis to reduce the
ability of a nearby LEU2 locus to serve as a recipient in
gene conversion events. In SKI,LEU2 is 17 kb from
HIS4 on chromosome ZZZ (M.LICHTEN,
unpublished
results). Inserts at CHAl and atMAT, located -70 and
-
100 kb from LEU2, respectively, had no effect on the
yield of Leu' recombinants (Table 3 ) .
DISCUSSION
Our experiments provide several examples of situations where entities outside the immediate vicinity of a
A
A ? +?
"
082 DED81
YSC
ARG483 84
I
rad5Os
B
TABLE 6
Effects of deleting break sitesin his4::arg4
on DSBs at the HIS4 promoter
"
full length
Insert
structure"
Strain
No insert
Full length
AL
AR
ALAR
MJL1083
MJLll78
MJL1248
MJL1384
MJL1515
Relative DSB
frequencyb
0.13
0.5
0.5
0.7
1
2 0.05 (4)
t 0.2 (4)
2 0.1 (4)
2 0.04 (4)
" Structures are as illustrated in Figure 1.
Double-strand break (DSB) frequencies (DSB/chromosome) relative to that at the HIS4 promoter in MJL1083, a
strain lacking inserts. Values are means t SDwith number
of independent determinations in parentheses. Meiotic DNA
was digested with NcoI (nt 69551 and 59752 on chromosome
III), displayed on Southern blots, and hybridized with an NcoISad (nt 69124-69551) fragment. DSBs at the HIS4 promoter
occur 1.2 kb from the NcoI site at nt 69551. The intensity
of bands produced by this DSB in lanes with the DNA from
h i s 4 : : u e strains was compared with the intensity ofDSB
bands in lanes containing DNA from MJL1083; band intensities were normalized to account for total lane DNA content
as described in MATERIALS AND METHODS. The frequency of
DSBs at theHIS4 promoter in MJLlO83, as determined in two
separate experiments, was 2 t 0.1% (data not shown).
-
C
RGURE5.-ARG4 promoter chromatin is DNase I hypersensitive at the normal ARG4 locus and at his4::arg4. The
three panels in this figure compare DSBs and DNase I hypersensitive sites at three loci: (A) Normal ARC4 locus; ( B )
his4::a@, full-length insert; ( C ) his4::a@, A I A R (all
pBR322 sequences deleted). In each panel, the top lane is
DNA from a meiotic culture of a rad5OS strain ( A and B,
MJL1170; C , MJL1382). The lower three lanes contains DNA
prepared from chromatin from a mitotic culture of the corresponding RAD50 strain (A and B, MJL1078; C, MJL1501) .
The upper chromatin lane contains a no-DNase 1 control
sample; in the lower two lanes, chromatin was digested with
increasing amounts of DNase I. In A, DNA was digested with
NcoI and probed with an NcoI-BglII fragment containing sequences from the normal ARG4 locus as previously described
(Wu and LICHTEN 1994)
; in B and C, DNA was digested with
XbaI and probed as described in Figure 2.
T.4. Wu and M. Lichten
64
TABLE 7
his4::ae inserts inhibit meiotic recombinationat LEU2
Strain
MJL1496
MJL1077, MJL1078
MJL1211
MJL1206
Insert type
None
Insert in cis to
-
LEU2 spores
total spores
X
lo4“
48 t 1
h 2 - 4 h2-R
h2-K
h2-R
17 t 2 (0.4)
Homozygous
22 t 7 (0.5)
Hemizygous
51 t 9 (1.1)
Hemizygous
a Frequency values expressed as means ? SD with values in parentheses relative to frequenciesin MJL1496,
a his4 auxotroph.
DSB site can alter both the frequency of breaks at that
site and the frequency of recombination in its vicinity.
In one example, the genomic location of an arg4containing plasmid insert significantly affected the frequency, but not thelocation, of DSBs within the insert.
This result is consistent with the presence in the yeast
genome of yet unidentified entities that can act over a
distance to modulate DSB frequencies. Because of the
extent of homology shared between insert loci, we conclude that the entities responsible for these locus-dependent position effects must act over distances >1.5
kb. The observation that his4::arg4 inserts inhibit recombination at the LEU2 locus, located 17 kb from
HIS4 on chromosome ZZZ,is consistent with action over
distances that are 10 times greater.
A second example of position effects is provided by
the examination of DSBsat his4::arg4. When a pBR322based plasmid construct containing a 3.3-kb arg4 fragment was inserted at HIS4, DSBs were lost from both
the insert-borne arg4 promoter and the nearby HIS4
promoter. The inhibition of breaks at these sites most
likely was due to the presence of strong DSB sites in
nearby plasmid sequences, because deleting these DSB
sites resulted in a significant increase in the frequency
of breaks at all remaining DSB sites. The distance between a deletion endpoint and the affected DSB site
could be as large as 8.8 kb. We will refer to this latter
phenomenon as “local suppression.” Although this
term is used to distinguish this type of position effect
from the locus-dependent effects described above, it is
possible that the two phenomena are the products of
the same basic mechanism. Testing this suggestion will
require both further analysis of the effect of plasmid
inserts on DSBs in flanking sequences and an analysis
of sequences flanking insert loci to identify the entities
responsible for locusdependent position effects.
What produces position effects? Both locusdependent position effects and local suppression indicate that
the frequency of DSBs at a given site is not determined
simply by the DNA sequence at thesite. Similar conclusions have been made elsewhere ( CAO et al. 1990;
Rocco et al. 1992; DE MASSY and NICOLAS 1993; GOYON
and LICHTEN1993). Several factors that are independent of DNA sequence have been proposed to affect
the frequency of breaks at a site; these include transcriptional interference ( ROCCOet al. 1992), changes in
chromatin structure ( CAo et al. 1990; DE MASSY and
NICOLAS 1993;
GOYON and LICHTEN
1993;Wu and LICHTEN 1994), and competition with nearby DSB sites for
limiting factors ( GOYON
and LICHTEN
1993). Below, we
discuss the role that each of these proposed mechanisms might play in producing position effects.
Transcripional interference: The ARG4 gene is flanked
by transcription terminators that stop adjacent transcripts from enteringtheARMpromoter
region.
ROCCO et al. (1992) used deletions and inversions to
remove these terminators and found that rearrangements that allowed transcription to pass through the
ARG4 promoter significantly reduced the frequency of
DSBs at that site. Is it possible that transcriptional interference is responsible for reducing DSB frequencies at
the ARG4 and HIS4 promoters on chromosomes with
full-length his4::arg4 inserts? The plasmid pBR322 a p
pears to contain functional yeast promoters in the regions removed by ARand AL( STRUHL
and DAVIS1981;
MARCZYNSKI and JAEHNINC 1985; KLEIN 1988). However, the ARM fragment
we used contains transcription
terminators on both sides of the gene that should prevent these transcripts from entering the promoter region ( ROCCOet al. 1992). In addition, when Northern
blots of RNA from meiotic cultures of a his4::arg4 diploid are probed with ARG4 coding sequences, only the
expected ARG4 transcript is detected (T.-C. WU and M.
LICHTEN,
unpublished results). For these reasons, we
believe it unlikely that transcriptional interference is
responsible for the observed lossof DSB from the ARG4
promoter site at his4 ::arg4.
Transcriptional interference might be partly responsible for the loss ofDSBs from the HIS4 promoter in
his4::arg4 strains, because transcription from a known
pBR322 promoter would be expected to pass through
this site ( MARCZYNSKI and JAEHNINC 1985). Even so, it
is likely that otherfactors are responsible for the insertinduced loss of breaks from the HIS4 promoter. The
frequency ofDSBs at this site was increased by AL,
which removes plasmid sequences between the URA3
and ARG4 portions of the insert. Transcription from
putative promoters between URA3 and ARG4 would
Position Effects in Yeast Meiosis
have to travel through almost 9 kb of DNA and atleast
two known terminator sequences before entering the
HIS4 promoter.
Changes in chromatin structure: We have presented evidence for a close correlation between the locations of
DNase I hypersensitive sitesin chromatin and DSB sites
and also have shown that changes in chromatin strucpH05 promoter areaccompanied by parallel
ture at the
changes in the distribution and frequency of DSBs at
that locus ( WU and LICHTEN1994). The observation
that DSB sites in pBR322 sequences are also DNase I
hypersensitive reinforces this finding and indicates that
this property is not unique to yeast sequences. Is it
possible that chromatin structure rearrangements are
responsible for the loss ofDSB from the HIS4 and ARM
promoters at his4::arg4? If this were the case, then the
hypothetical entities responsible for these changes must
be capable of acting over several kilobases. Two other
examples of such long distance effects in yeast are the
silencing of promoters at the silent mating type loci
and in the vicinity of telomeres ( LAURENSON and RINE
1992; RENAULD et al. 1993). Both are accompanied by
observable and significant changes in chromatin structure within the affected regions (NASMYTH
1982;
GO-ITSCHLING
1992). In contrast, our findings indicate
that an open chromatin configuration, as revealed by
DNase I hypersensitivity, is necessary but not sufficient
for DSB to occur at a site. Inserts with reduced DSB
frequencies at the ARM and HIS4 promoters retained
normal patterns of DNase I hypersensitivity ofchromatin at these sites, and deletions that restored DSB to
these sites did not appear to alter their
DNase I sensitivity. For this reason, we believe it unlikely that major
changes in chromatin structure are responsible for either theloss ofDSB in the ARG4 promoter at his4::arg4
or the recovery of DSB at that site in the ALAR insert.
OHTAand coworkers (1994) have shown that chromatin at several DSB sites becomes more sensitive to micrococcal nuclease during meiosis and suggest that this
reflects an alteration in chromatin structure that is essential for DSB formation This change is not detected
when DNase I is used as a probe of chromatin structure
(K. OHTAand T. SHIBATA,
personal communication),
and thus remains a possible way to account for the
position effects we observe.
Inhibition by competition: Both locus-specific position
effects and local suppression might be the result of
competition between nearby DSB sites; the loss ofDSBs
from the HIS4 and ARM promoters in his4 ::arg4 strains
mightbe due to presence of stronger DSB sites in
pBR322 which successfullycompete forlimiting factors
necessary for break formation. Similarly, locus-specific
position effects might be due to competition between
DSB sites within the plasmid insert and sites in flanking
chromosomal sequences. This interpretation isvalid
only if these postulated factors are ratelimiting for DSB
formation and are not freely diffusible throughout the
65
nucleus. Two possible models that meet these criteria
are discussed below.
The first is derived from a mechanism proposed for
the interaction of the RecBCD enzyme with its target
site x (TAYLOR
and SMITH1992). In our modification
of this model, the binding of a DSB-forming activity
that then moves along the chromosome in search of a
potential DSB sites is postulated to be rate limiting.
The area over which competition could occur would
be determinedby a combination of the location of entry
points, the rate of enzyme travel, and the probability
that, upon encountering a potential DSB site, the enzyme becomes unavailable for cutting other sites.
A second hypothesis suggests that, at thetime of DSB
formation,both chromosomes and DSB-forming enzymes are organized into structures that contain domains analogous to transcriptional regulatory domains
in metazoan organisms ( EISSENBERG
and ELGIN 1991
).
In this model, structural features serve as a source of
DSB-forming activitiesand create boundaries thatfunctionally isolate adjacent regions from one another. The
region over which competition could occur would be
determined by domain size and by the location of domain boundaries, and the frequency of breaks at a site
would be affected by competition only with other sites
within the same domain.
DSBs form in the absence of homology: In yeast, h e
mologous chromosomes transiently associate with each
other early in meiosis, before DSBs are formed (WEINER
and KLECKNER 1994). It has been suggested that DSBs
form within biparental structures, perhaps those that mediate this association ( HAWLEY and ARBEL 1993). We
find that hemizygous and homozygous his4::arg4 inserts
display similar DSB distributions and frequencies, indicating that pairing between homologues at a DSB site
itself is not required for DSB formation. Similar results
have been obtained in RAD50 strains (T.G. Wu and M.
LICHTEN,
unpublished results) and in haploid strains
undergoing meiosis (DE MASSEY et al. 1994; GILBERTSON
and STAHL1994; FANet al. 1995) . These results rule out
recombination models in which DSBs are formed via
the processing of a previously formed recombination
intermediate and provide further supportfor the suggestion that DSBs are the primary lesion responsible for
initiating meiotic recombination.
We thank J. E. HABER and N. KLECKNER for yeast strains and plasA. GOLDMAN,
J.E. HABER, A. SEGALL,T.
mids, and D. CHATTORAJ,
SHIBATA,
F.W.STAHL, C. VINSONand M. YARMOLINSKY
for helpful
discussions that improved the manuscript. Some of thedatapresented here have been published elsewhere in a preliminary form
(Wu and LICHTEN1993).
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and N. KLECKNER, 1990 Analysis of wild-type
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G. KOHLHAWand P. SCHIM-I,
66
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MEL,1984 Yeast LEU2. Repression of mRNAlevelsby leucine
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Corresponding editor: S. JINKS-ROBERTSON