Mutation Research 451 Ž2000.

91–105
www.elsevier.comrlocatermolmut
Community address: www.elsevier.comrlocatermutres

Review

Damage-induced recombination in the yeast

Saccharomyces cereÕisiae
Martin Kupiec )
Department of Molecular Microbiology and Biotechnology, Tel AÕiÕ UniÕersity, Ramat AÕiÕ 69978, Israel
Received 25 October 1999; received in revised form 27 December 1999; accepted 18 January 2000

Abstract

Prokaryotic and eukaryotic cells have developed a network of DNA repair systems that restore genomic integrity
following DNA damage from endogenous and exogenous genotoxic sources. One of the mechanisms used to repair damaged
chromosomes is genetic recombination, in which information present as a second chromosomal copy is used to repair a
damaged region of the genome.
In this review, I summarized what is known about the molecular and cellular mechanisms by which various
DNA-damaging agents induce recombination in yeast. The yeast Saccharomyces cereÕisiae has served as an excellent model
organism to study the induction of recombination. It has helped to define the basic phenomenology and to isolate the genes
involved in the process. Given the evolutionary conservation of the various DNA repair systems in eukaryotes, it is likely
that the knowledge gathered about induced recombination in yeast is applicable to mammalian cells and thus to humans.
Many carcinogens are known to induce recombination and to cause chromosomal rearrangements. An understanding of the
mechanisms, by which genotoxic agents cause increased levels of recombination will have important consequences for the
treatment of cancer, and for the assessment of risks arising from exposure to genotoxic agents in humans. q 2000 Elsevier
Science B.V. All rights reserved.

Keywords: DNA Repair; Eukaryotic cell; Saccharomyces cereÕisiae; Genetic recombination

1. Introduction original DNA structure. These include the base exci-

The maintenance of genome integrity is a major
task for all living cells. DNA damage can occur as a
consequence of the normal cellular metabolism or as
a result of exposure to chemical or physical agents.
In the presence of damaged DNA, various repair
mechanisms are mobilized in order to restore the
)
Tel.: q972-3-640-9031; fax: q972-3-640-9407.
E-mail address: martin@ccsg.tau.ac.il ŽM. Kupiec..
sion repair ŽBER., the nucleotide excision repair
ŽNER., the mutagenic repair, and the recombina-
tional repair mechanisms Žreviewed in Ref. w38x.. In
the present review, I shall concentrate on the induc-
tion of recombinational repair by treatment with
DNA damaging agents. For recent reviews on related
subjects, such as spontaneous recombination and on
radiation-induced DNA processes, see Refs.
w22,88,92x and other chapters of this issue. A detailed
account of the different mechanisms of recombina-
tion and the models proposed to explain them can be

0027-5107r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 2 7 - 5 1 0 7 Ž 0 0 . 0 0 0 4 2 - 7
92 M. Kupiecr Mutation Research 451 (2000) 91–105
found in Ref. w88x. Below, I summarize in brief the
main points pertinent to this review.
2. Monitoring mitotic recombination
Recombination takes place between sequences that
share homology, and results in the transfer of genetic
information from one DNA molecule to the other
Žgene conversion. and in the reciprocal exchange of
DNA fragments between chromosomes Žcrossing-
over.. Gene conversion and crossing-over events
show a nonrandom association with each other; this
has lead to the assumption that they are mechanisti-
cally related. This association is stronger Ž; 50%. in
meiotic recombination, but can also be detected in
mitotic cells Žreviewed in Ref. w88x.. The frequency
of recombination at any determined region of the
genome is relatively low. Although nonselective
schemes have been successfully tried w39,46x, se-
lectable genetic systems are normally used in order
to be able to detect such rare events. As an experi-
mental system, yeast has been pivotal in the develop-
ment of genetics systems able to monitor specific
types of recombination events w2,29,34,61,65,
76,77,98,102,128,129x. A number of such selectable
assays for recombination are discussed below.
Gene conversion is responsible for the great ma-
jority of intragenic recombination events w83,102x. In
diploid yeast strains, gene conversion between two
different alleles of a nutritional marker can be conve-
niently measured by plating cells on the appropriate
selective medium. Mitotic crossing-over events are
usually scored in diploids by the segregation of
recessive homozygotes from heterozygous markers
w128,129x.
Recombination usually occurs between sequences
located at similar positions on homologous chromo-
somes Žallelic recombination. or sister chromatids;
however, homologous sequences located at different
positions in the genome can also interact Žectopic
recombination.. Allelic and ectopic recombination
occur at similar frequencies, implying that homology
is searched throughout the genome and not exclu-
sively on the homologues. This fact has been ex-
ploited in order to measure recombination in haploid
cells. Various genetic schemes selecting for gene
conversion between artificial or naturally occurring
repeated sequences have been used w59,72,75x. In
addition, one can select for recombination events
between truncated genes located on different chro-
mosomes, thus creating specific translocations w33–
35x.
Other popular systems used to follow recombina-
tion monitor events between two tandemly located
sequences in the genome in either direct or inverted
orientation. These are easily created by the integra-
tion of the appropriate plasmids w2,41,67,98,121x.
Direct repeat recombination ŽDRR. is probably car-
ried out by several different mechanisms, including
replication slippage, unequal sister chromatid recom-
bination, intra-chromatidal crossing-over, single-
strand annealing, etc. Žreviewed in Refs. w90,98x..
Similarly, inverted repeat recombination ŽIRR. can
occur by crossing-over or by gene conversion be-
tween sister chromatids w11x. Finally, specific sub-
strates have been designed to monitor unequal sister
chromatid recombination ŽUSCR. between tandem
repeats w34,61,112x.
In summary, many different assays are used to
monitor spontaneous and induced mitotic recombina-
tion in yeast. Analysis of the effect of recombination
mutants on the various recombination substrates
shows that different recombinational mechanisms are
responsible for the recombination observed in the
different substrates. Accordingly, not all types of
recombination are equally inducible.
3. Induction of recombination by DNA damage
Mitotic recombination is one of the mechanisms
used by the cell to repair spontaneous DNA damage.
Such damage is believed to result from the natural
metabolism of the cells, such as the activity of
reactive oxygen species or of DSBs created during
DNA replication and transcription w38x. In addition, a
number of DNA damaging agents elevate the level
of homologous recombination w8,30,96,97,101,
122,128x. This recombination results from repair pro-
cesses that restore the integrity of damaged DNA
regions.
When a lesion is created at the DNA, it may
remain unrepaired, leading to cell death, or can be
repaired Žor bypassed. in a process that involves
recombination. Many different DNA-damaging
 M. Kupiecr Mutation Research 451 (2000) 91–105
agents cause a dose-dependent increase in the fre-
quency of recombination. These include ionizing and
UV-irradiation, and chemicals such as cisplatin,
MMS, MNNG, 4-NQO, EMS, Aflatoxin B, etc.
w8,30,33,51,97,101,128x. Most of the recombinogenic
damaging agents also induce mutagenesis; this im-
plies that the lesions caused by the agent can also be
repaired by other DNA repair or tolerance mecha-
nisms w51,128x. Some carcinogens, however, are neg-
ative in mutagenesis tests, but induce recombination,
suggesting that the mechanism of carcinogenesis in-
volves the process of recombination. The good corre-
lation between carcinogenesis and the ability to in-
duce recombination has led to the development of
yeast-based carcinogenesis detection assays w33,
97,129x.
The level of induction of recombination depends
on the genetic system used to detect it and on the
damaging agent used. Some recombinational sub-
strates are poorly induced by DNA damaging agents,
whereas others can be induced to levels comparable
to those observed during meiosis. As a general rule,
recombination substrates that give very low levels of
spontaneous recombination are induced to larger ex-
tent than substrates that give a higher basal level. As
discussed below, these results may imply the exis-
tence of two independent mechanisms for sponta-
neous and induced recombination.
The most extensively studied type of recombino-
genic DNA lesions are double strand breaks ŽDSBs..
DSBs occur quite often, either as spontaneous errors
during replication, or as a consequence of the action
of radiation and chemicals. It is well established
today that in yeast, meiotic recombination is initiated
by DSBs w127x. The creation of DSBs in vegetatively
growing cells by site-specific nucleases also leads to
high levels of recombination Žsummarized in Ref.
w88x.. Unrepaired DSBs are lethal; the ability of the
cells to use homology present in the genome to
repair the break is essential for their survival. Linear
double-stranded DNA molecules are extremely re-
combinogenic, a property widely exploited for yeast
manipulations. These findings have led to the cur-
rently held recombination models, which assume that
the essential recombinogenic substrate in DNA is a
DSB w88,110x. The recombinational repair of DSBs
in mitotic and meiotic cells is described in other
chapters of this special issue.
93
A great number of noxious agents have been
tested in yeast, with various effects Že.g.: see Ref.
w128x.. A summary of the effects on recombination
of so many compounds is beyond the scope of the
present review. Instead, I will summarize below
what is known about the induction of recombination
in yeast by four different DNA-damaging agents for
which the types of lesions created have been well
characterized. A great deal is also known about the
mechanisms responsible for their repair; however,
many questions remain to be answered regarding the
way by which they induce mitotic recombination.
3.1. Ionizing radiation
Ionizing radiation induces several types of DNA
lesions, including single- and double-strand breaks,
DNA-protein crosslink and base damage. However,
it is the occurrence of DSBs that determines cell
survival after ionizing radiation w37,94x. Consistent
with the idea that DSBs directly initiate recombina-
tion, analysis of dose–response kinetics showed that
at sublethal doses, X-rays induce heteroallelic con-
version with linear kinetics w80x, whereas other
sources of damage, such as UV, are nonlinear w23x.
This has been interpreted to mean that UV-created
lesions need to be processed in order to initiate
recombination, in contrast to DSBs. In addition,
mutants that are defective in DSB repair Že.g. rad52 .
show pure exponential survival curves when irradi-
ated. For these cases, it has been calculated that one
DSB per cell corresponds to one lethal hit w36,37x.
Thus, for ionizing radiation, the observed increase
in recombination reflects more recombination reac-
tions initiated by DSBs. The group of genes respon-
sible for this recombinational repair Žthe RAD52
group. has been well characterized w45x. A mutation
in any of the genes in this group leads both to a great
sensitivity to ionizing radiation, and to the abolish-
ment of induced recombination w45,56x.
3.2. UV irradiation
The main two types of lesions created by UV
irradiation are pyrimidine dimers and pyrimidine 6–4
lesions. These lesions are efficiently repaired in yeast
cells by the NER group of genes Žreviewed in Ref.
w38x.. In contrast, recombination plays only a minor
94 M. Kupiecr Mutation Research 451 (2000) 91–105
role in UV damage repair and mutations in genes of
the RAD52 group confer a slight UV sensitivity, and
only at high doses. The induction of recombination
observed after UV treatment is probably a conse-
quence of the processing of primary lesions left after
NER into secondary lesions that are recombinogenic.
However, how this is accomplished is still not clear,
despite extensive studies on the subject. Unrepaired
lesions, such as DNA adducts and single-strand
breaks, may cause stalling of the replication machin-
ery during S-phase. Replication may be resumed at
another replicon downstream, thus creating a single-
strand gap. Such gaps can be easily converted into
recombinogenic DSBs w20,64x. It has been suggested
that UV-induced damage can persist in the DNA
through several cell divisions, to be later converted
into DSBs w44x. In addition, at high UV doses, the
excision of pyrimidine dimers in close proximity on
opposite strands can lead to the creation of DSBs.
Consistent with this notion, most mutants of the
RAD52 group of recombination genes show UV
sensitivity at high UV doses w51x.
If pyrimidine dimers have to be removed in order
to initiate recombination, one would predict that
inactivation of the NER system would prevent in-
duced recombination. However, mutations in NER
that prevent removal of the initial lesions have been
reported to have various effects on recombination.
For example in one study w62x, it was shown that
mutations in the RAD1 gene caused a reduction in
diploid heteroallelic recombination, with a concomi-
tant increase in sister-chromatid recombination. In
another study, it was shown that mutations in the
RAD3 gene impair induced heteroallelic gene con-
version, but increase induced crossing-over w65x. In a
third study, while mutations in RAD2 caused a
significant increase in the induction of gene conver-
sion at two different loci, mutations at the RAD1,
RAD3, and RAD4 genes caused only a mild increase
over the wild type levels w55x. These results probably
testify to the heterogeneity of mechanisms that can
lead to recombination, but also demonstrate that we
are far from understanding some of the basic aspects
of induced recombination. A true comparison of
results is complicated by the fact that different
recombination substrates were used in the different
studies. In addition, some of these studies were
carried out with strains carrying leaky alleles. One
additional complication is that some of the genes
required for dimer excision, such as RAD1, may
actually participate in recombination w99x.
Some types of recombination, such as DRR, may
actually be induced by a mechanism that does not
require DNA breakage and rejoining. For example,
lesions in the DNA may cause replication stalling;
this in turn may facilitate slippage by dissociation of
replicated strands and reassociation with a second
copy of the repeat w5,78x. One study of DRR showed
that this type of recombination was induced by UV
irradiation in dividing cells, but not in G1- or G2-
arrested cells w43x. This result implies a dependence
on DNA replication, although it is not yet clear
whether in order to allow slippage or for the conver-
sion of primary lesions into DSBs.
3.3. Alkylating agents
A large number of chemical compounds are able
to alkylate DNA w38x. Although the direct effect of
alkylation has been quite accurately defined both in
vitro and in vivo, it is still unclear why some alkylat-
ing agents, such as ethyl methanesulfonate ŽEMS.
are potent mutagens, but do not induce recombina-
tion, while others, such as the similar compound
methyl methanesulfonate ŽMMS. readily induce re-
combination. MMS is usually referred as ‘‘IR-radio-
mimetic’’, since it has an effect very similar to that
of ionizing radiation w109x. However, we are far from
understanding the mechanism by which MMS treat-
ment promotes recombination. Mutations that affect
DSB repair and recombination show a great sensitiv-
ity to MMS w16,38,124,125x. However, the main
effect of MMS appears to be the methylation of
bases in the DNA creating 7-deoxyguanine and 3-
methyladenine Ž3MeA., and not DSBs formation.
3MeA, the main biologically relevant lesion created
by MMS, differs from UV-induced lesions and bulky
adducts in that it is in the minor groove and does not
significantly alter the DNA double helix conforma-
tion. The repair of alkylation damage has been exten-
sively investigated in yeast. Alkylation lesions are
mainly repaired by the BER mechanism, in which a
DNA glycosylase removes the damaged base, fol-
lowed by cleavage at the abasic site by an
apurinicrapyrimidinic ŽAP. endonuclease w38,124,
125x. The radiomimetic properties of MMS could be
 M. Kupiecr Mutation Research 451 (2000) 91–105
explained by assuming that the primary lesions cre-
ated by MMS are processed by the BER pathway in
a way that converts them into DSBs. The results of
DNA analysis by neutral and alkaline gradient could
be consistent with this idea w57x. However, the analy-
sis of single- and double-mutants does not fit this
explanation: mutants in recombination genes are more
sensitive to MMS than mutants in the 3 MeA glyco-
sylase gene Ž MAG1.; inactivation of the BER path-
way does not suppress this sensitivity; on the con-
trary, a synergistic effect is seen Žw125x; Jablonovich
and Kupiec, unpublished.. These results are further
complicated by the genetic evidence for a role of the
NER and error-prone repair enzymes in alkylating
repair w16,125x.
3MeA apparently blocks DNA synthesis, and is
thus considered a major lethal lesion w6,25x. It is
therefore possible that the lesions created by MMS
cause a block in replication, which is then acted
upon by the recombinational repair group of genes
w64x. Alternatively, the passage through S transforms
the lesions into fully recombinational ones. A check-
point that slows down replication in the presence of
low MMS concentrations is known to operate w91x.
The cells continue to cycle, until finally they arrest at
the G2rM checkpoint w1,71,91x. It is possible that a
correlation exists between the S checkpoint and the
processing of primary MMS lesions. Eventually,
these are converted into DSBs, eliciting the G2rM
checkpoint, and inducing the recombinational repair
mechanism.
3.4. Crosslinking agents
Bifunctional agents that can react with two differ-
ent nucleophilic centers in DNA have the potential to
form intrastrand and interstrand DNA crosslinks.
Several different types of crosslinking agents, includ-
ing psoralens, mitomycin C, and cisplatin have been
analyzed in yeast. Crosslinking agents are exten-
sively used in cancer chemotherapy Že.g. Refs.
w93,108x., as they are supposed to preferentially af-
fect DNA replication and transcription in rapidly
growing cancer cells. Interstrand crosslinks can, in
principle, prevent DNA strand separation and there-
fore may block DNA replication and transcription.
Interestingly, it has been lately shown that cisplatin
causes a cell cycle arrest in yeast cells at the G2
checkpoint, and does not elicit the S phase check-
95
point expected from a treatment that block DNA
polymerases w47x. These results may suggest an alter-
native mechanism of action of bifunctional agents.
Treatment with crosslinking agents induces mitotic
recombination in a process that requires a specialized
group of genes Ž PSO genes. whose precise biochem-
ical role remains to be elucidated. The PSO genes
seem to be completely specific for crosslinks; ac-
cordingly, some of them are needed for induced
recombination by crosslinking agents w81,85x. In con-
trast to what happens in bacteria w13x, DSB formation
is an obligatory intermediate in the repair of inter-
strand crosslinks in eukaryotes w18x, and thus the
induction of recombination is likely to result from
the repair of such breaks w4x. The precise mechanism
by which the Pso proteins transform the crosslinks
into DSBs remains to be elucidated. Interestingly,
crosslinking agents such as cisplatin can sensitize
prokaryotic and eukaryotic cells to killing by ioniz-
ing radiation, by a mechanism not yet understood,
that seems to involve cisplatin inhibition of DSB
repair after ionizing radiation w21x.
To summarize, although it is clear that DSBs are
repaired in yeast preferentially by recombination,
and that DSBs are the substrates for the recombina-
tional repair of ionizing radiation damage, much less
is known about the conversion of other types of
lesions into recombinogenic substrates Žpresumably
DSBs.. A picture is starting to emerge, in which
many different types of lesions are acted upon by the
recombinational machinery during DNA replication,
either by the conversion of the initial lesion into
DSBs, or by the coupling of the processes of replica-
tion and recombination. Consistent with this picture,
many damaging agents require cell division in order
to become recombinogenic Že.g. Refs. w42,43x. and
polymerase Delta is needed for this process w49,117x.
In addition to a link to replication, evidence has
been accumulating lately for a similar link to tran-
scription: in some cases, increased transcription may
induce recombination Žreviewed in Ref. w86x.. Such
an association between transcription and repair fac-
tors has been well established for NER. The stalling
of RNA polymerase on a damaged template activates
the NER machinery, which is physically linked to
the transcription machinery w38x. A similar link to
recombination may exist, although direct biochemi-
cal evidence for this is still lacking.
96 M. Kupiecr Mutation Research 451 (2000) 91–105
4. Effect of the cell cycle on induced recombina-
tion
In meiotic cells, recombination takes place after
DNA replication, at the four-strand stage. In vegeta-
tive cells, many studies have been carried out to
determine at what stage of the cell cycle recombina-
tion takes place. Half-sectored colonies w96x, similar
to ‘‘twin spots’’ in other organisms, provide evi-
dence that recombination in vegetative cells can take
place at the G2 stage. Unequal reciprocal sister
chromatid recombination, which can take place only
after DNA replication, has also been detected in
natural and artificial tandem repeats w34,61,112x. On
the other hand, pedigree analysis of X-ray induced
mitotic recombination showed that most of the cells
recombined before DNA replication w120x. Finally,
elegant genetic studies have demonstrated that spon-
taneous and induced recombination can occur in
vegetative cells arrested at many different points in
the cell cycle w26–28,60x.
The induction of recombination varies throughout
the cell cycle. The increase of both gene conversion
and crossing over by nonlethal doses of UV and
X-rays in synchronized diploid cultures is maximal
at the G1 stage, and minimal at G2 w19,23,24,28x.
The low level of recombination detected is not due
to failure to repair the damage, since G2 diploids are
able to survive DNA damage at the G2 stage better
than at G1 w10,28,45x. The interpretation to these
results is that in cells that have replicated their
chromosomes, the damage is preferentially repaired
by recombination with the sister chromatids w28x.
This hypothesis was then directly tested using a
strain in which both homolog and sister chromosome
recombination could be measured w61x. This study
not only showed that X-rays preferentially induced
sister chromatid recombination in G2 cells; it also
suggested that sister chromatids, for reason still to be
understood, have the capacity to repair more DNA
damage than do homologs w61x.
5. Effect of the mating type on induced recom-
bination
In addition to the effect of ploidity in the effi-
ciency of DNA repair, yeast cells may be of three
different mating types: a,a or ara. The cell type of
each individual cell affects the efficiency of DNA
repair, and the induction of recombination. In this
respect, yeast cells provide a good model system to
study how DNA repair mechanisms are influenced
by cell type.
In nature, yeast cells are ara only if they are
diploids. Cells with a heterozygous configuration at
their mating type locus show higher levels of ra-
dioresistance, and higher frequencies of radiation-as-
sociated recombination events than isogenic ara and
ara cells w40,31,32,52x. Genetic analysis has shown
that this effects requires functional a1 and a 2 gene
products of the MAT locus and is independent of the
transcriptional regulator RME1, which plays a role in
initiating meiosis w52,53x. The exact mechanism by
which mating type heterozygosity affects recombina-
tion is not known, although it has been proposed that
mating type heterozygosity enhances the efficiency
of pairing Žand thus of recombination. between ho-
mologs, thus improving the chances of survival of
diploid cells, which are usually ara w61x. This ex-
planation, however, does not seem to hold since Ž1.
all the results gathered to date point to the fact that
pairing between the homologous sequences is not a
limiting step in recombination w59,72,75x; Ž2. mating
type heterozygosity confers radioresistance to hap-
loid strains as well w52,100,126x; and Ž3. mating type
heterozygosity also increases the efficiency of
DSBs-initiated intrachromosomal recombination in
haploid cells, which does not require pairing between
homologs w31x.
Alternative explanations for the effect of mating
type heterozygosity are that either diploid-specific
recombination systems are expressed in ara cells
w52,66x or rate-limiting recombination proteins may
be induced at higher levels in ara cells compared to
their isogenic ara and ara cells w31,32,66x. These
explanations are not necessarily exclusive, and both
agree with the observation that in addition to in-
crease recombinational repair, mating type heterozy-
gosity decreases mutagenic repair w52x. Recombina-
tional repair should be of advantage to diploids,
which carry an additional set of chromosomes, but
haploid cells favor non-recombinational repair, even
if it is mutagenic, since attempts to recombine in
haploids may lead to chromosomal rearrangements
and cell death.
 M. Kupiecr Mutation Research 451 (2000) 91–105
6. The mechanism of induction of recombination
by DNA damage
There are two different mechanisms through which
DNA damage can induce recombination. On one
hand, the damage causes an activation of the genes
encoding enzymes involved in recombination Ži.e.
induction of trans-acting factors.. On the other hand,
DNA damage also provides the DNA lesions which
serve to initiate the process of recombination Ž cis-
acting factors.. The coupling between the two mech-
anisms ensures that enough recombination enzymes
will be available to cope with increasing DNA dam-
age levels.
Although evidence proving that both modes of
induction operate in yeast has been gathered Žsee
below., it is not always obvious which one is respon-
sible for most of the induction. Is the enzymatic
activity present in the cell in the absence of external
damage sufficient to cope with most insults, or is the
transcriptional induction the main mechanism re-
sponsible for the increase in recombination? Since
the experiments carried out to answer this question
are not always easy to interpret, and the relationship
between these two ‘‘modes’’ of induction is far from
being understood, below I present arguments in favor
and against the supremacy of each one of them.
6.1. DNA damage induces recombination by increas-
ing transcription of recombination genes
In an elegant series of experiments, Fabre and
Roman w29x gathered information supporting the ex-
istence of a signal for increased levels of recombina-
tion. A mating-capable diploid strain, heteroallelic at
the ADE6 locus Ž ade6-21rade6-45 ., was mated
with haploid cells of a double mutant Ž ade6-21, 45 .
strain, that was subjected to irradiation. The results
showed a dose-dependent induction of recombination
in the selected triploids created by the mating of the
diploid and the irradiated haploid. Since this last
strain carried the same mutations as the diploid, it
could not contribute genetic information. The authors
therefore concluded that the recombination events
did not involve the participation of the irradiated
chromosome Ži.e. cis-acting lesions., and that the
induction was caused by the release of certain
97
factorŽs. elicited by the lesions in the DNA caused
by the radiation w29x.
Since these seminal experiments were carried out,
it has become clear that both prokaryotic and eukary-
otic cells respond to DNA damage by inducing the
transcription of a large set of genes, which probably
facilitate cell cycle arrest, DNA repair, or adaptation
to the insult Žfor a review, see Ref. w22x..
In bacteria, the inducible SOS repair system has
been extensively studied. In response to DNA dam-
age, a regulon comprising more than 20 genes con-
trolling DNA replication, recombination, mutation,
etc., is expressed at higher levels Žreviewed in Ref.
w38x.. The damage–response system of Escherichia
coli has served as a paradigm for many years. In
yeast, the existence of an inducible ‘‘error prone’’
mechanism of repair has been suggested by experi-
ments carried out with temperature-sensitive mutants
w7,104x. However, evidence for the existence of simi-
lar damage-induced system for recombination re-
mains more controversial.
Lately, the RAD9 checkpoint gene has been shown
to activate, upon DNA damage, a great regulon of
genes that include repair, recombination, and replica-
tion genes. This transcriptional activation is indepen-
dent of the cell-cycle-arrest role of RAD9 in the
checkpoint w1x. Similarly, in mammals, DNA damage
leads to the transcriptional activation of certain genes
through the actions of the ATM and p53 tumor
suppressor genes w9x. The yeast homolog of ATM,
MEC1, is responsible for most of the damage-in-
duced transcriptional induction in yeast. Following
DNA damage, a phosphorylation cascade that in-
cludes the Rad53 and Dun1 protein kinases leads to
cell cycle arrest and to phosporylation of the Crt1
repressor. This repressor, usually bound to DNA
damage-inducible genes, loses its ability to bind to
the regulated promoters upon phosphorylation. Thus,
phosphorylation of Crt1 leads to the activation of the
inducible genes w54,111x.
The number of genes activated by DNA damage
in yeast is very large. For example, in a recent
genomic study w58x, as many as 325 genes showed at
least fourfold increased transcription levels after
treatment with the alkylating agent MMS. This rep-
resents ; 5% of the yeast genome, and gives a
conservative estimate, since many more genes
showed significant induction levels that were below
98 M. Kupiecr Mutation Research 451 (2000) 91–105
the fourfold artificial threshold. As expected, many
of these genes are involved in DNA replication
andror repair; however, these were only a minority
of the genes induced, and the relevance of the tran-
scriptional induction in protecting cells against dam-
age remains to be determined w58x.
Although the idea that recombination genes should
be induced as a response to the presence of DNA
damage in order to increase recombinational repair is
aesthetically pleasing, the experimental results do not
always support this logic. The RAD54 recombina-
tion gene, which has been thoroughly studied, is a
good case study. The RAD54 gene is transcription-
ally induced by DNA damage. Different types of
DNA damaging agents, including UV, X-rays, MMS,
or overexpression of a restriction enzyme, are able to
elicit this response. Cells synchronized at the G1
stage of the cell cycle are still inducible, implying
that the induction is not due to the accumulation of
cells at a certain stage of the cell cycle at which the
level of expression is high w3,12,14x. An analysis of
the promoter, and its comparison to other damage-in-
ducible genes reveals the presence of an element,
termed damage–responsive element ŽDRE. fully re-
sponsible for the transcriptional induction. A similar
element is present, for example, upstream of the
UV-inducible excision repair gene RAD2. Deletion
of the DRE from these genes, however, does not
confer enhanced radiation sensitivity after exposure
to UV or ionizing radiation, respectively w15,105x.
Moreover, strains carrying an allele of RAD54 inca-
pable of transcriptional induction are still fully capa-
ble of showing induced recombination upon expo-
sure to DNA-damaging agents w15x. Thus, although
the gene is induced and more protein has been
shown to be produced following DNA damage, it is
still not clear that this induction is biologically sig-
nificant, or that contributes in any way to the en-
hanced levels of mitotic recombination observed af-
ter irradiation.
It should also be noted that while some genes
which physically interact with RAD54 during recom-
bination are also damage-induced Že.g. RAD51., oth-
ers, such as the central RAD52 gene, are not in-
ducible w14x. It is possible to rationalize these results
by assuming that the Rad52 protein is more abundant
in the cell, or is needed at lower concentrations, than
Rad51p and Rad54p. If this is so, only the later two
would be limiting for recombination and would re-
quire induction. To complicate the issue, it has been
recently shown that the transcriptional induction of
RAD51 is RAD52-dependent w4x. It is still possible
that the noninducible allele of RAD54 is defective in
some aspect of induced recombination still to be
defined.
A strong argument against the supremacy of an
inducible recombination repair system comes from
split-dose experiments. If induction of recombination
involves two components: the induction of the
recombination enzymes on one hand, and the cre-
ation of the recombination substrate on the other,
exposure to a dose that induces the enzymatic ma-
chinery should elevate the level of recombination
caused by a subsequent UV dose. Split-dose experi-
ments of this type have uncovered adaptive re-
sponses protecting against alkylating damage in
prokaryotes and eukaryotes Žreviewed in Ref. w82x..
Similar experiments, however, failed to show any
induction of recombination beyond the additive ef-
fect expected from both doses w107x. On the other
hand, other authors have deduced the existence of a
DSB-inducible repair mechanism from experiments
in which cells subjected to low doses of ionizing
radiation or cisplatin treatment showed lower levels
of mutagenesis, when challenged later by MNNG.
These results were interpreted to mean that the first
treatment induced an Žerror free. recombination sys-
tem in the cells, able to repair the MNNG damage
without introducing mutations w7,21x. Biochemical
confirmation for the existence of such an inducible
recombination system, however, is still lacking.
6.2. DNA damage induces recombination by proÕid-
ing additional substrates
The second mechanism of induction is by facili-
tating the initial step in recombination, which is
believed to be the creation of a DSB w88,110x. A
certain level of activity of the recombination en-
zymes is present in cells not exposed to genotoxic
agents, but the lesions in the DNA constitute the
limiting factor for recombination. The level of induc-
tion reached by treatment with DNA damaging agents
is usually lower than that seen in meiotic cells. It has
now become established that meiotic recombination
is induced in yeast by the creation of DSBs at many
 M. Kupiecr Mutation Research 451 (2000) 91–105
different places in the genome w68,127x. Mutations in
the SPO11 and RAD50 genes, which prevent the
creation of the DSBs, completely eliminate meiotic
recombination w68x. DSBs created by X-rays or site-
specific endonucleases are able to induce meiotic
recombination in spo11 or rad50 mutant cells
w79,115x, showing that the creation of DSBs is the
limiting step in the induction of meiotic recombina-
tion.
A direct test of the hypothesis that most induced
recombination depends on increased availability of
recombination substrates, was carried out using plas-
mid transformation w106x. In these experiments,
which are formally similar to those carried out by
Fabre and Roman w29x described above, a diploid
yeast strain heteroallelic at the HIS3 locus was
transformed with minichromosomes Žcentromeric
plasmids. carrying homology to the HIS3 gene, but
containing the same two mutations present in the
chromosomes. When a DSB was introduced in the
region of homology, an increase in the recombina-
tion frequency between the heteroalleles was ob-
served, although the plasmid was unable to donate
wild-type information. This induction of recombina-
tion was dependent on the presence of homology
between the broken minichromosomes and the diploid
chromosomes. Thus, recombination was not induced
by the presence of DSBs in regions of nonhomology,
but it was induced by the presence of DSBs in
homologous regions, even if these were unable to
transfer wild type information. These experiments
are consistent with a model in which recombination
is initiated locally by the recruitment to the broken
chromosome of the recombination machinery, which
can be later transferred to the other chromosomes to
promote their recombination. Evidence for tripartite
events, involving the plasmid and the two homolo-
gous chromosomes, was presented w106x. According
to this interpretation, the authors re-assessed the
results obtained by Fabre and Roman w29x in the
following way: irradiation of the haploid created
lesions in its genome. Recruitment of recombination
proteins on the doubly mutated ade6 allele was
followed by their transfer to the homologous regions
in the heteroalleles. One way to decide between the
two alternative interpretations would be to repeat the
Fabre and Roman experiments using cells carrying a
complete deletion of the ADE6 gene. A technical
99
caveat for such an experiment is that the deletion
should be large enough to ensure that recombination
proteins would not nucleate at homologous regions
bordering the ADE6 gene in the haploid strain.
Another argument in favor of a model in which
induction results from the repair of more lesions,
rather than as a response to a global signal, comes
from studies on recombination of Ty elements. These
repeated sequences, which constitute ; 3% of the
yeast genome, are randomly dispersed in the chro-
mosomes. Recombination between members of the
Ty family occurs spontaneously at low rates, despite
their large number w69,70x. DNA damage-stimulated
ectopic recombination between Ty elements has not
been observed even when the same treatments ŽUV,
MMS, or X-rays. induce other types of recombina-
tion in the same cells w72,89x. However, when a DSB
is introduced at a Ty, it is efficiently repaired by
recombination w90x. These results are more consistent
with a local, rather than global, view of induction,
and can be explained by assuming that some special
feature of Ty elements, such as chromatin configura-
tion, makes them refractory to receive spontaneous
or damage-induced lesions. Alternatively, DNA
damage in Ty elements is either not usually pro-
cessed into DSBs and leads to cell death, or is
preferentially repaired by a ‘‘silent’’ mechanism,
such as sister chromatid recombination. When a DSB
is forcibly created locally, however, it is readily
repaired by recombination, implying that the DNA is
accessible to the recombination machinery w90x.
It should be noted that the two modes of induction
described are not necessarily exclusive. A DSB can
initiate recombination locally, and also cause the
transcriptional induction of DNA repair and recom-
bination genes. Such an arrangement should be ad-
vantageous for the cell. One may assume that under
low damage levels, enough recombination proteins
are present in the cells to allow some repair. The
transcriptional induction of DNA repair genes may
allow the cell to be prepared to cope with increasing
levels of damage. In the last years, most of the
proteins involved in recombination have been cloned
and expressed, and assays to measure recombination
activity in vitro are being developed. These improve-
ments should allow in the near future to directly
measure the concentration of relevant proteins and
the recombinational activity in cells following expo-
100 M. Kupiecr Mutation Research 451 (2000) 91–105
sure to damaging agents. In this way, it will be
possible to evaluate the relative importance of the
two modes of induction.
7. The relationship between spontaneous and in-
duced recombination
Spontaneous recombination results from the repair
of lesions created by the normal metabolism of the
cell. These include damage caused by reactive oxy-
gen species, as well as DSBs created during DNA
replication and transcription w38x. As explained
above, it is usually the case that recombination sub-
strates that yield low spontaneous recombination lev-
els can be induced to greater degrees by DNA
damaging agents, than those that give a high sponta-
neous rate.
Several explanations may be given to this obser-
vation. Ž1. Following the mechanism of induction
presented in Section 6.1, one may assume that the
substrate that gives a high spontaneous recombina-
tion rate may cause the cells to be already at an
induced state, so that exogenous damage does not
provoke the induction seen with other substrates, and
therefore has no effect. Ž2. According to the mecha-
nism presented in Section 6.2, the induction depends
upon the creation of new lesions, and the substrate
may already provide a very high level of those
lesions, such that the recombinational machinery is
already at its maximal activity level and additional
lesions may have no further effect. Ž3. The lesions
created by the DNA damaging agent may be qualita-
tively different from the spontaneous ones, and may
initiate recombination by a different mechanism. If
many spontaneous lesions are created, or the lesions
are very recombinogenic, compared to those induced
by the damaging agent, only high doses of the
noxious agent will produce any detectable effect,
over the high spontaneous background. Analysis of
the induction of DRR by several DNA damaging
agents, using such a substrate with high basal recom-
bination level, supports the third interpretation. At
low doses, no increase in recombination is seen over
the background, but at higher doses a proportional
induction can be detected Žw72x; Liefshitz, Steinlauf
and Kupiec, unpublished results..
8. Concluding remarks and human implications
The integrity of the genome is constantly being
challenged by endogenous and exogenous DNA
damaging agents. Eukaryotic cells have evolved a
variety of mechanisms that allow them to cope with
this danger. The significance of DNA repair for
humans and the close link between genome stability
and cancer is illustrated by the extreme phenotypes
of patients with hereditary diseases caused by defects
in repair processes, such as Xeroderma pigmento-
sum, Cockayne’s syndrome, Ataxia telangiectasia,
and Nijmegen breakage syndrome w119x.
Recombination is one of the many pathways that
help the cell to cope with lesions in its chromo-
somes. Many chemical and environmental carcino-
gens are recombinogenic, and cause genomic rear-
rangements, such as gene amplification w116x, and
chromosomal rearrangements w48x. Since mitotic re-
combination can lead to the homozygosity of reces-
sive alleles, it plays a pivotal role in the multistep
process of carcinogenesis w48,123x. Exposure to ion-
izing radiation is correlated with an increase in the
incidence of acute myelocytic leukemia, tyroid can-
cer, and other forms of neoplasias w50,73,84x. In
addition, the risk of exposure to harmful UV radia-
tion is gradually increasing, with the erosion of the
stratospheric ozone layer w17x. Greater exposure to
UV is likely to result in a general increase in the
level of cancer incidence in the population. The
understanding of the mechanisms of induced mitotic
recombination in mammalian cells is therefore of
capital importance.
Yeast cells have played a pivotal role in the
understanding of basic DNA repair and recombina-
tion mechanisms. Numerous studies over the years
have served to define the basic concepts and to
identify the main players. Given the high genetic
conservation of DNA repair mechanisms and genes
between all eukaryotes, it is not surprising that our
understanding of the systems operating in yeast have
lately started to be applied to the study of recombina-
tion in mammalian cells Žfor a recent review, see
Ref. w63x.. One expected difference between yeast
and mammalian cells is the relative weight of homol-
ogous recombination versus non-homologous end-
joining ŽNHEJ. mechanisms. Whereas the proteins
that participate in these two competing mechanisms
 M. Kupiecr Mutation Research 451 (2000) 91–105
are conserved from yeast to humans, and play very
similar biochemical roles w63,88x; in yeast most DSBs
are preferentially repaired by homologous recom-
bination, whereas NHEJ plays a main role in human
cells. Whether NHEJ is DNA damage-inducible is
still a matter of debate w22x.
A radio-adaptive response ŽRAR. has been de-
scribed in mammalian cells, in which a first low
radiation dose protects against a subsequent large
dose w22,87x. In addition, it has been known for a
long time that DNA-damaging agents induce sister
chromatid exchanges w48x. However, direct evidence
for induced homologous recombination in humans is
scant w74x. This situation has started to change, with
the isolation of the human counterparts of the yeast
recombination genes, which has allowed the creation
of mammalian cell lines and transgenic animals de-
fective in homologous recombination genes Že.g.
Refs. w113,114x.. At the same time, new recombina-
tion substrates, shaped after those developed for
yeast studies Že.g. Refs. w95,103x. have become avail-
able. This combination of biochemical, genetic, and
molecular biology techniques has lately brought to a
better understanding of the relationship between the
different genes and proteins involved in the process
of recombination, and rapid progress is being made
lately in deciphering the biochemical activities of the
various players Žw63,88,118x.. A future challenge is
to try to understand the mechanisms involved in the
induction of recombination. Such an understanding
will have important consequences in cancer treat-
ment, and in the assessment of risks arising from
exposure to genotoxic agents.
Acknowledgements
Due to the concise format of this review, I was
prevented from referring to many publications, com-
ing from many different laboratories, that bear on the
subject of induced recombination. I therefore apolo-
gize to all colleagues whose work was not cited. I
thank Batia Liefshitz and Sigal Ben-Yehuda for
comments on the manuscript, two anonymous re-
viewers for advice, and Francis Fabre for numerous
and fruitful discussions. This work was supported
from a grant from the Israeli Science Foundation.
101
References
w1x A. Aboussekhra, J.E. Vialard, D.E. Morrison, M.A. Torre-
Ruiz, L. Cernakova, F. Fabre, N.F. Lowndes, A novel role
for the budding yeast RAD9 checkpoint gene in DNA
damage-dependent transcription, EMBO J. 15 Ž1996. 3912–
3922.
w2x A. Aguilera, H.L. Klein, Genetic control of intrachromoso-
mal recombination in Saccharomyces cereÕisiae: I. Isola-
tion and genetic characterization of hyper-recombination
mutations, Genetics 119 Ž1988. 779–790.
w3x D. Averbeck, S. Averbeck, Induction of the genes RAD54
and RNR2 by various DNA damaging agents in Saccha-
romyces cereÕisiae, Mutat. Res. 315 Ž1994. 123–138.
w4x D. Averbeck, S. Averbeck, DNA photodamage, repair, gene
induction and genotoxicity following exposures to 254 nm
UV and 8-methoxyposoralen plus UVA in a eukaryotic cell
system, Photochem. Photobiol. 68 Ž1998. 289–295.
w5x H. Bierne, B. Michel, When replication forks stop, Mol.
Microbiol. 13 Ž1994. 17–23.
w6x S. Boiteaux, O. Huisman, J. Laval, 3-Methyladenine residues
in DNA induce the SOS function sfiA in Escherichia coli,
EMBO J. 3 Ž1984. 2569–2573.
w7x D.R. Boreham, R.E. Mitchel, DNA lesions that signal the
induction of radioresistance and DNA repair in yeast, Ra-
diat. Res. 128 Ž1991. 19–28.
w8x R.J. Brennan, B.E.P. Swoboda, R.H. Schiestl, Oxidative
mutagens induce intrachromosomal recombination in yeast,
Mutat. Res. 308 Ž1994. 159–167.
w9x J. Brugarolas, C. Chandrasekaran, J.I. Gordon, D. Beach, T.
Jacks, G.J. Hannon, Radiation-induced cell cycle arrest
compromised by p21 deficiency, Nature 377 Ž1995. 552–
557.
w10x G. Brunborg, M. Resnick, D.H. Williamson, Cell cycle-
specific repair of DNA DSBs in Saccharomyces cereÕisiae,
Radiat. Res. 82 Ž1980. 547–558.
w11x W. Chen, S. Jinks-Robertson, Mismatch repair proteins
regulate heteroduplex formation during mitotic recombina-
tion in yeast, Mol. Cell. Biol. 18 Ž1998. 6525–6537.
w12x B. Clever, J. Schmuckli-Maurer, M. Sigrist, B.J. Glassner,
W.D. Heyer, Specific negative effects resulting from ele-
vated levels of the recombinational repair protein Rad54p in
Saccharomyces cereÕisiae, Yeast 15 Ž1999. 721–740.
w13x R.S. Cole, Repair of DNA containing interstrand crosslinks
in E. coli: sequential excision and recombination, Proc.
Natl. Acad. Sci. U. S. A. 70 Ž1973. 1064–1068.
w14x G.M. Cole, D. Schild, S.T. Lovett, R.K. Mortimer, Regula-
tion of RAD54 and RAD52-lacZ gene fusions in S. cere-
Õisiae in response to DNA damage, Mol. Cell. Biol. 7
Ž1987. 1078–1084.
w15x G.M. Cole, S.T. Lovett, D. Schild, R.K. Mortimer, Failure
to induce a DNA repair gene, RAD54, in Saccharomyces
cereÕisiae does not affect DNA repair or recombination
pathways, Mol. Cell. Biol. 9 Ž1989. 3314–3322.
w16x A.J. Cooper, R. Waters, A complex pattern of sensitivity to
simple monofunctional alkylating agents exists amongst the
102 M. Kupiecr Mutation Research 451 (2000) 91–105
rad mutants of Saccharomyces cereÕisiae, Mol. Gen. Genet.
209 Ž1987. 142–148.
w17x P.J. Crutzen, Ultraviolet on the increase, Nature 356 Ž1992.
104–105.
w18x M. Dardalhon, D. Averbeck, Pulsed-field gel electrophore-
sis analysis of the repair of psoralen plus UVA induced
DNA photoadducts in Saccharomyces cereÕisiae, Mutat.
Res. 336 Ž1995. 49–60.
w19x P.J. Davies, R.S. Tippins, J.M. Parry, Cell cycle variation in
the inducibility of lethality and mitotic recombination after
treatment with UV and nitrous acid in the yeast Saccha-
romyces cereÕisiae, Mutat. Res. 51 Ž1978. 327–346.
w20x L. DiCaprio, B.S. Cox, The effect of UV irradiation on the
molecular weight of preexisting and newly-synthesized DNA
of yeast, Mutat. Res. 82 Ž1981. 69–85.
w21x J.A. Dolling, D.R. Boreham, D.L. Brown, G.P. Raaphorst,
R.E. Mitchel, Cisplatin-modification of DNA repair and
ionizing radiation lethality in yeast Saccharomyces cere-
Õisiae, Mutat. Res. 433 Ž1999. 127–136.
w22x F. Eckardt-Schupp, C. Klaus, Radiation inducible DNA
repair processes in eukaryotes, Biochimie 81 Ž1999. 161–
171.
w23x M.S. Esposito, J.E. Wagstaff, Mechanisms of mitotic
recombination, in: J.N. Strathern, E.W. Jones, J.R. Broach
ŽEds.., The Molecular Biology of the Yeast Saccharomyces,
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY,
1981, pp. 341–370.
w24x R.E. Esposito, Genetic recombination in synchronized cul-
tures of Saccharomyces cereÕisiae, Genetics 59 Ž1968.
191–202.
w25x G. Evensen, E. Seeberg, Adaptation to alkylation resistance
involves the induction of a DNA glycosylase, Nature 296
Ž1982. 773–775.
w26x F. Fabre, Induced intragenic recombination in yeast can
occur during the G1 mitotic phase, Nature 272 Ž1978.
795–798.
w27x F. Fabre, Mitotic recombination and repair in relation to cell
cycle, in: D. von Wettstein, J. Friis, M. Kielland-Brandt, A.
Stenderup ŽEds.., Molecular Genetics of Yeast, Munskgaad,
Copenhagen, 1981, pp. 399–409.
w28x F. Fabre, A. Boulet, H. Roman, Gene conversion at differ-
ent points in the mitotic cycle of Saccharomyces cereÕisiae,
Mol. Gen. Genet. 195 Ž1984. 139–143.
w29x F. Fabre, H. Roman, Genetic evidence for the inducibility
of recombination competence in yeast, Proc. Natl. Acad.
Sci. U. S. A. 74 Ž1977. 1667–1671.
w30x R. Fahrig, Evidence that induction and suppression of muta-
tions and recombinations by chemical mutagens in S. cere-
Õisiae during mitosis are jointly correlated, Mol. Gen. Genet.
168 Ž1979. 125–139.
w31x M. Fasullo, T. Bennet, P. Dave, Expression of Saccha-
romyces cereÕisiae MATa and MATa enhances the HO
endonuclease-stimulation of chromosomal rearrangements
directed by his3 recombinational substrates, Mutat. Res. 433
Ž1999. 33–44.
w32x M. Fasullo, P. Dave, Mating type regulates the radiation-as-
sociated stimulation of reciprocal translocation events in
Saccharomyces cereÕisiae, Mol. Gen. Genet. 243 Ž1994.
63–70.
w33x M.T. Fasullo, P. Dave, R. Rothstein, DNA damaging agents
stimulate the formation of directed reciprocal translocations
in Saccharomyces cereÕisiae, Mutat. Res. 314 Ž1994. 121–
133.
w34x M.T. Fasullo, R.W. Davis, Recombinational substrates de-
signed to study recombination between unique and repeti-
tive sequences in vivo, Proc. Natl. Acad. Sci. U. S. A. 84
Ž1987. 6215–6219.
w35x M.T. Fasullo, R.W. Davis, Direction of chromosome rear-
rangements in Saccharomyces cereÕisiae by use of his3
recombinational substrates, Mol. Cell. Biol. 8 Ž1988. 4370–
4380.
w36x D. Frankenberg, M. Frankenberg-Schwager, D. Blocher, R.
Harbich, Evidence for DNA DSBs as the critical lesions in
yeast cells irradiated with sparsely or densely ionizing
radiation under oxic and anoxic conditions, Radiat. Res. 88
Ž1981. 524–532.
w37x M. Frankenberg-Schwager, D. Frankenberg, DNA double-
strand breaks: their repair and relationship to cell killing in
yeast, Intl. J. Radiat. Biol. 58 Ž1990. 569–575.
w38x E.C. Friedberg, G.C. Walker, W. Siede, DNA Repair and
Mutagenesis, American Society for Microbiology, Washing-
ton, DC, 1995.
w39x A.A. Friedl, M. Kiechle, B. Fellerhoff, F. Eckardt-Schupp,
Radiation-induced chromosome aberrations in Saccha-
romyces cereÕisiae: influence of DNA repair pathways,
Genetics 148 Ž1998. 975–988.
w40x J. Friis, H. Roman, The effect of the mating type alleles on
intragenic recombination in yeast, Genetics 59 Ž1968. 33–
36.
w41x A. Galli, R.H. Schiestl, On the mechanism of UV and
Gamma-ray induced intrachromosomal recombination in
yeast cells synchronized in different stages of the cell cycle,
Mol. Gen. Genet. 248 Ž1995. 301–310.
w42x A. Galli, R.H. Schiestl, Effects of DNA double-strand and
single-strand breaks on intrachromosomal recombination
events in cell cycle-arrested yeast cells, Genetics 149 Ž1998.
1235–1250.
w43x A. Galli, R.H. Schiestl, Cell division transforms mutagenic
lesions into deletion-recombinogenic lesions in yeast cells,
Mutat. Res. 429 Ž1999. 13–26.
w44x S.M. Galloway, Chromosome aberrations induced in vitro:
mechanism, delayed expression, and intriguing questions,
Environ. Mol. Mutagen. 23 Ž1994. 44–53.
w45x J.C. Game, DNA double-strand breaks and the RAD50-
RAD57 genes in Saccharomyces cereÕisiae, Sem. Cancer
Biol. 4 Ž1993. 73–83.
w46x J.C. Game, K.C. Sitney, V.E. Cook, R.K. Mortimer, Use of
a ring chromosome and pulse-field gels to study interho-
molog recombination, double-strand DNA breaks and
sister-chromatid exchange in yeast, Genetics 123 Ž1989.
695–713.
w47x K.F. Grossman, J. Brown, R.E. Moses, Cisplatin DNA
 M. Kupiecr Mutation Research 451 (2000) 91–105
cross-links do not inhibit S-phase and cause only a G2rM
arrest in Saccharomyces cereÕisiae, Mutat. Res. 434 Ž1999.
29–39.
w48x L. Hagmar, S. Bonassi, U. Stromberg, A. Brogger, L.E.
Knudsen, H. Norppa, C. Reuterwall, Chromosomal aberra-
tions in lymphocytes predict human cancer: a report from
the European Study Group on Cytogenic Biomarkers and
Health ŽESCH., Cancer Res. 58 Ž1998. 4117–4121.
w49x A. Halas, H. Baranowska, Z. Policinska, W.J. Jachymczyk,
Involvement of the RE V3 gene in the methylated base-ex-
cision repair system. Co-operation of two DNA poly-
merases, delta and Rev3p, in the repair of MMS-induced
lesions in the DNA of Saccharomyces cereÕisiae, Curr.
Genet. 31 Ž1997. 292–301.
w50x P. Hall, L.E. Holm, Radiation-associated thyroid cancer —
facts and fiction, Acta Oncol. 37 Ž1998. 325–330.
w51x R.H. Haynes, B.A. Kunz, DNA repair and mutagenesis, in:
J.N. Strathern, E.W. Jones, J.R. Broach ŽEds.., The Molecu-
lar Biology of the Yeast Saccharomyces, Cold Spring Har-
bor Laboratory, Cold Spring Harbor, NY, 1981, pp. 371–
414.
w52x M. Heude, F. Fabre, ara control of DNA repair in the
yeast Saccharomyces cereÕisiae: genetic and physiological
aspects, Genetics 133 Ž1993. 489–498.
w53x A.K. Hopper, J. Kirsch, B.D. Hall, Mating type and sporu-
lation in yeast: II. Meiosis, recombination and radiation-
sensitivity in an a ra diploid with altered sporulation
control, Genetics 80 Ž1975. 61–76.
w54x M. Huang, Z. Zhou, S.J. Elledge, The DNA replication and
damage checkpoint pathways induce transcription by inhibi-
tion of the Crt1 repressor, Cell 94 Ž1998. 595–605.
w55x E.G. Hunnable, B.S. Cox, The genetic control of dark
recombination in yeast, Mutat. Res. 13 Ž1971. 297–309.
w56x Z. Jablonovich, B. Liefshitz, R. Steinlauf, M. Kupiec, Char-
acterization of the role played by the RAD59 gene of
Saccharomyces cereÕisiae in ectopic recombination, Curr.
Genet. 36 Ž1999. 13–20.
w57x W.J. Jachymczyk, E. Chlebowicz, Z. Swietlinska, J. Zuk,
Alkaline sucrose sedimentation studies of MMS-induced
DNA single-strand breakage and rejoining in the wild type
and in UV-sensitive mutants of Saccharomyces cereÕisiae,
Mutat. Res. 43 Ž1977. 1–10.
w58x S.A. Jelinsky, L.D. Samson, Global response of Saccha-
romyces cereÕisiae to an alkylating agent, Proc. Nat. Acad.
Sci. U. S. A. 96 Ž1999. 1486–1491.
w59x S. Jinks-Robertson, T.D. Petes, Chromosomal translocations
generated by high frequency meiotic recombination be-
tween repeated yeast genes, Genetics 114 Ž1986. 731–752.
w60x L.H. Johnston, A.L. Johnson, Induced recombination in the
mitotic cell cycle of the yeast Saccharomyces cereÕisiae,
Genet. Res. Camb. 43 Ž1983. 1–10.
w61x L.C. Kadyk, L.H. Hartwell, Sister chromatids are preferred
over homologs as substrates for recombinational repair in
Saccharomyces cereÕisiae, Genetics 132 Ž1992. 387–402.
w62x L.C. Kadyk, L.H. Hartwell, Replication-dependent sister
chromatid recombination in rad1 mutants of Saccha-
romyces cereÕisiae, Genetics 133 Ž1993. 469–487.
w63x R. Kanaar, J.H.J. Hoeijmakers, D.C. van Gent, Molecular
103
mechanisms of double-strand break repair, Trends Cell
Biol. 8 Ž1998. 483–489.
w64x W.K. Kaufmann, R.S. Paules, DNA damage and cell cycle
checkpoints, FASEB J. 10 Ž1996. 238–247.
w65x R. Kern, F.K. Zimmermann, The influence of defects in
excision and error prone repair on spontaneous and induced
mitotic recombination and mutation in Saccharomyces cere-
Õisiae, Mol. Gen. Genet. 161 Ž1978. 81–88.
w66x H.L. Klein, RDH54, a RAD54 homologue in Saccha-
romyces cereÕisiae, is required for mitotic diploid-specific
recombination and repair and for meiosis, Genetics 147
Ž1997. 1533–1543.
w67x H.L. Klein, T.D. Petes, Intrachromosomal gene conversion
in yeast, Nature 289 Ž1981. 144–148.
w68x M. Kupiec, B. Byers, R.E. Esposito, A.P. Mitchell, Meiosis
and sporulation in Saccharomyces cereÕisiae, in: The
Molecular Biology of the Yeast Saccharomyces, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, 1997,
pp. 889–1036.
w69x M. Kupiec, T.D. Petes, Meiotic recombination between
repeated transposable elements in Saccharomyces cere-
Õisiae, Mol. Cell Biol. 8 Ž1988. 2942–2954.
w70x M. Kupiec, T.D. Petes, Allelic and ectopic recombination
between Ty elements in yeast, Genetics 119 Ž1998. 549–
559.
w71x M. Kupiec, G. Simchen, Arrest of the mitotic cell cycle and
of meiosis in Saccharomyces cereÕisiae by MMS, Mol.
Gen. Genet. 201 Ž1985. 558–564.
w72x M. Kupiec, R. Steinlauf, Damage-induced ectopic recom-
bination in the yeast Saccharomyces cereÕisiae, Mutat. Res.
384 Ž1997. 33–44.
w73x P. Leder, J. Battey, G. Lenoir, C. Moulding, W. Murphy, H.
Porter, T. Stewart, T. Taub, Translocations among antibody
genes and human cancer, Science 222 Ž1983. 765–770.
w74x S. Lehnert, T.Y. Chow, Low doses of ionizing radiation
induce nuclear activity in human tumor cell lines which
catalyzes homologous double-strand recombination, Radiat.
Environ. Biophys. 36 Ž1997. 67–70.
w75x M. Lichten, J.E. Haber, Position effects in ectopic and
allelic mitotic recombination in Saccharomyces cereÕisiae,
Genetics 123 Ž1989. 261–268.
w76x B. Liefshitz, A. Parket, R. Maya, M. Kupiec, The role of
DNA repair genes in recombination between repeated se-
quences in yeast, Genetics 140 Ž1995. 1199–1211.
w77x B. Liefshitz, R. Steinlauf, A. Friedl, F. Eckardt-Schupp, M.
Kupiec, Genetic interactions between mutants of the
‘‘error-prone’’ repair group of Saccharomyces cereÕisiae
and their effect on recombination and mutagenesis, Mutat.
Res. 407 Ž1998. 135–145.
w78x S.T. Lovett, P.T. Drapkin, V.A. Sutera, T.J. Gluckman-
Peskind, A sister-strand exchange mechanism for recA-in-
dependent deletion of repeated DNA sequences in E. coli,
Genetics 135 Ž1993. 631–642.
w79x A. Malkova, L. Ross, D. Dawson, M.F. Hoekstra, J.E.
Haber, Meiotic recombination initiated by a double-strand
break in rad50 delta yeast cells otherwise unable to initiate
meiotic recombination, Genetics 143 Ž1996. 741–754.
w80x T.R. Manney, R.K. Mortimer, Allelic mapping in yeast by
104 M. Kupiecr Mutation Research 451 (2000) 91–105
X-ray induced mitotic reversion, Science 143 Ž1964. 581–
583.
w81x L.B. Meira, M.B. Fonseca, D. Averbeck, A.C.G. Schen-
berg, J.A.P. Henriques, The pso4-1 mutation reduces spon-
taneous mitotic gene conversion and reciprocal recombina-
tion in Saccharomyces cereÕisiae, Mol. Gen. Genet. 235
Ž1992. 311–316.
w82x S. Mitra, B. Kaina, Regulation of repair of alkylation
damage in mammalian genomes, in: Progr. Nucleic Acid
Res. Mol. Biol. vol. 44 Academic Press, New York, 1993,
pp. 109–142.
w83x R.K. Mortimer, S. Fogel, Genetic interference and gene
conversion, in: R.F. Grell ŽEd.., Mechanisms in Recombina-
tion, Plenum, New York, 1969, pp. 263–275.
w84x C. Mothersill, C.B. Seymour, Mechanisms and implications
of genomic instability and other delayed effects of ionizing
radiation exposure, Mutagenesis 13 Ž1998. 421–426.
w85x E. Moustacchi, C. Cassier, R. Chanet, N. Magana-
Schwenke, T. Saeki, J.A.P. Henriques, Biological role of
photo-induced crosslinks and monoadducts in yeast DNA:
genetic control and steps involved in their repair, in: E.C.
Friedberg, B.A. Bridges ŽEds.., Cellular Responses to DNA
Damage, A.R. Liss, New York, 1983, pp. 87–106.
w86x Y. Nevo-Caspi, M. Kupiec, Transcriptional induction of Ty
recombination in yeast, Proc. Natl. Acad. Sci. U. S. A. 91
Ž1994. 12711–12715.
w87x G. Olivieri, J. Bodycote, S. Wolff, Adaptive response of
human lymphocytes to low concentrations of radioactive
thymidine, Science 223 Ž1984. 594–597.
w88x F. Paques, J.E. Haber, Multiple pathways of recombination
induced by double-strand breaks in Saccharomyces cere-
Õisiae, Microbiol. Mol. Biol. Rev. 63 Ž1999. 349–404.
w89x A. Parket, M. Kupiec, Ectopic recombination between Ty
elements in Saccharomyces cereÕisiae is not induced by
DNA damage, Mol. Cell. Biol. 12 Ž1992. 4441–4448.
w90x A. Parket, O. Inbar, M. Kupiec, Recombination of Ty
elements in yeast can be induced by a double-strand break,
Genetics 140 Ž1995. 67–77.
w91x A.G. Paulovich, R.U. Margulies, B.M. Garvik, L.H.
Hartwell, RAD9, RAD17 and RAD24 are required for S
phase regulation in Saccharomyces cereÕisiae in response
to DNA damage, Genetics 145 Ž1997. 45–62.
w92x T.D. Petes, R.E. Malone, L.S. Symington, Recombination
in yeast, in: J.R. Broach, J.R. Pringle, E.W. Jones ŽEds..,
Molecular and Cellular Biology of the Yeast Saccha-
romyces cereÕisiae, Cold Spring Harbor Lab. Press, Plain-
view, NY, 1991, pp. 407–521.
w93x G.P. Raaphorst, G. Wang, D. Stewart, C.E. Ng, Concomi-
tant low dose–rate irradiation and cisplatin treatment in
ovarian carcinoma cell lines sensitive and resistant to cis-
platin treatment, Int. J. Radiat. Biol. 69 Ž1996. 623–631.
w94x M.A. Resnick, P. Martin, The repair of double-strand breaks
in the nuclear DNA of Saccharomyces cereÕisiae and its
genetic control, Mol. Gen. Genet. 143 Ž1976. 119–129.
w95x C. Richardson, M.E. Moynahan, M. Jasin, Double-strand
break repair by interchromosomal recombination: suppres-
sion of chromosomal translocations, Genes Dev. 12 Ž1998.
3831–3842.
w96x H. Roman, F. Jacob, A comparison of spontaneous and
UV-induced allelic recombination with reference to the
recombination of outside markers, Cold Spring Harbor
Symp. Quant. Biol. 23 Ž1959. 155–160.
w97x R.H. Schiestl, Nonmutagenic carcinogens induce intrachro-
mosomal recombination in yeast, Nature 337 Ž1989. 285–
288.
w98x R.H. Schiestl, S. Igarashi, P.J. Hastings, Analysis of the
mechanism for reversion of a disrupted gene, Genetics 119
Ž1988. 237–247.
w99x R.H. Schiestl, S. Prakash, RAD1, an excision repair gene of
Saccharomyces cereÕisiae, is also involved in recombina-
tion, Mol. Cell. Biol. 8 Ž1988. 3619–3626.
w100x D. Schild, Suppression of a new allele of the yeast RAD52
gene by overexpression of RAD51, mutations in srs2 and
ccr4 or mating type heterozygosity, Genetics 140 Ž1996.
1150127.
w101x C. Sengstag, B. Weibel, M. Fasullo, Genotoxicity of Afla-
toxin B1: evidence for a recombination-mediated mecha-
nism in Saccharomyces cereÕisiae, Cancer Res. 56 Ž1996.
5457–5465.
w102x F. Sherman, H. Roman, Evidence for two types of allelic
recombination in yeast, Genetics 48 Ž1963. 253–271.
w103x M.J. Shulman, C. Collins, A. Connor, L.R. Read, M.D.
Baker, Interchromosomal recombination is suppressed in
mammalian somatic cells, EMBO J. 14 Ž1995. 4102–4107.
w104x W. Siede, F. Eckardt, Indications for an inducible compo-
nent of error-prone DNA repair in yeast, Br. J. Cancer 6
Ž1984. 103–106.
w105x W. Siede, G.W. Robinson, D. Kalainov, T. Malley, E.C.
Friedberg, Regulation of the RAD2 gene of Saccharomyces
cereÕisiae, Mol. Microbiol. 3 Ž1989. 1697–1698.
w106x R. Silberman, M. Kupiec, Plasmid-mediated induction of
recombination in yeast, Genetics 137 Ž1994. 41–48.
w107x J.R. Simon, P.D. Moore, Induction of homologous recom-
bination in Saccharomyces cereÕisiae, Mol. Gen. Genet.
214 Ž1988. 37–41.
w108x K. Skov, S. MacPhail, Interaction of platinum drugs with
clinically relevant X-ray doses in mammalian cells: a com-
parison of cisplatin, carboplatin, iproplatin, and teraplatin,
Int. J. Radiat. Oncol., Biol., Phys. 20 Ž1991. 221–225.
w109x R. Snow, C.T. Korch, Alkylation induced gene conversion
in yeast: use in fine structure mapping, Mol. Gen. Genet.
107 Ž1970. 201–208.
w110x F. Stahl, Meiotic recombination in yeast: coronation of the
double-strand-break repair model, Cell 87 Ž1996. 965–968.
w111x Z. Sun, D.S. Fay, F. Marini, D.F. Stern, Spk1rRad53 is
regulated by Mec1-dependent protein phosphorylation in
DNA replication and damage checkpoint, Genes Dev. 10
Ž1996. 395–406.
w112x J.W. Szostak, R. Wu, Unequal crossing-over in the riboso-
mal DNA of Saccharomyces cereÕisiae, Nature 284 Ž1980.
426–430.
w113x M. Takata, M.S. Sasaki, E. Sonoda, C. Morrison, M.
Hashimoto, H. Utsumi, Y. Yamaguchi-Iwai, A. Shinohara,
S. Takeda, Homologous recombination and non-homolo-
gous end-joining pathways of DNA double-strand break
repair have overlapping roles in the maintenance of chromo-
 M. Kupiecr Mutation Research 451 (2000) 91–105
somal integrity in vertebrate cells, EMBO J. 17 Ž1998.
5497–5508.
w114x T.L.R. Tan, J. Essers, E. Citterio, S.M.A. Swagemakers, J.
de Wit, F.E. Benson, J.H.J. Hoeijmakers, R. Kanaar, Mouse
Rad54 affects DNA conformation and DNA-damage-in-
duced Rad51 foci formation, Curr. Biol. 9 Ž1999. 325–328.
w115x L.W. Thorne, B. Byers, Stage-specific effects of X-irradia-
tion on yeast meiosis, Genetics 134 Ž1993. 29–42.
w116x T.D. Tlsty, P.C. Brown, R.T. Schimke, UV-irradiation facil-
itates methotrexate resistance and amplification of the di-
hidrofolate reductase gene in cultured 3T6 cells, Mol. Cell.
Biol. 4 Ž1984. 1040–1056.
w117x C.A. Torres-Ramos, S. Prakash, L. Prakash, Requirement of
yeast DNA polymerase Delta in Post-replicational repair of
UV-damaged DNA, J. Biol. Chem. 272 Ž1997. 25445–
25448.
w118x E. Van Dyck, A.Z. Stasiak, A. Stasiak, S.C. West, Binding
of double-strand breaks in DNA by human Rad52 protein,
Nature 398 Ž1999. 728–731.
w119x G. Weeda, J. de Boer, I. Donker, J. de Wit, S.B. Winkler,
G.T. van der Horst, W. Vermeulen, D. Bootsma, J.H.
Hoeijmakers, Molecular basis of DNA repair mechanisms
and syndromes, Rec. Results Cancer Res. 154 Ž1998. 147–
155.
w120x J. Wildenberg, The relation of mitotic recombination to
DNA replication in yeast pedigrees, Genetics 66 Ž1970.
291–304.
w121x K.K. Willis, H.L. Klein, Intrachromosomal recombination
in Saccharomyces cereÕisiae: reciprocal exchange in an
inverted repeat and associated gene conversion, Genetics
117 Ž1987. 633–643.
105
w122x D. Wilkie, D. Lewis, The effect of UV light on recombina-
tion in yeast, Genetics 48 Ž1963. 1701–1716.
w123x F.E. Wurgler, Recombination and gene conversion, Mutat.
Res. 284 Ž1992. 3–14.
w124x W. Xiao, B.L. Chow, Synergism between yeast nucleotide
and base excision repair pathways in the protection against
DNA methylation damage, Curr. Genet. 3 Ž1998. 92–99.
w125x W. Xiao, B.L. Chow, L. Rathgeber, The repair of DNA
methylation damage in Saccharomyces cereÕisiae, Curr.
Genet. 30 Ž1996. 461–468.
w126x Y.X. Yan, R.H. Schiestl, L. Prakash, mating-type suppres-
sion of the DNA-repair defect of the yeast rad6D mutation
requires the activity of genes in the RAD52 epistasis group,
Curr. Genet. 28 Ž1995. 8–12.
w127x D. Zenvirth, T. Arbel, A. Sherman, M. Goldway, S. Klein,
G. Simchen, Multiple sites for double-strand breaks in
whole meiotic chromosomes of Saccharomyces cereÕisiae,
EMBO J. 11 Ž1992. 3441–3447.
w128x F.K. Zimmermann, R.C. von Borstel, E.S. von Halle, J.M.
Parry, D. Siebert, G. Zetterberg, R. Barale, N. Loprieno,
Testing of chemicals for genetic activity with Saccha-
romyces cereÕisiae: a report of the U.S. Environmental
protection agency gene-tox program, Mutat. Res. 133 Ž1984.
199–244.
w129x F.K. Zimmermann, R. Kern, H. Rasenberger, A yeast strain
for simultaneous detection of induced mitotic crossing over,
mitotic gene conversion and reverse mutation, Mutat. Res.
28 Ž1975. 381–390.