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Mutation Research 451 Ž2000.

91–105
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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-


sion repair ŽBER., the nucleotide excision repair
The maintenance of genome integrity is a major ŽNER., the mutagenic repair, and the recombina-
task for all living cells. DNA damage can occur as a tional repair mechanisms Žreviewed in Ref. w38x.. In
consequence of the normal cellular metabolism or as the present review, I shall concentrate on the induc-
a result of exposure to chemical or physical agents. tion of recombinational repair by treatment with
In the presence of damaged DNA, various repair DNA damaging agents. For recent reviews on related
mechanisms are mobilized in order to restore the subjects, such as spontaneous recombination and on
radiation-induced DNA processes, see Refs.
w22,88,92x and other chapters of this issue. A detailed
)
Tel.: q972-3-640-9031; fax: q972-3-640-9407. account of the different mechanisms of recombina-
E-mail address: martin@ccsg.tau.ac.il ŽM. Kupiec.. 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 repeated sequences have been used w59,72,75x. In
main points pertinent to this review. addition, one can select for recombination events
between truncated genes located on different chro-
mosomes, thus creating specific translocations w33–
2. Monitoring mitotic recombination 35x.
Other popular systems used to follow recombina-
Recombination takes place between sequences that tion monitor events between two tandemly located
share homology, and results in the transfer of genetic sequences in the genome in either direct or inverted
information from one DNA molecule to the other orientation. These are easily created by the integra-
Žgene conversion. and in the reciprocal exchange of tion of the appropriate plasmids w2,41,67,98,121x.
DNA fragments between chromosomes Žcrossing- Direct repeat recombination ŽDRR. is probably car-
over.. Gene conversion and crossing-over events ried out by several different mechanisms, including
show a nonrandom association with each other; this replication slippage, unequal sister chromatid recom-
has lead to the assumption that they are mechanisti- bination, intra-chromatidal crossing-over, single-
cally related. This association is stronger Ž; 50%. in strand annealing, etc. Žreviewed in Refs. w90,98x..
meiotic recombination, but can also be detected in Similarly, inverted repeat recombination ŽIRR. can
mitotic cells Žreviewed in Ref. w88x.. The frequency occur by crossing-over or by gene conversion be-
of recombination at any determined region of the tween sister chromatids w11x. Finally, specific sub-
genome is relatively low. Although nonselective strates have been designed to monitor unequal sister
schemes have been successfully tried w39,46x, se- chromatid recombination ŽUSCR. between tandem
lectable genetic systems are normally used in order repeats w34,61,112x.
to be able to detect such rare events. As an experi- In summary, many different assays are used to
mental system, yeast has been pivotal in the develop- monitor spontaneous and induced mitotic recombina-
ment of genetics systems able to monitor specific tion in yeast. Analysis of the effect of recombination
types of recombination events w2,29,34,61,65, mutants on the various recombination substrates
76,77,98,102,128,129x. A number of such selectable shows that different recombinational mechanisms are
assays for recombination are discussed below. responsible for the recombination observed in the
Gene conversion is responsible for the great ma- different substrates. Accordingly, not all types of
jority of intragenic recombination events w83,102x. In recombination are equally inducible.
diploid yeast strains, gene conversion between two
different alleles of a nutritional marker can be conve-
niently measured by plating cells on the appropriate 3. Induction of recombination by DNA damage
selective medium. Mitotic crossing-over events are
usually scored in diploids by the segregation of Mitotic recombination is one of the mechanisms
recessive homozygotes from heterozygous markers used by the cell to repair spontaneous DNA damage.
w128,129x. Such damage is believed to result from the natural
Recombination usually occurs between sequences metabolism of the cells, such as the activity of
located at similar positions on homologous chromo- reactive oxygen species or of DSBs created during
somes Žallelic recombination. or sister chromatids; DNA replication and transcription w38x. In addition, a
however, homologous sequences located at different number of DNA damaging agents elevate the level
positions in the genome can also interact Žectopic of homologous recombination w8,30,96,97,101,
recombination.. Allelic and ectopic recombination 122,128x. This recombination results from repair pro-
occur at similar frequencies, implying that homology cesses that restore the integrity of damaged DNA
is searched throughout the genome and not exclu- regions.
sively on the homologues. This fact has been ex- When a lesion is created at the DNA, it may
ploited in order to measure recombination in haploid remain unrepaired, leading to cell death, or can be
cells. Various genetic schemes selecting for gene repaired Žor bypassed. in a process that involves
conversion between artificial or naturally occurring recombination. Many different DNA-damaging
M. Kupiecr Mutation Research 451 (2000) 91–105 93

agents cause a dose-dependent increase in the fre- A great number of noxious agents have been
quency of recombination. These include ionizing and tested in yeast, with various effects Že.g.: see Ref.
UV-irradiation, and chemicals such as cisplatin, w128x.. A summary of the effects on recombination
MMS, MNNG, 4-NQO, EMS, Aflatoxin B, etc. of so many compounds is beyond the scope of the
w8,30,33,51,97,101,128x. Most of the recombinogenic present review. Instead, I will summarize below
damaging agents also induce mutagenesis; this im- what is known about the induction of recombination
plies that the lesions caused by the agent can also be in yeast by four different DNA-damaging agents for
repaired by other DNA repair or tolerance mecha- which the types of lesions created have been well
nisms w51,128x. Some carcinogens, however, are neg- characterized. A great deal is also known about the
ative in mutagenesis tests, but induce recombination, mechanisms responsible for their repair; however,
suggesting that the mechanism of carcinogenesis in- many questions remain to be answered regarding the
volves the process of recombination. The good corre- way by which they induce mitotic recombination.
lation between carcinogenesis and the ability to in-
duce recombination has led to the development of 3.1. Ionizing radiation
yeast-based carcinogenesis detection assays w33,
97,129x. Ionizing radiation induces several types of DNA
The level of induction of recombination depends lesions, including single- and double-strand breaks,
on the genetic system used to detect it and on the DNA-protein crosslink and base damage. However,
damaging agent used. Some recombinational sub- it is the occurrence of DSBs that determines cell
strates are poorly induced by DNA damaging agents, survival after ionizing radiation w37,94x. Consistent
whereas others can be induced to levels comparable with the idea that DSBs directly initiate recombina-
to those observed during meiosis. As a general rule, tion, analysis of dose–response kinetics showed that
recombination substrates that give very low levels of at sublethal doses, X-rays induce heteroallelic con-
spontaneous recombination are induced to larger ex- version with linear kinetics w80x, whereas other
tent than substrates that give a higher basal level. As sources of damage, such as UV, are nonlinear w23x.
discussed below, these results may imply the exis- This has been interpreted to mean that UV-created
tence of two independent mechanisms for sponta- lesions need to be processed in order to initiate
neous and induced recombination. recombination, in contrast to DSBs. In addition,
The most extensively studied type of recombino- mutants that are defective in DSB repair Že.g. rad52 .
genic DNA lesions are double strand breaks ŽDSBs.. show pure exponential survival curves when irradi-
DSBs occur quite often, either as spontaneous errors ated. For these cases, it has been calculated that one
during replication, or as a consequence of the action DSB per cell corresponds to one lethal hit w36,37x.
of radiation and chemicals. It is well established Thus, for ionizing radiation, the observed increase
today that in yeast, meiotic recombination is initiated in recombination reflects more recombination reac-
by DSBs w127x. The creation of DSBs in vegetatively tions initiated by DSBs. The group of genes respon-
growing cells by site-specific nucleases also leads to sible for this recombinational repair Žthe RAD52
high levels of recombination Žsummarized in Ref. group. has been well characterized w45x. A mutation
w88x.. Unrepaired DSBs are lethal; the ability of the in any of the genes in this group leads both to a great
cells to use homology present in the genome to sensitivity to ionizing radiation, and to the abolish-
repair the break is essential for their survival. Linear ment of induced recombination w45,56x.
double-stranded DNA molecules are extremely re-
combinogenic, a property widely exploited for yeast 3.2. UV irradiation
manipulations. These findings have led to the cur-
rently held recombination models, which assume that The main two types of lesions created by UV
the essential recombinogenic substrate in DNA is a irradiation are pyrimidine dimers and pyrimidine 6–4
DSB w88,110x. The recombinational repair of DSBs lesions. These lesions are efficiently repaired in yeast
in mitotic and meiotic cells is described in other cells by the NER group of genes Žreviewed in Ref.
chapters of this special issue. 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 additional complication is that some of the genes
the RAD52 group confer a slight UV sensitivity, and required for dimer excision, such as RAD1, may
only at high doses. The induction of recombination actually participate in recombination w99x.
observed after UV treatment is probably a conse- Some types of recombination, such as DRR, may
quence of the processing of primary lesions left after actually be induced by a mechanism that does not
NER into secondary lesions that are recombinogenic. require DNA breakage and rejoining. For example,
However, how this is accomplished is still not clear, lesions in the DNA may cause replication stalling;
despite extensive studies on the subject. Unrepaired this in turn may facilitate slippage by dissociation of
lesions, such as DNA adducts and single-strand replicated strands and reassociation with a second
breaks, may cause stalling of the replication machin- copy of the repeat w5,78x. One study of DRR showed
ery during S-phase. Replication may be resumed at that this type of recombination was induced by UV
another replicon downstream, thus creating a single- irradiation in dividing cells, but not in G1- or G2-
strand gap. Such gaps can be easily converted into arrested cells w43x. This result implies a dependence
recombinogenic DSBs w20,64x. It has been suggested on DNA replication, although it is not yet clear
that UV-induced damage can persist in the DNA whether in order to allow slippage or for the conver-
through several cell divisions, to be later converted sion of primary lesions into DSBs.
into DSBs w44x. In addition, at high UV doses, the
excision of pyrimidine dimers in close proximity on 3.3. Alkylating agents
opposite strands can lead to the creation of DSBs.
Consistent with this notion, most mutants of the A large number of chemical compounds are able
RAD52 group of recombination genes show UV to alkylate DNA w38x. Although the direct effect of
sensitivity at high UV doses w51x. alkylation has been quite accurately defined both in
If pyrimidine dimers have to be removed in order vitro and in vivo, it is still unclear why some alkylat-
to initiate recombination, one would predict that ing agents, such as ethyl methanesulfonate ŽEMS.
inactivation of the NER system would prevent in- are potent mutagens, but do not induce recombina-
duced recombination. However, mutations in NER tion, while others, such as the similar compound
that prevent removal of the initial lesions have been methyl methanesulfonate ŽMMS. readily induce re-
reported to have various effects on recombination. combination. MMS is usually referred as ‘‘IR-radio-
For example in one study w62x, it was shown that mimetic’’, since it has an effect very similar to that
mutations in the RAD1 gene caused a reduction in of ionizing radiation w109x. However, we are far from
diploid heteroallelic recombination, with a concomi- understanding the mechanism by which MMS treat-
tant increase in sister-chromatid recombination. In ment promotes recombination. Mutations that affect
another study, it was shown that mutations in the DSB repair and recombination show a great sensitiv-
RAD3 gene impair induced heteroallelic gene con- ity to MMS w16,38,124,125x. However, the main
version, but increase induced crossing-over w65x. In a effect of MMS appears to be the methylation of
third study, while mutations in RAD2 caused a bases in the DNA creating 7-deoxyguanine and 3-
significant increase in the induction of gene conver- methyladenine Ž3MeA., and not DSBs formation.
sion at two different loci, mutations at the RAD1, 3MeA, the main biologically relevant lesion created
RAD3, and RAD4 genes caused only a mild increase by MMS, differs from UV-induced lesions and bulky
over the wild type levels w55x. These results probably adducts in that it is in the minor groove and does not
testify to the heterogeneity of mechanisms that can significantly alter the DNA double helix conforma-
lead to recombination, but also demonstrate that we tion. The repair of alkylation damage has been exten-
are far from understanding some of the basic aspects sively investigated in yeast. Alkylation lesions are
of induced recombination. A true comparison of mainly repaired by the BER mechanism, in which a
results is complicated by the fact that different DNA glycosylase removes the damaged base, fol-
recombination substrates were used in the different lowed by cleavage at the abasic site by an
studies. In addition, some of these studies were apurinicrapyrimidinic ŽAP. endonuclease w38,124,
carried out with strains carrying leaky alleles. One 125x. The radiomimetic properties of MMS could be
M. Kupiecr Mutation Research 451 (2000) 91–105 95

explained by assuming that the primary lesions cre- point expected from a treatment that block DNA
ated by MMS are processed by the BER pathway in polymerases w47x. These results may suggest an alter-
a way that converts them into DSBs. The results of native mechanism of action of bifunctional agents.
DNA analysis by neutral and alkaline gradient could Treatment with crosslinking agents induces mitotic
be consistent with this idea w57x. However, the analy- recombination in a process that requires a specialized
sis of single- and double-mutants does not fit this group of genes Ž PSO genes. whose precise biochem-
explanation: mutants in recombination genes are more ical role remains to be elucidated. The PSO genes
sensitive to MMS than mutants in the 3 MeA glyco- seem to be completely specific for crosslinks; ac-
sylase gene Ž MAG1.; inactivation of the BER path- cordingly, some of them are needed for induced
way does not suppress this sensitivity; on the con- recombination by crosslinking agents w81,85x. In con-
trary, a synergistic effect is seen Žw125x; Jablonovich trast to what happens in bacteria w13x, DSB formation
and Kupiec, unpublished.. These results are further is an obligatory intermediate in the repair of inter-
complicated by the genetic evidence for a role of the strand crosslinks in eukaryotes w18x, and thus the
NER and error-prone repair enzymes in alkylating induction of recombination is likely to result from
repair w16,125x. the repair of such breaks w4x. The precise mechanism
3MeA apparently blocks DNA synthesis, and is by which the Pso proteins transform the crosslinks
thus considered a major lethal lesion w6,25x. It is into DSBs remains to be elucidated. Interestingly,
therefore possible that the lesions created by MMS crosslinking agents such as cisplatin can sensitize
cause a block in replication, which is then acted prokaryotic and eukaryotic cells to killing by ioniz-
upon by the recombinational repair group of genes ing radiation, by a mechanism not yet understood,
w64x. Alternatively, the passage through S transforms that seems to involve cisplatin inhibition of DSB
the lesions into fully recombinational ones. A check- repair after ionizing radiation w21x.
point that slows down replication in the presence of To summarize, although it is clear that DSBs are
low MMS concentrations is known to operate w91x. repaired in yeast preferentially by recombination,
The cells continue to cycle, until finally they arrest at and that DSBs are the substrates for the recombina-
the G2rM checkpoint w1,71,91x. It is possible that a tional repair of ionizing radiation damage, much less
correlation exists between the S checkpoint and the is known about the conversion of other types of
processing of primary MMS lesions. Eventually, lesions into recombinogenic substrates Žpresumably
these are converted into DSBs, eliciting the G2rM DSBs.. A picture is starting to emerge, in which
checkpoint, and inducing the recombinational repair many different types of lesions are acted upon by the
mechanism. recombinational machinery during DNA replication,
either by the conversion of the initial lesion into
3.4. Crosslinking agents
DSBs, or by the coupling of the processes of replica-
Bifunctional agents that can react with two differ- tion and recombination. Consistent with this picture,
ent nucleophilic centers in DNA have the potential to many damaging agents require cell division in order
form intrastrand and interstrand DNA crosslinks. to become recombinogenic Že.g. Refs. w42,43x. and
Several different types of crosslinking agents, includ- polymerase Delta is needed for this process w49,117x.
ing psoralens, mitomycin C, and cisplatin have been In addition to a link to replication, evidence has
analyzed in yeast. Crosslinking agents are exten- been accumulating lately for a similar link to tran-
sively used in cancer chemotherapy Že.g. Refs. scription: in some cases, increased transcription may
w93,108x., as they are supposed to preferentially af- induce recombination Žreviewed in Ref. w86x.. Such
fect DNA replication and transcription in rapidly an association between transcription and repair fac-
growing cancer cells. Interstrand crosslinks can, in tors has been well established for NER. The stalling
principle, prevent DNA strand separation and there- of RNA polymerase on a damaged template activates
fore may block DNA replication and transcription. the NER machinery, which is physically linked to
Interestingly, it has been lately shown that cisplatin the transcription machinery w38x. A similar link to
causes a cell cycle arrest in yeast cells at the G2 recombination may exist, although direct biochemi-
checkpoint, and does not elicit the S phase check- 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- different mating types: a,a or ara. The cell type of
tion each individual cell affects the efficiency of DNA
repair, and the induction of recombination. In this
In meiotic cells, recombination takes place after respect, yeast cells provide a good model system to
DNA replication, at the four-strand stage. In vegeta- study how DNA repair mechanisms are influenced
tive cells, many studies have been carried out to by cell type.
determine at what stage of the cell cycle recombina- In nature, yeast cells are ara only if they are
tion takes place. Half-sectored colonies w96x, similar diploids. Cells with a heterozygous configuration at
to ‘‘twin spots’’ in other organisms, provide evi- their mating type locus show higher levels of ra-
dence that recombination in vegetative cells can take dioresistance, and higher frequencies of radiation-as-
place at the G2 stage. Unequal reciprocal sister sociated recombination events than isogenic ara and
chromatid recombination, which can take place only ara cells w40,31,32,52x. Genetic analysis has shown
after DNA replication, has also been detected in that this effects requires functional a1 and a 2 gene
natural and artificial tandem repeats w34,61,112x. On products of the MAT locus and is independent of the
the other hand, pedigree analysis of X-ray induced transcriptional regulator RME1, which plays a role in
mitotic recombination showed that most of the cells initiating meiosis w52,53x. The exact mechanism by
recombined before DNA replication w120x. Finally, which mating type heterozygosity affects recombina-
elegant genetic studies have demonstrated that spon- tion is not known, although it has been proposed that
taneous and induced recombination can occur in mating type heterozygosity enhances the efficiency
vegetative cells arrested at many different points in of pairing Žand thus of recombination. between ho-
the cell cycle w26–28,60x. mologs, thus improving the chances of survival of
The induction of recombination varies throughout diploid cells, which are usually ara w61x. This ex-
the cell cycle. The increase of both gene conversion planation, however, does not seem to hold since Ž1.
and crossing over by nonlethal doses of UV and all the results gathered to date point to the fact that
X-rays in synchronized diploid cultures is maximal pairing between the homologous sequences is not a
at the G1 stage, and minimal at G2 w19,23,24,28x. limiting step in recombination w59,72,75x; Ž2. mating
The low level of recombination detected is not due type heterozygosity confers radioresistance to hap-
to failure to repair the damage, since G2 diploids are loid strains as well w52,100,126x; and Ž3. mating type
able to survive DNA damage at the G2 stage better heterozygosity also increases the efficiency of
than at G1 w10,28,45x. The interpretation to these DSBs-initiated intrachromosomal recombination in
results is that in cells that have replicated their haploid cells, which does not require pairing between
chromosomes, the damage is preferentially repaired homologs w31x.
by recombination with the sister chromatids w28x. Alternative explanations for the effect of mating
This hypothesis was then directly tested using a type heterozygosity are that either diploid-specific
strain in which both homolog and sister chromosome recombination systems are expressed in ara cells
recombination could be measured w61x. This study w52,66x or rate-limiting recombination proteins may
not only showed that X-rays preferentially induced be induced at higher levels in ara cells compared to
sister chromatid recombination in G2 cells; it also their isogenic ara and ara cells w31,32,66x. These
suggested that sister chromatids, for reason still to be explanations are not necessarily exclusive, and both
understood, have the capacity to repair more DNA agree with the observation that in addition to in-
damage than do homologs w61x. crease recombinational repair, mating type heterozy-
gosity decreases mutagenic repair w52x. Recombina-
tional repair should be of advantage to diploids,
5. Effect of the mating type on induced recom- which carry an additional set of chromosomes, but
bination haploid cells favor non-recombinational repair, even
if it is mutagenic, since attempts to recombine in
In addition to the effect of ploidity in the effi- haploids may lead to chromosomal rearrangements
ciency of DNA repair, yeast cells may be of three and cell death.
M. Kupiecr Mutation Research 451 (2000) 91–105 97

6. The mechanism of induction of recombination factorŽs. elicited by the lesions in the DNA caused
by DNA damage by the radiation w29x.
Since these seminal experiments were carried out,
There are two different mechanisms through which it has become clear that both prokaryotic and eukary-
DNA damage can induce recombination. On one otic cells respond to DNA damage by inducing the
hand, the damage causes an activation of the genes transcription of a large set of genes, which probably
encoding enzymes involved in recombination Ži.e. facilitate cell cycle arrest, DNA repair, or adaptation
induction of trans-acting factors.. On the other hand, to the insult Žfor a review, see Ref. w22x..
DNA damage also provides the DNA lesions which In bacteria, the inducible SOS repair system has
serve to initiate the process of recombination Ž cis- been extensively studied. In response to DNA dam-
acting factors.. The coupling between the two mech- age, a regulon comprising more than 20 genes con-
anisms ensures that enough recombination enzymes trolling DNA replication, recombination, mutation,
will be available to cope with increasing DNA dam- etc., is expressed at higher levels Žreviewed in Ref.
age levels. w38x.. The damage–response system of Escherichia
Although evidence proving that both modes of coli has served as a paradigm for many years. In
induction operate in yeast has been gathered Žsee yeast, the existence of an inducible ‘‘error prone’’
below., it is not always obvious which one is respon- mechanism of repair has been suggested by experi-
sible for most of the induction. Is the enzymatic ments carried out with temperature-sensitive mutants
activity present in the cell in the absence of external w7,104x. However, evidence for the existence of simi-
damage sufficient to cope with most insults, or is the lar damage-induced system for recombination re-
transcriptional induction the main mechanism re- mains more controversial.
sponsible for the increase in recombination? Since Lately, the RAD9 checkpoint gene has been shown
the experiments carried out to answer this question to activate, upon DNA damage, a great regulon of
are not always easy to interpret, and the relationship genes that include repair, recombination, and replica-
between these two ‘‘modes’’ of induction is far from tion genes. This transcriptional activation is indepen-
being understood, below I present arguments in favor dent of the cell-cycle-arrest role of RAD9 in the
and against the supremacy of each one of them. checkpoint w1x. Similarly, in mammals, DNA damage
leads to the transcriptional activation of certain genes
through the actions of the ATM and p53 tumor
6.1. DNA damage induces recombination by increas- suppressor genes w9x. The yeast homolog of ATM,
ing transcription of recombination genes MEC1, is responsible for most of the damage-in-
duced transcriptional induction in yeast. Following
In an elegant series of experiments, Fabre and DNA damage, a phosphorylation cascade that in-
Roman w29x gathered information supporting the ex- cludes the Rad53 and Dun1 protein kinases leads to
istence of a signal for increased levels of recombina- cell cycle arrest and to phosporylation of the Crt1
tion. A mating-capable diploid strain, heteroallelic at repressor. This repressor, usually bound to DNA
the ADE6 locus Ž ade6-21rade6-45 ., was mated damage-inducible genes, loses its ability to bind to
with haploid cells of a double mutant Ž ade6-21, 45 . the regulated promoters upon phosphorylation. Thus,
strain, that was subjected to irradiation. The results phosphorylation of Crt1 leads to the activation of the
showed a dose-dependent induction of recombination inducible genes w54,111x.
in the selected triploids created by the mating of the The number of genes activated by DNA damage
diploid and the irradiated haploid. Since this last in yeast is very large. For example, in a recent
strain carried the same mutations as the diploid, it genomic study w58x, as many as 325 genes showed at
could not contribute genetic information. The authors least fourfold increased transcription levels after
therefore concluded that the recombination events treatment with the alkylating agent MMS. This rep-
did not involve the participation of the irradiated resents ; 5% of the yeast genome, and gives a
chromosome Ži.e. cis-acting lesions., and that the conservative estimate, since many more genes
induction was caused by the release of certain showed significant induction levels that were below
98 M. Kupiecr Mutation Research 451 (2000) 91–105

the fourfold artificial threshold. As expected, many would be limiting for recombination and would re-
of these genes are involved in DNA replication quire induction. To complicate the issue, it has been
andror repair; however, these were only a minority recently shown that the transcriptional induction of
of the genes induced, and the relevance of the tran- RAD51 is RAD52-dependent w4x. It is still possible
scriptional induction in protecting cells against dam- that the noninducible allele of RAD54 is defective in
age remains to be determined w58x. some aspect of induced recombination still to be
Although the idea that recombination genes should defined.
be induced as a response to the presence of DNA A strong argument against the supremacy of an
damage in order to increase recombinational repair is inducible recombination repair system comes from
aesthetically pleasing, the experimental results do not split-dose experiments. If induction of recombination
always support this logic. The RAD54 recombina- involves two components: the induction of the
tion gene, which has been thoroughly studied, is a recombination enzymes on one hand, and the cre-
good case study. The RAD54 gene is transcription- ation of the recombination substrate on the other,
ally induced by DNA damage. Different types of exposure to a dose that induces the enzymatic ma-
DNA damaging agents, including UV, X-rays, MMS, chinery should elevate the level of recombination
or overexpression of a restriction enzyme, are able to caused by a subsequent UV dose. Split-dose experi-
elicit this response. Cells synchronized at the G1 ments of this type have uncovered adaptive re-
stage of the cell cycle are still inducible, implying sponses protecting against alkylating damage in
that the induction is not due to the accumulation of prokaryotes and eukaryotes Žreviewed in Ref. w82x..
cells at a certain stage of the cell cycle at which the Similar experiments, however, failed to show any
level of expression is high w3,12,14x. An analysis of induction of recombination beyond the additive ef-
the promoter, and its comparison to other damage-in- fect expected from both doses w107x. On the other
ducible genes reveals the presence of an element, hand, other authors have deduced the existence of a
termed damage–responsive element ŽDRE. fully re- DSB-inducible repair mechanism from experiments
sponsible for the transcriptional induction. A similar in which cells subjected to low doses of ionizing
element is present, for example, upstream of the radiation or cisplatin treatment showed lower levels
UV-inducible excision repair gene RAD2. Deletion of mutagenesis, when challenged later by MNNG.
of the DRE from these genes, however, does not These results were interpreted to mean that the first
confer enhanced radiation sensitivity after exposure treatment induced an Žerror free. recombination sys-
to UV or ionizing radiation, respectively w15,105x. tem in the cells, able to repair the MNNG damage
Moreover, strains carrying an allele of RAD54 inca- without introducing mutations w7,21x. Biochemical
pable of transcriptional induction are still fully capa- confirmation for the existence of such an inducible
ble of showing induced recombination upon expo- recombination system, however, is still lacking.
sure to DNA-damaging agents w15x. Thus, although
the gene is induced and more protein has been 6.2. DNA damage induces recombination by proÕid-
shown to be produced following DNA damage, it is ing additional substrates
still not clear that this induction is biologically sig-
nificant, or that contributes in any way to the en- The second mechanism of induction is by facili-
hanced levels of mitotic recombination observed af- tating the initial step in recombination, which is
ter irradiation. believed to be the creation of a DSB w88,110x. A
It should also be noted that while some genes certain level of activity of the recombination en-
which physically interact with RAD54 during recom- zymes is present in cells not exposed to genotoxic
bination are also damage-induced Že.g. RAD51., oth- agents, but the lesions in the DNA constitute the
ers, such as the central RAD52 gene, are not in- limiting factor for recombination. The level of induc-
ducible w14x. It is possible to rationalize these results tion reached by treatment with DNA damaging agents
by assuming that the Rad52 protein is more abundant is usually lower than that seen in meiotic cells. It has
in the cell, or is needed at lower concentrations, than now become established that meiotic recombination
Rad51p and Rad54p. If this is so, only the later two is induced in yeast by the creation of DSBs at many
M. Kupiecr Mutation Research 451 (2000) 91–105 99

different places in the genome w68,127x. Mutations in caveat for such an experiment is that the deletion
the SPO11 and RAD50 genes, which prevent the should be large enough to ensure that recombination
creation of the DSBs, completely eliminate meiotic proteins would not nucleate at homologous regions
recombination w68x. DSBs created by X-rays or site- bordering the ADE6 gene in the haploid strain.
specific endonucleases are able to induce meiotic Another argument in favor of a model in which
recombination in spo11 or rad50 mutant cells induction results from the repair of more lesions,
w79,115x, showing that the creation of DSBs is the rather than as a response to a global signal, comes
limiting step in the induction of meiotic recombina- from studies on recombination of Ty elements. These
tion. repeated sequences, which constitute ; 3% of the
A direct test of the hypothesis that most induced yeast genome, are randomly dispersed in the chro-
recombination depends on increased availability of mosomes. Recombination between members of the
recombination substrates, was carried out using plas- Ty family occurs spontaneously at low rates, despite
mid transformation w106x. In these experiments, their large number w69,70x. DNA damage-stimulated
which are formally similar to those carried out by ectopic recombination between Ty elements has not
Fabre and Roman w29x described above, a diploid been observed even when the same treatments ŽUV,
yeast strain heteroallelic at the HIS3 locus was MMS, or X-rays. induce other types of recombina-
transformed with minichromosomes Žcentromeric tion in the same cells w72,89x. However, when a DSB
plasmids. carrying homology to the HIS3 gene, but is introduced at a Ty, it is efficiently repaired by
containing the same two mutations present in the recombination w90x. These results are more consistent
chromosomes. When a DSB was introduced in the with a local, rather than global, view of induction,
region of homology, an increase in the recombina- and can be explained by assuming that some special
tion frequency between the heteroalleles was ob- feature of Ty elements, such as chromatin configura-
served, although the plasmid was unable to donate tion, makes them refractory to receive spontaneous
wild-type information. This induction of recombina- or damage-induced lesions. Alternatively, DNA
tion was dependent on the presence of homology damage in Ty elements is either not usually pro-
between the broken minichromosomes and the diploid cessed into DSBs and leads to cell death, or is
chromosomes. Thus, recombination was not induced preferentially repaired by a ‘‘silent’’ mechanism,
by the presence of DSBs in regions of nonhomology, such as sister chromatid recombination. When a DSB
but it was induced by the presence of DSBs in is forcibly created locally, however, it is readily
homologous regions, even if these were unable to repaired by recombination, implying that the DNA is
transfer wild type information. These experiments accessible to the recombination machinery w90x.
are consistent with a model in which recombination It should be noted that the two modes of induction
is initiated locally by the recruitment to the broken described are not necessarily exclusive. A DSB can
chromosome of the recombination machinery, which initiate recombination locally, and also cause the
can be later transferred to the other chromosomes to transcriptional induction of DNA repair and recom-
promote their recombination. Evidence for tripartite bination genes. Such an arrangement should be ad-
events, involving the plasmid and the two homolo- vantageous for the cell. One may assume that under
gous chromosomes, was presented w106x. According low damage levels, enough recombination proteins
to this interpretation, the authors re-assessed the are present in the cells to allow some repair. The
results obtained by Fabre and Roman w29x in the transcriptional induction of DNA repair genes may
following way: irradiation of the haploid created allow the cell to be prepared to cope with increasing
lesions in its genome. Recruitment of recombination levels of damage. In the last years, most of the
proteins on the doubly mutated ade6 allele was proteins involved in recombination have been cloned
followed by their transfer to the homologous regions and expressed, and assays to measure recombination
in the heteroalleles. One way to decide between the activity in vitro are being developed. These improve-
two alternative interpretations would be to repeat the ments should allow in the near future to directly
Fabre and Roman experiments using cells carrying a measure the concentration of relevant proteins and
complete deletion of the ADE6 gene. A technical 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 8. Concluding remarks and human implications
possible to evaluate the relative importance of the
two modes of induction. 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
7. The relationship between spontaneous and in- this danger. The significance of DNA repair for
duced recombination humans and the close link between genome stability
and cancer is illustrated by the extreme phenotypes
Spontaneous recombination results from the repair of patients with hereditary diseases caused by defects
of lesions created by the normal metabolism of the in repair processes, such as Xeroderma pigmento-
cell. These include damage caused by reactive oxy- sum, Cockayne’s syndrome, Ataxia telangiectasia,
gen species, as well as DSBs created during DNA and Nijmegen breakage syndrome w119x.
replication and transcription w38x. As explained Recombination is one of the many pathways that
above, it is usually the case that recombination sub- help the cell to cope with lesions in its chromo-
strates that yield low spontaneous recombination lev- somes. Many chemical and environmental carcino-
els can be induced to greater degrees by DNA gens are recombinogenic, and cause genomic rear-
damaging agents, than those that give a high sponta- rangements, such as gene amplification w116x, and
neous rate. chromosomal rearrangements w48x. Since mitotic re-
Several explanations may be given to this obser- combination can lead to the homozygosity of reces-
vation. Ž1. Following the mechanism of induction sive alleles, it plays a pivotal role in the multistep
presented in Section 6.1, one may assume that the process of carcinogenesis w48,123x. Exposure to ion-
substrate that gives a high spontaneous recombina- izing radiation is correlated with an increase in the
tion rate may cause the cells to be already at an incidence of acute myelocytic leukemia, tyroid can-
induced state, so that exogenous damage does not cer, and other forms of neoplasias w50,73,84x. In
provoke the induction seen with other substrates, and addition, the risk of exposure to harmful UV radia-
therefore has no effect. Ž2. According to the mecha- tion is gradually increasing, with the erosion of the
nism presented in Section 6.2, the induction depends stratospheric ozone layer w17x. Greater exposure to
upon the creation of new lesions, and the substrate UV is likely to result in a general increase in the
may already provide a very high level of those level of cancer incidence in the population. The
lesions, such that the recombinational machinery is understanding of the mechanisms of induced mitotic
already at its maximal activity level and additional recombination in mammalian cells is therefore of
lesions may have no further effect. Ž3. The lesions capital importance.
created by the DNA damaging agent may be qualita- Yeast cells have played a pivotal role in the
tively different from the spontaneous ones, and may understanding of basic DNA repair and recombina-
initiate recombination by a different mechanism. If tion mechanisms. Numerous studies over the years
many spontaneous lesions are created, or the lesions have served to define the basic concepts and to
are very recombinogenic, compared to those induced identify the main players. Given the high genetic
by the damaging agent, only high doses of the conservation of DNA repair mechanisms and genes
noxious agent will produce any detectable effect, between all eukaryotes, it is not surprising that our
over the high spontaneous background. Analysis of understanding of the systems operating in yeast have
the induction of DRR by several DNA damaging lately started to be applied to the study of recombina-
agents, using such a substrate with high basal recom- tion in mammalian cells Žfor a recent review, see
bination level, supports the third interpretation. At Ref. w63x.. One expected difference between yeast
low doses, no increase in recombination is seen over and mammalian cells is the relative weight of homol-
the background, but at higher doses a proportional ogous recombination versus non-homologous end-
induction can be detected Žw72x; Liefshitz, Steinlauf joining ŽNHEJ. mechanisms. Whereas the proteins
and Kupiec, unpublished results.. that participate in these two competing mechanisms
M. Kupiecr Mutation Research 451 (2000) 91–105 101

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