Download The dual nature of homologous recombination in plants

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TRENDS in Genetics Vol.21 No.3 March 2005
The dual nature of homologous
recombination in plants
David Schuermann, Jean Molinier*, Olivier Fritsch and Barbara Hohn
Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland
Homologous recombination creates covalent linkages
between DNA in regions of highly similar or identical
sequence. Recent results from several laboratories,
many of them based on forward and reverse genetics
in Arabidopsis, give insights into the mechanisms of the
enzymatic machinery and the involvement of chromatin
in somatic and meiotic DNA recombination. Also,
signaling pathways and interconnections between
repair pathways are being discovered. In addition,
recent work shows that biotic and abiotic influences
from the environment can dramatically affect plant
genomes. The resulting changes in the DNA sequence,
exerted at the level of somatic or meiotic tissue, might
contribute to evolution.
Finally, many specific mutation screens can be used to
identify genes of interest, including screens for radiation
sensitivity and for altered levels of HR (see below).
In this review, we will concentrate on newer findings in
HR in meiotic and in somatic tissue. A section is devoted to
influences of the environment on the regulation of the HR
frequency, with special reference to the possible effects of
pathogens on the stability of the plant genome. Because
not all aspects of HR can be covered in this short article,
we refer the interested reader to a previous review on
HR in plants [2], to general reviews on mechanisms of
HR [3,4], to a general description of the uses of
Arabidopsis as a DNA repair model [5] and to accounts
of gene targeting [6,7] (Box 1).
Introduction
Homologous recombination (HR) is one of the fundamental
processes of life. It has a dual function through its activities
both in meiosis and in somatic cells. It creates new linkages
in the genetic material in meiosis and in many organisms
it is even required for fertility. Indeed, HR substantially
contributes to evolution. HR is also important for repairing
damaged DNA in somatic tissue. At the same time HR
between repeated genes must be tightly controlled to avoid
unwanted gene rearrangements.
There are several important reasons for studying HR
and other repair processes in plants: one of them is the fact
that unlike in animal systems, functional depletion of
several repair genes is not lethal, thus enabling the study
of their replacement activities (see below). Another lies in
the fact that the germline is created late in development.
Therefore, genomic changes in somatic tissue have a
certain probability of being transmitted to the next generation. This special aspect of plant development enables
natural selection to act on new genetic traits in somatic or
even in gametophytic tissue.
The recent development of genetic and genomic tools
for Arabidopsis thaliana is one of the reasons why
analysis of HR and other repair processes in this plant
has gained an enormous boost. The completion of
Arabidopsis genome sequencing in 2000 [1] is the basis
for gene discovery and through the use of T-DNA and
transposon insertions, multiple publicly available gene
knockout libraries of this plant have been produced.
Meiotic recombination
HR maintains the integrity of the genome in somatic cells;
conversely, in meiotic cells it creates genetic variability by
reciprocal crossovers and DNA exchanges. This results in
Corresponding author: Hohn, B. ([email protected]).
* Present address: CNRS UMR6547, BIOMOVE, 24 Avenue des Landais, F-63177
Aubière, France.
Available online 19 January 2005
Box 1. Homologous recombination and plant breeding
Genetic improvement of crop plants, executed for many thousands
of years, depends on HR. The classical breeding procedure involves
crossing of chosen parental lines and subsequently selecting the
offspring for the desired trait. An understanding of the mechanism of
HR and its control might help to achieve this more quickly. One could
imagine that the use of hyper-recombination mutants like those
described in Table 2 and mutants directly affecting meiotic recombination could increase the frequency of mHR. This could be
exploited in breeding programs, because the time needed for
crossing and backcrossing would be considerably reduced.
Resistance to stresses, such as plant pathogens, drought and
salinity, frequently has to be introduced into inbred lines. Apart from
classical breeding, which is limited to the same or closely related
species at best, transgenesis-aided breeding could be the option for
the future in many cases. Already this is common practice for the
introduction of genes from more distantly related organisms. The
ability to produce targeted changes in plant genes would have a
tremendous impact on fundamental research and on molecular
breeding. However, the frequency of one targeted event per 104–106
transformants is too low to be routinely used [62,63] (discussed in
Refs [6,7,64]). Stimulation of HR in the cells used in transgenesis
protocols might increase the chance of obtaining targeted integration events. Stimulation of HR might be achieved by using
radiation or chemical agents or genetically by employing mutants,
as discussed above. The only reported attempt to increase the
targeting frequency using the bacterial recombinase RecA has failed
[65]. The use of two counter-selectable markers flanking the
targeting gene is a valuable approach because it yielded clean
targeted events only [62]. The combination of this approach with
genetic upregulation of HR in the required tissue could lead to
improved gene replacement frequencies.
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Review
TRENDS in Genetics Vol.21 No.3 March 2005
random combinations of alleles and traits, which are
subjected to natural selection and therefore contribute to
the evolution of an organism. Meiosis is a key event in the
life cycle of all sexually reproducing eukaryotic organisms,
initiating the transition from the diploid to the haploid
phase. Differentiated cells undergo a reductional meiotic
division characterized by a single round of DNA replication and chromosome condensation. In most species the
induction of double-strand breaks (DSBs), the alignment
of the maternal and paternal chromosomes and the
recombination between homologous sequences are mechanistically linked resulting in full synapsis (see Glossary).
The haploid gametes are the products of two subsequent
rounds of meiotic chromosome segregation. Surprisingly,
recent cytological, genetic and molecular investigations
have revealed many species-specific variations in the
meiotic process previously thought to be highly conserved
(reviewed in Refs [8–11]). The behavior of plant mutants
in different phases of meiosis – from the differentiation of
the meiocytes, the arrangement of sister chromatids and
chromosomes in the synaptonemal complex, to DNA
recombination, the formation of bivalents and finally the
separation of chromosomes – has been extensively
described elsewhere [12,13]. Herein we focus on the recent
findings concerning Arabidopsis gene products that
participate mechanistically in the process of meiotic HR
(mHR) and compare them with components that are active
(a) DSB induction
End-processing
SPO11-2 SPO11-3 and
and TOP6B
TOP6B
? ?
SPO11-1
Recombinase
loading
RAD51
paralogs?
MRE11–
RAD50–
?
173
Glossary
Bivalent: a distinct chromosomal figure in prophase I of meiosis, consisting of
the fully condensed homologous chromosomes linked by chiasmata.
Chiasma (plural chiasmata): a meiotic X-shaped structure seen at the
separation of the bivalents; resulting from the sequence exchange between
homologous chromosomes by HR.
Crossover: DNA exchange between homologous chromosomes.
Gene cluster: multiple similar genes at a given locus, arising from gene
duplications and recombination.
Holliday junction (HJ): an intermediate four-stranded branched DNA structure
in the HR process whose resolution results in crossover/noncrossover of the
flanking markers (see Figures 1 and 2).
Meiocyte: a differentiated cell subsequently undergoing a meiotic division.
Polyad: an aberrant product of meiotic division observed in some mutants,
forming an atypical number of spores with random chromosome number and
DNA content.
Synapsis: a complete lengthwise association of the homologous chromosomes during early prophase I of meiosis.
Synaptonemal complex (SC): a tripartite proteinaceous structure, formed
between aligned homologous chromosomes.
Tetrad: a product of meiotic division with four equally distributed haploid sets
of chromosomes.
Univalent: a chromosomal figure observed in mutants that fail to synapse and
to form bivalents.
in mHR in the budding yeast Saccharomyces cerevisiae
(Figure 1 and Table 1).
Meiotic recombination is initiated by the induction of
DSBs. In yeast, the endonuclease Spo11 introduces DNA
DSBs in meiotic chromosomes, triggering strand exchange
and crossover, which is essential for synapsis in some
organisms [10]. Spo11 shows homology to the A subunit of
Strand invasion
RAD51C
Resolvases?
ATM
XRCC3
?
BRCA2
Branch migration
HJ resolution
RAD51C
RAD54-L ?
MSH4–
MSH5?
?
XRCC3
SC
(b)
?
RAD51 and DMC1
?
MLH1–
MLH3?
RAD54-like ?
Mre11–
Rad50–
Nbs1
Spo11
Mre11–
Rad50–
Nbs1
(c)
Rad52
Rad55
Rad57
Msh4–
Msh5
?
Rad54
Mms4–
Mus81
SC
Rad51 and Dmc1
(d)
Mec1
Leptotene
Chromosome
condensation
Zygotene
Pairing of chromosomes
Initation of SC
Pre-synapsis
Mlh1, Mlh3 and others
Pachytene
Bivalents formed
Synapsis
Rad54
Diplotene
Chromosome separation
Disassembly of SC
chiasmata
Diakinesis
Chromosome
separation and
condensation
Post-synapsis
TRENDS in Genetics
Figure 1. Mechanism and proteins involved in meiotic homologous recombination embedded in the choreography of meiosis. Important processes of HR (a) and the proteins
whose functions were assigned to meiotic HR in Arabidopsis (b) and in budding yeast (c) are placed into the temporal progression of meiotic prophase I and its typical
chromosomal figures (d). The involvement of proteins in meiotic HR was reported (colored and filled shapes), suggested (empty shapes) or unknown (dashed shapes). Note
that sister chromatids are only shown in the first and last phases.
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Review
TRENDS in Genetics Vol.21 No.3 March 2005
Table 1. Homologous recombination genes in Arabidopsis, yeast and mammalsa
Arabidopsis
ATM
Refs
[29]
ATR
BRCA2c
BRU
Centrin2
DMC1
?
ERCC1
INO80
MIM
MLH1
MRE11
MSH4
?
?
RAD1
RAD9
RAD17
RAD25 (also known as
XPB1)
RAD50
RAD51
RAD51C
?
RAD54L?
SNM1
SPO11-1
SPO11-2
SPO11-3
XRCC3
[72]
[22]
[35]
[46]
[20]
[73]
[47]
[34,74]
[26]
[17]
[27]
[75–78]
[39]
[39]
[79]
[18,19]
[21]
[23]
[44]
[14,16]
[14,15]
[15]
[22]
Proposed functionb
Double-strand break (DSB) signaling, cell-cycle
checkpoint
DSB signaling, cell-cycle checkpoint
Strand invasion
Chromatin state, silencing
Nucleotide excision repair (NER)
Meiotic interstrand invasion
DSB repair?
NER
DSB repair, homologous recombination (HR)
DNA repair and recombination
Promotion of crossovers in meiosis
DSB end processing
Promotion and stabilization of meiotic HR
DNA damage signaling?
DSB end processing?
NER, nonhomologous overhang end removal
Cell-cycle checkpoint
Cell-cycle checkpoint
NER
Budding yeast
Tel1
Mammals
ATM
Mec1
–
?
?
Dmc1
–
Rad10
Ino80
Smc6?
Mlh1
Mre11
Msh4
–
Xrs2
Rad1
Rad9
Rad17
Rad25
ATR
BRCA2
?
Centrin2
DMC1
DNA-PK
ERCC1
INO80
SMC6?
MLH1
MRE11
MSH4
p53
NBS1
XPF
RAD9
RAD17
XPB
DSB end processing
Strand invasion
Holliday junction (HJ) resolution?
HR?
HR, perhaps through chromatin events?
Interstrand crosslink repair, recombination
Induction of meiotic DSB
?
?
HJ resolution?
Rad50
Rad51
–
Rad52
Rad54
Snm1?
Spo11
–
–
Rad57
RAD50
RAD51
RAD51C
RAD52
RAD54
SNM1?
SPO11
–
–
XRCC3
a
The main factors involved in or potentially affecting somatic and meiotic HR are listed in alphabetical order with their function. Known homologs in budding yeast and
mammals are shown. Yeast or mammalian genes for which no obvious homologs exist in Arabidopsis are highlighted in bold.
b
Refers to function in Arabidopsis.
c
Two homologs in Arabidopsis.
the archaebacterial topoisomerase VI complex, which
induces transient DSBs to disentangle DNA. In contrast
to most other organisms, Arabidopsis possesses three
paralogs of Spo11 and a homolog of the topoisomerase VIB
subunit (TOP6B) [14,15] (Table 1). In an Arabidopsis
mutant of the SPO11-1 gene the formation of chiasmata
and bivalents was severely reduced, and synapsis could
not be observed [14,16]. Meiotic division proceeded in the
spo11-1 background, resulting in ‘polyads’ of random DNA
content instead of the typical tetrads. It remains to be
elucidated whether production of residual DSBs and the
reduced formation of chiasmata are due to alternative
pathways or a result of the function of the two other
SPO11 paralogs. However, both of them but not SPO11-1
were shown to interact with the homologs of the B subunit,
suggesting a eukaryotic topoisomerase-like function [15].
The budding yeast Mre11–Rad50–Nbs1 complex is
assumed to participate in the Spo11-dependent induction
of DSBs and in the processing of the ends generating
3 0 -protruding single stranded DNA (ssDNA). Mutants
of the Arabidopsis orthologs of Mre11 [17] and Rad50
[18,19] were isolated and analyzed for their meiotic
phenotypes. Both mutants exhibited chromosomal fragmentation and failure of synapsis, supporting a role
for the Arabidopsis MRE11–RAD50 complex in early
steps of meiotic HR, presumably in processing the DNA
ends. In contrast to the situation in budding yeast, the
Arabidopsis protein complex is not required for induction
of the SPO11-mediated DSBs, because the chromosome
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fragmentation and the complete sterility of mre11 could be
partially suppressed in a spo11-1 background [17].
In the subsequent step of yeast mHR, Rad51 (the homolog
of the bacterial RecA recombinase) and its meiotic paralog
Dmc1 assemble on the ssDNA to form a nucleoprotein
complex. This complex searches for homologous sequences
and finally triggers strand invasion to form a joint
molecule between parental chromosomes, a prerequisite
for synapsis. Consistent with this, Arabidopsis mutants in
the DMC1 [20] and RAD51 [21] genes were severely
affected in mHR. Their chromosomes clearly failed to
synapse and to form bivalents, but unlike in yeast,
chromosome fragmentation caused by persisting meiotic
DSBs was observed only in rad51 mutants. This suggests
a function of DMC1 in the selective invasion of the
homologous chromosome rather than in the repair of
the SPO11-induced DSBs. These could be repaired by
RAD51-mediated HR between sister chromatids or by an
alternative pathway. Interestingly, a DMC1 homolog could
not be identified in Drosophila melanogaster nor in
Caenorhabditis elegans, in both of which the synaptonemal complex is formed independently of SPO11-induced
DSBs [11]. Recently, the function of the two Arabidopsis
recombinases RAD51 and DMC1 was suggested to be
dependent on their genetic and molecular interaction with
the two paralogs of BRCA2. The absence of synapsis and
the chromosome fragmentation phenotype in RNAi-brca2
plants was reminiscent of that of the double-mutant
dmc1/RNAi-rad51 [22], and the BRCA2 paralogs
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TRENDS in Genetics Vol.21 No.3 March 2005
interacted with both DMC1 and RAD51 in a yeast-twohybrid analysis. With the same technique interactions
within the RAD51 paralogs RAD51 and XRCC3, and
between XRCC3 and RAD51C were detected [23]. In
agreement with these findings, xrcc3 mutant plants
revealed severe meiotic defects and a sterility phenotype
[24]. In contrast to the previously described mutants,
synapsis in xrcc3 appeared to be unaffected and bivalents
were formed normally. As meiosis proceeded chromosome
bridges and fragmentation were observed suggesting a
distinct role of XRCC3 in a later phase of mHR; most
probably, branch migration and resolution of Holliday
junctions (HJ) are dependent on both XRCC3 and
RAD51C [25].
Some eukaryotic homologs of the bacterial mismatch
repair proteins MutS and MutL are thought to promote
the crossover, branch migration and the resolution of the
HJs in mHR. An Arabidopsis mlh1 mutant was described
that lacked an obvious meiotic or somatic phenotype [26],
although the nature of this allele did not permit a final
conclusion. A mutation in the Arabidopsis MSH4 gene led
to a reduction of chiasmata number and to delayed and
incomplete synapsis resulting in univalents [27]. This
supports a role of MSH4 in mHR, whereby it probably
acts in a heterodimeric complex with MSH5 as a sliding
clamp, keeping together the homologous chromosomes
engaged in HJs, as was recently proposed for the human
MSH4–MSH5 complex [28].
An interesting aspect of plant meiosis is the lack of a
checkpoint, which controls the progression of meiosis,
provoking meiotic arrest in yeast and apoptosis in
animals. All of the mutants described above proceed
through meiosis despite containing aberrant chromosome
figures, enabling more extensive studies on meiotic
chromosome dynamics. Nevertheless, the Arabidopsis
homolog of Ataxia Telangiectasia-Mutated (ATM), which
signals from DSB to checkpoint and repair pathways in
other eukaryotes, was shown to be involved in somatic
DNA damage response and clearly has a function in
175
meiotic recombination; mutants in this gene reveal a
chromosome fragmentation phenotype similar to that of
the xrcc3 mutant [29]. This suggests that Arabidopsis
ATM controls mHR at SPO11 induced DSBs, but not the
integrity of meiotic chromosomes.
It is to be expected that more genes acting in mHR will
be identified with time and that careful analyses of
multiple mutations will decipher plant meiosis. For
instance, some of the mutants isolated in a screen for
X-ray sensitivity had also changed levels of meiotic
recombination [30,31], but the genes responsible for this
phenotype have not been isolated yet (Table 2).
Somatic recombination
DSBs are among the most dangerous forms of DNA
damage; a single unrepaired DSB in yeast, even in a
dispensable gene, can result in cell death [32]. DSBs are
caused by both cell-external and internal factors: ionizing
radiation is an example of an external factor, DNA
replication across a nick is an internal factor, whereas
reactive oxygen species can accumulate in plant cells as a
consequence of pathogen attack and/or of intrinsic
metabolic activities. Numbers are not available for plants,
but it has been estimated that 5–10% of first passage
primary fibroblasts from mice or humans have a chromosome break (discussed in Ref. [33]). In plants and
vertebrates most of these breaks are repaired by nonhomologous-end joining (NHEJ), an error-prone process.
HR enables repair in a precise fashion. However, HR in
somatic tissue is not only active in repair, but also in
changing the copy number of genes, as is already evident
in recombination products originating from HR in transgenic marker genes (see Figure I in Box 2). Repair will
have to be fast and efficient, whereas changes in copy
number will have to be modulated in a subtle way. The
repair of DNA damage in somatic cells has to be understood in terms of the repair pathways involved and in
terms of modulation of these pathways as influenced by
development and the environment.
Box 2. Marker genes for assays of homologous recombination
Mapped phenotypic markers (such as color markers in maize kernels
or the classic pea markers used by Mendel) or molecular markers are
usually employed for the analysis of mHR frequencies in plants,
recombination being assayed in the recombinant progeny. To enable
the measurement and visualization of somatic and meiotic HR events
up to the resolution of a single cell, special substrates were developed.
The basis of the technique is the insertion into the plant genome
(using Agrobacterium tumefaciens) of transgenes permitting selectable or screenable detection of HR events. Markers based on selection
(for instance involving the creation by recombination of antibiotic
resistance) are more difficult to use and require cell culture of plant
tissue (reviewed in Ref. [2]). The most valuable constructs enable the
analysis of HR at the plant level using a visible reporter. Such a system
was recently developed, consisting of an endogenous cluster of
Ribulose-Bisphosphate-Carboxylase small subunit (RBCS) genes,
one of which is fused to a promoterless Luciferase reporter gene
(Figure Ia). Recombination between the RBCS repeats enables the
luciferase gene to switch from an inactive state to an active screenable
state. This locus-specific integrated recombination substrate can reveal
meiotic intergenic unequal crossing-over between sister chromatids
[66,67]. Moreover, somatic recombination events, occurring in late
flower or very early embryo development, can also be identified.
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The analysis of HR in somatic plant tissue was previously restricted
to the use of ill-characterized natural systems [68,69]. The use of new
marker genes has revolutionized the research field within the past
15 years. Currently used reporter-based constructs consist of tandem
repeats of a disrupted b-glucuronidase (GUS) or a Luciferase (LUC)
gene (Figure Ib–d; schemes shown for GUS). Different orientations,
relative to each other, of the disrupted genes enable the scoring of
intramolecular (Figure Ib,c) or intermolecular HR events (Figure Id). In
the second case a chromosomal homolog or a sister chromatid serves
as recombination partner [2,70]. Thereby, different types of recombination processes could lead to a functional reporter gene: pop-out,
unequal reciprocal exchange and gene conversion [70], which is –
shown in Figure Id – possibly the most frequent [71]. The structure of
the recombination substrate trap could not be designed to enable a
distinction between interchromatid or interchromosomal recombination. Only the use of molecular markers flanking the repeats of the
disrupted marker gene would permit this. It should be noted that in
plants containing the intramolecular constructs (Figure Ib,c) HR can
also occur between sister chromatids and homologous chromosomes
although at a lower frequency than expected for intramolecular
recombination.
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176
TRENDS in Genetics Vol.21 No.3 March 2005
(a)
RBCS1
T
P RBCS2
T
P RBCS3
T
LUC
T
P RBCS2
P RBCS3
T
T
LUC
RBCS1
RBCS3/1
LUC
LUC
LUC+
Direct repeats
U′
U′
Indirect repeats
U′
H
G
(c)
S
H
G
(b)
P
U′
S
P
G
U′
U′
G
G
U′
S
U′
S
U
S
GUS+
H
U′
S
G
U′
U S
GUS+
(d)
U′
G
(e)
U′
P
U′
S
G U′
U′
U′
S
S
G
U′
G U S
G
U′
G
U′
GUS+
U′
S
TRENDS in Genetics
Figure I. Reporter-based homologous recombination substrates. (a) A synthetic RBCSB gene cluster containing a promoter-less firefly Luciferase gene [67]. Unequal
crossover events between sister chromatids that contain the RBCSB cluster enables the expression of the Luciferase gene, which can be scored by live-imaging using a
sensitive CCD camera. RBCS1, RBCS2 and RBCS3 are repeats of Ribulose-Bisphosphate-Carboxylase small subunit gene; P and T indicate the endogenous promoter and
terminator for gene expression. (b,c) Intrachromosomal HR reporter constructs with direct and indirect repeats. The constructs consist of two inactive fragments
(GU’, U’S) of a b-glucuronidase (GUS) gene sharing an identical stretch of 618 bp (U’). Intramolecular HR events between the repeats restore a functional gene (GUSC)
with different molecular products: the direct repeats (GU’-U’S) give rise to deletion of the sequence between the two repeats, resulting in a probably short-lived
nonreplicative circular molecule. HR between indirect repeats (U’G-U’S) results in inversion and conservation of the sequence separating the repeats. Black triangles
represent the borders of the T-DNA; P indicates the CaMV viral promoter driving GUS expression; H is the hygromycin resistance gene. (d) Reporter construct used to
visualize intermolecular HR events. The major pathway that gives rise to the restoration of the GUS gene has been found to be gene conversion (depicted), but unequal
reciprocal recombination between sister chromatids or allelic chromosomes and ‘pop-out’ events were also reported [70,71]. (e) In planta detection of recombination events in
reporter plants harboring a stably integrated HR construct, as in (b). Histochemical GUS staining of reporter plants visualizes cells in which the GUS gene was restored by HR.
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TRENDS in Genetics Vol.21 No.3 March 2005
177
Table 2. Plant mutants with altered somatic homologous recombination frequencya,b
Mutantc
Gene (AGI
Pathway
access number)
Genetic screens for genotoxic stress sensitivity
xrs4
ND
ND
Assay
Repair and recombination phenotype
Refs
X-ray sensitivity, genetic screen
[30,31]
xrs9
ND
ND
X-ray sensitivity, genetic screen
xrs11
ND
ND
X-ray sensitivity, genetic screen
Hyrecd
ND
HR
Spontaneous
Mim
At5 g61460
Chromatin
MMS sensitivity, genetic screen
Bru
At3 g18730
ND
MMS sensitivity, genetic screen
X-ray, MMS and MMC sensitivity;
decreased somatic HR and increased
mHR
X-ray and MMS sensitivity; decreased
somatic HR and mHR
X-ray and MMC sensitivity; defective in
X-ray-mediated somatic HR induction
g-ray resistant, increased interhomologs HR, intrachromosomal HR
unchanged
MMS, UV-C, MMC and X-ray sensitivity;
decreased somatic HR
MMS, MMC, UV-C and bleomycin
sensitivity; increased somatic HR, TGS
release
Genetic screens for altered HR phenotypes using chromosomal HR substrates
Centrin2
At4 g37010
NER
Increased HR genetic screen
ino80
At3 g57300
HR
Reverse genetics and functional genomics
rad50
At2 g31970
NHEJ
Altered HR genetic screen
Homology to known protein
rad17
At5 g66130
Cell-cycle
checkpoint
Homology to known protein
rad9
At3 g05480
Cell-cycle
checkpoint
Homology to known protein
ercc1
At3 g05210
NER
Homology to known protein
snm1
At3 g26680
HR?
Functional genomics
[30,31]
[37]
[34,74]
[35]
UV-C sensitivity; increased somatic HR,
defective in repair of UV-damaged DNA
Decreased somatic HR
[46]
MMS sensitivity; sterility, telomeric
defect, increased somatic HR
Bleomycin and MMC sensitivity;
increased somatic HR, rapid DSB repair
defect
Bleomycin and MMC sensitivity;
increased somatic HR, rapid DSB repair
defect
UV-C sensitivity; ECR with nonhomologous overhangs reduced, decreased
chromosomal HR
Bleomycin and H2O2 sensitivity;
impaired induction of HR by H2O2 and
flagellin but not bleomycin, MMC and
MMS
[18,38,40]
Mutants previously isolated for other function
rad1 (uvh1,
At5 g41150
NER
xpf)
Known UV-C-sensitive mutant
uvr2 (phr1)
At1 g12370
Photorepair
Known UV-B-sensitive mutant
cim3
ND
Plant defense
Uncharacterized defense mutant
vtc1 (soz1)
At2 g39770
Vitamin C
Known UV-B-sensitive mutant
Tt4 (chs)
At5 g13930
Flavonoid
Known UV-B-sensitive mutant
UV-B sensitivity; chalcone synthase
deficient, increased somatic HR
Tt5 (chi)
At3 g55120
Flavonoid
Known UV-B-sensitive mutant
UV-B sensitivity; chalcone isomerase
deficient, increased somatic HR
a
[30,31]
UV-B, UV-C, g-ray and cisplatin sensitivity; ECR with nonhomologous overhangs reduced, impaired HR induction
by bleomycin
UV-B sensitivity; defective in CPDs
photorepair, slightly increased somatic
HR
Constitutively activated systemic
acquired resistance, increased somatic
HR
UV-B and H2O2 sensitivity; ascorbicacid deficient, increased somatic HR
[47]
[39]
[39]
[73]
[44]
[75–78,80]
[41,53,81]
[43]
[82,83], (G. Ries,
PhD thesis,
Basel University,
1999)
[82,84], (G. Ries,
PhD thesis,
Basel University,
1999)
[82,84], (G. Ries,
PhD thesis,
Basel University,
1999)
Abbreviations: CPDs, cyclobutane pyrimidine dimers; ECR, extrachromosomal HR; HR, homologous recombination; mHR, meiotic HR; MMC, mitomycin C; MMS,
methylmethane sulfonate; ND, not determined; NER, nucleotide excision repair; NHEJ, nonhomologous end-joining; TGS, transcriptional gene silencing.
Within the past few years, the use of various strategies proved to be successful and complementary in identifying a large number of mutants involved in somatic HR in
plants. Such mutants are listed in the top part of the table with the expected impaired pathway and a summary of their phenotypes. These include: (i) mutants originating
from indirect genetic screens (i.e. by searching for mutations resulting in altered sensitivity to genotoxic stress and by subsequently testing their effect on HR); (ii) mutants
obtained through direct genetic screening of mutagenized Arabidopsis lines carrying a chromosomal HR reporter construct; (iii) mutants originating from reverse genetic
approaches (i.e. testing publicly available Arabidopsis mutants in genes homologous to recombination or repair genes known from other organisms) or genes inferred from
functional genomics studies; and (iv) some additional previously characterized mutants are listed in the lower part of the table. These were identified for other phenotypes
and only subsequently shown to affect HR.
c
Alternative names are indicated in parenthesis.
d
Hyrec is in Nicotiana tabacum; all other mutants are in Arabidopsis thaliana.
b
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178
TRENDS in Genetics Vol.21 No.3 March 2005
DSB
ATM
Signaling
ATR
Chromatin events
INO80 ?
BRU ?
?
RAD50
KU70
KU80
NHEJ
MRE11
MIM ?
Interconnection
with other repair
SNM1
pathways
(i) 5′- 3′ resection
RAD51 ?
Pathway
determination
Centrin
RAD17
RAD54L ?
DSB repair model
RAD9
SDSA model
(ii) Strand invasion
(vii) First strand invasion
and synthesis
(iii) New DNA synthesis
Branch migration
(viii) Second strand invasion
and synthesis
RAD1
ERCC1
(iv) Holliday junction
resolution
(ix) New strands annealing
and synthesis
(x) Gene conversion with
unaffected template
(v) No crossing-over
(vi) Crossing-over of
flanking markers
TRENDS in Genetics
Figure 2. The DSB and synthesis-dependent strand-annealing (SDSA) repair models of HR in plants. (i–vi) DSB repair model for HR. (i) Initially the DSB is resected in 5 0 -to-3 0
direction, producing 3 0 single-stranded DNA ends. (ii) The 3 0 ends invade a homologous DNA duplex forming a DNA crossover, or Holliday junction (HJ), and providing
primers to initiate new DNA synthesis. (iii) The region of heteroduplex is extended through branch migration of the HJ away from the initial crossover site. (iv) HJs are
resolved by cleavage of either the crossed (green arrows) or the non-crossed (black arrows) strands of the junction. Resolution in the same orientation does not affect the
flanking markers (v), whereas a mixed resolution of the two HJs does (vi). (vii–x) The SDSA model for HR. (vii) One of the 3 0 single-stranded tails invades the homologous
duplex, priming DNA synthesis. (viii) The other 3 0 single-stranded tail can also subsequently invade the homologous duplex and prime synthesis. After displacement from the
donor duplex (ix) the nascent strand pairs with the other 3 0 single-stranded tail and DNA synthesis and ligation complete repair (x). Factors known to be involved in HR in
plants, most of which are discussed in the main text, are depicted.
Recently, the combination of various approaches to
isolate mutants in somatic HR has led to a dramatic
increase in our knowledge about plant HR, its pathways,
its components and its regulation (Table 2 and Figure 2).
Plant mutants screened for sensitivity to UV, methylmethane sulfonate (MMS) and X-rays were tested for
HR; publicly available random insertional mutants in
genes known from other organisms to be involved in HR
and in genes inferred from functional genomic approaches
were analyzed. Finally, direct screens for altered levels
of HR in populations of mutagenized recombination
tester-lines were conducted. All approaches yielded HR
mutants, and proved to be complementary (Table 2).
Strikingly, genetic screen approaches yielded many novel
genes or genes not expected to be involved in the
regulation of HR.
Some of the X-ray-sensitive mutants were found to be
changed at the level of somatic recombination, some at
www.sciencedirect.com
the level of mHR and some at both levels (xrs mutants in
Table 2). The screens for MMS sensitivity yielded the two
mutants mim [34] and bru [35]. The MIM gene encodes a
protein closely related to the structural maintenance of
chromosomes (SMC) family of proteins, which have a
central role in chromosome organization and dynamics in
budding yeast. The yeast MIM homolog, Smc6, has
recently been implicated in interchromosomal and sisterchromatid recombination [36]. In the bru mutant, in
which the stability of heterochromatin is affected, levels of
somatic HR are increased. It is therefore entirely possible
that the elevated HR levels in bru plants reflect an
increased sensitivity to DNA-damaging agents or an
improved accessibility to the sites of damage for repair
factors rather than a direct involvement in the repair
machinery. It is of interest to mention the only described
hyper-recombinogenic mutant isolated in a non-Arabidopsis
species: a tobacco mutant obtained in a transposon
Review
TRENDS in Genetics Vol.21 No.3 March 2005
mutagenesis approach exhibited a 1000-fold increased
frequency of mitotic recombination between homologous
chromosomes, as measured by the endogenous ‘sulfur’
system, while leaving intrachromosomal HR unaffected.
Unfortunately, the mutation could not be characterized [37].
In the first reverse genetic approach mentioned above,
that is, searching for and characterizing publicly available
insertional mutants in specific genes, Arabidopsis
mutants in the genes RAD50, RAD9 and RAD17 could
be obtained that exhibited increased levels of HR, linked
with defects in DSB repair and hypersensitivity to
genotoxic stress [38,39]. RAD9 and RAD17 were shown
to be involved in the cell-cycle checkpoint in both yeast
and Arabidopsis and might point to a connection between
DNA damage signaling and HR regulation (Figure 2). In
Arabidopsis both rad50 mutants, which are hypersensitive to DNA damage and hyper-recombinogenic in somatic
tissue, and rad51 mutant plants [21] grow normally but
are sterile. This is in sharp contrast to the situation in
mouse where mutations in both genes abolish cell viability
(as discussed in Refs [21,40]). Mammalian cells obviously
require the activity of both genes for cell cycle progression.
The influence of the rad50, rad9 and rad17 mutations
on the level of HR seems to be indirect, which could point
to a regulated balance between repair activities and
altered availability of damaged sites to repair machineries. A similar explanation could hold for plants mutated
in the photolyase gene [41], impaired in scavenging of
reactive oxygen species [42] and in plants upregulated for
pathogen defense [43]: there, increased HR could be due to
increased levels of DNA damage.
The snm1 mutant was obtained by searching for
insertional mutants in genes inferred from functional
genomic studies to be differentially regulated by genotoxic
stress. It is defective in the induction of somatic HR by
specific oxidative stress (Table 2) and could unravel the
existence in plants of a specific recombinational repair
pathway for oxidatively induced DNA damage [44,45].
The approaches to identifying plant genes involved in
HR by knocking out candidate genes will of course not
yield new and unsuspected genes; only a screen directly
testing for HR will permit this. In two different screens
employing the recombination substrate lines shown in
Figure Ic and Id in Box 2, a multitude of mutants changed
in HR levels were identified and isolated (O. Fritsch et al.,
unpublished). Also, in one of these mutants the reason for
the strongly induced frequency of HR seems to be indirect:
a T-DNA-mediated knockout of the Centrin2 gene, encoding a protein involved in recognition of DNA damage that
can be repaired by the nucleotide excision repair (NER)
pathway, led to a UV-C-sensitive, hyper-recombinogenic
mutant. This points to a novel connection between an
early step of NER and HR [46]. Another mutant, with a
reduced level of HR, was mutated in a gene coding for
INO80, a member of the SWI–SNF ATPase family [47].
The efficiency of NHEJ seemed unaffected in the mutant.
In vitro interaction of this protein with nucleosomes
suggested that INO80 acts through modification of
chromatin structure, possibly at the site of DNA repair.
Interestingly, the yeast Ino80 protein, as part of the
INO80 complex, has recently been shown to be recruited to
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179
DSBs, in a manner dependent on phosphorylated histone
H2A [48]. Further studies are necessary to find out if the
Arabidopsis INO80 – or the potential complex that
contains it – is also recruited to sites of DNA damage,
and if such recruitment leads to the specific engagement of
the HR machinery.
The influence of the environment on somatic HR
The in planta assays described (Box 2) enable the
quantitative assessment of HR events, thus permitting
the analysis of the influence of abiotic and biotic agents on
HR. The effects of gamma radiation, UV radiation, heavy
metals and pathogens on the physiological behavior of
plants have been amply described. However, the impact
of these environmental factors on HR in genomes of the
affected plants, discussed in more general terms by
McClintock [49], has not been thoroughly studied until
recently. Exposure of plants to gamma radiation – either
resulting from accidents in atomic power stations or in an
experimental setup – was expected to lead to higher levels
of DSBs. Indeed, the use of plants carrying recombination
reporter constructs yielded HR frequencies that directly
reflected the level of radioactive contamination [50].
Especially interesting was the fact that even low levels
of gamma radiation could be recorded with surprising
accuracy, opening the possibility for environmental biomonitoring regimes [51]. Other analyses of abiotic influences on HR in Arabidopsis have included tests with UV-B
[52], heavy metals [53], X-rays, heat shock and UV-C
(reviewed in Ref. [2]). The mechanisms by which the
measured increases in rates of HR are established are not
known, but direct induction of DSBs, the activity of
reactive oxygen species and the upregulation of DNA
repair activities are likely to contribute.
The influence of biotic factors such as pathogens on
plants has been studied from various aspects, but only a
few studies focus on the possible effects of pathogens on
the genomes of plants. Elevated mutation rates in maize
following virus infection have been reported, but molecular data are lacking [54]. Inoculation of Arabidopsis lines
containing an HR reporter gene with Peronospora parasitica led to stimulation of somatic HR. The same effect
was observed when pathogen defense mechanisms were
chemically or genetically activated in the absence of a
pathogen [43]. Infection of tobacco plants carrying
recombination markers with tobacco mosaic virus (TMV)
yielded increased recombination rates not only in inoculated leaves but also in noninoculated leaves [55].
Treatment of plants with UV-B or with TMV led to an
increased rate of HR-mediated genetically fixed restoration of the functional reporter gene (Box 2) in the
offspring [52,55]: the number of seedlings that were
completely b-glucuronidase- or luciferase-positive was
greater in the offspring of treated plants than in those of
untreated plants. It is unclear, however, whether these
induced events were due to somatic HR events late in
plant development, that is, before or after meiosis, or due
to enhanced rates of mHR.
An increase in either somatic or meiotic HR could
facilitate evolutionary adaptation of plant populations
to stressful environments. Possible substrates for
180
Review
TRENDS in Genetics Vol.21 No.3 March 2005
recombination events in plants are the numerous disease
resistance genes spread in clusters throughout the
genome [56]. New resistance genes can be created by
sequence exchange between genes in the same or different
clusters, as was reported for the maize Rp1 complex
[57,58] and the tomato Cf-4 and Cf-9 loci ([59] and Refs
therein). Infection of plants with pathogens can therefore
lead to an increased frequency of rearrangements between
resistance genes and thereby possibly to an adaptive
advantage in pathogen defense. It can thus be proposed
that abiotic and biotic environmental influences affect the
genotypes of plants on two levels: (i) the level of mutation
(so far shown for HR and point mutation [60]) enabling a
large genetic diversity in the population; and (ii) the level
of selection in the offspring.
Concluding remarks
Recent forward and reverse genetics of Arabidopsis and
other plants has permitted the isolation of numerous
genes involved in meiotic and somatic HR. Some of these
were expected to have a role in HR based on the roles of
homologous genes in other organisms, but in other cases
their role in plants deviates in some aspect from that of
their homologs in animals and yeast. It is predicted that
many more functions will be unraveled in the not too
distant future.
Specific questions for researchers include: (i) are there
functional homologs of RAD52 and other repair proteins in
plants (Table 1); (ii) which other proteins are directly
involved in the mechanics of meiotic and somatic HR in
plants and; (iii) how does HR work on the level of
chromatin? Examples for the contribution of chromatin
in HR have been cited (MIM, INO80 and perhaps BRU),
and additional proteins acting on chromatin at the stage of
formation of SPO11-dependent DSBs can be expected for
plants, as has been shown for C. elegans [61]. Finally, how
is somatic HR regulated, or more specifically, how are
different repair activities orchestrated?
Another crucial aspect for plants is the influence of the
environment on the plant genome: ‘illustrations from
nature.support the conclusion that stress, and the
genome’s reaction to it, may underlie many formations of
new species’ [49]. We will have to analyze how this stress
is perceived, how signals are sent through cells and
organisms, and how and with what specificity changes are
introduced into the genomes. Are these changes genetically stable, are they epigenetic in nature or are there
epigenetic intermediates?
Acknowledgements
We thank the members of our group for stimulating discussions and the
Novartis Research Foundation for financial support. J.M. was funded by
the EU project PLANTREC N8 QLG2-CT-2001–01397.
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