Download Transcription-associated recombination in eukaryotes: link between

Mutagenesis vol. 24 no. 3 pp. 203–210, 2009
Advance Access Publication 12 January 2009
doi:10.1093/mutage/gen072
REVIEW
Transcription-associated recombination in eukaryotes: link between transcription,
replication and recombination
Ponnari Gottipati1 and Thomas Helleday1,2,*
1
Gray Institute for Radiation Oncology and Biology, University of Oxford,
Oxford OX3 7DQ, UK and 2Department of Genetics Microbiology and
Toxicology, Stockholm University, S-106 91 Stockholm, Sweden
Homologous recombination (HR) is an important DNA
repair pathway and is essential for cellular survival. It
plays a major role in repairing replication-associated
lesions and is functionally connected to replication.
Transcription is another cellular process, which has
emerged to have a connection with HR. Transcription
enhances HR, which is a ubiquitous phenomenon referred
to as transcription-associated recombination (TAR). Recent evidence suggests that TAR plays a role in inducing
genetic instability, for example in the THO mutants (Tho2,
Hpr1, Mft1 and Thp2) in yeast or during the development
of the immune system leading to genetic diversity in
mammals. On the other hand, evidence also suggests that
TAR may play a role in preventing genetic instability in
many different ways, one of which is by rescuing replication
during transcription. Hence, TAR is a double-edged sword
and plays a role in both preventing and inducing genetic
instability. In spite of the interesting nature of TAR, the
mechanism behind TAR has remained elusive. Recent
advances in the area, however, suggest a link between TAR
and replication and show specific genetic requirements for
TAR that differ from regular HR. In this review, we aim to
present the available evidence for TAR in both lower and
higher eukaryotes and discuss its possible mechanisms, with
emphasis on its connection with replication.
Introduction
To maintain genomic stability, cells possess multiple mechanisms of DNA repair that remove damage and in most cases
restore the original DNA molecule. DNA repair mechanisms
are complex, often interlinked, and their failure can result in
tumorigenesis arising from sequential accumulation of unrepaired DNA damage, both at the nucleotide and chromosomal
levels (1). DNA repair mechanisms are also associated with
cellular processes like transcription and replication to ensure
genome integrity at every stage of the cell cycle. DNA repair is
more efficient in transcribed regions and it is directly associated
with transcription in a process called transcription-coupled repair
(TCR) (2,3). Furthermore, specific DNA repair processes, e.g.
homologous recombination (HR) and translesion synthesis
(TLS), occur at replication forks to ensure proper replication
and prevent genomic instability (4). In higher organisms, DNA
repair pathways are exploited to generate genetic diversity
during development of the immune system. Non-homologous
end joining (NHEJ) is involved in V(D)J recombination, an early
process in antibody maturation, where as both NHEJ and base
excision repair as well as many DNA damage response factors
are important for class switch recombination (CSR), a late
process in antibody maturation (5). Similarly, error-prone DNA
repair processes are suggested to play a role in somatic
hypermutation (SHM), another process involved in the late
stages of antibody maturation (6).
In this review, we discuss transcription-associated recombination (TAR), a ubiquitous phenomenon that associates HR
with the cellular process, transcription. It has long been
established that transcription enhances HR (7), though the
mechanism remains to be elucidated. Here, we also discuss the
possible mechanisms behind this association with emphasis on
the involvement of replication-associated lesions as substrates
for TAR.
HR and replication lesions
HR is required to repair replication-associated DNA lesions
and DNA double-strand breaks (DSBs), is generally error free
and occurs mainly during late S and G2 phases of the cell cycle
(8,9).
DSBs are produced endogenously by various sources
including topoisomerases or exogenously by sources like
ionizing radiation and chemicals that generate reactive oxygen
species [reviewed in (10)]. DSBs also normally occur during
V(D)J recombination and immunoglobulin class-switching
processes and also during replication as a consequence of
replication fork arrest and collapse.
HR plays an important role in the repair of damage during
replication in all organisms (11,12). In mammalian cells,
replication forks collapse when they collide with unrepaired
DNA single-strand breaks (SSBs) triggering HR (13,14).
During normal cell cycle, restarting replication when DNA
replication forks stall due to damage in the DNA template can
involve generation of a DSB intermediate (15). HR has been
suggested to repair such naturally occurring DSBs arising
during the S phase of the cell cycle that lead to collapsed
replication forks (16,17). There is increasing evidence to show
that HR in mammalian cells has an important role in the repair
of stalled replication forks caused by a variety of lesions (e.g.
breaks, DNA adducts and chromatin-bound proteins) [reviewed
in (8)]. Replication may bypass the block either by TLS or by
re-priming downstream of the block and the remaining gaps
can then be filled by template switching via HR (8). Replication block bypass can also take place by template
switching via HR which leads to the uncoupling of leading
and lagging strand synthesis and fork regression forming
a ‘chicken-foot’ structure. HR is then required to rescue and
restart replication [reviewed in (8,18)]. The interesting feature
about stalled replication forks is that they may also occur in the
absence of detectable DSBs (12), suggesting that HR repairs
*To whom correspondence should be addressed. Tel: þ44 1865 617 324; Fax: þ44 1865 857 127; Email: [email protected]
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a variety of lesions that occur at replication forks and not just
DSBs. Though the substrates for HR in this case remain elusive,
it has been proposed that fork reversal and formation of chickenfoot structures as discussed above serve as substrates for HR,
which can occur without the cleavage of the Holliday junction
with in the chicken foot and can therefore occur in the absence
of DSBs (8) as is the case in bacteria [reviewed in (8,19)].
It is possible that transcription may result in such replicationassociated lesions due to interference with replication when
both cellular processes occur on the same DNA substrate at the
same time, perhaps resulting in TAR.
TAR in yeast
Evidence for enhanced recombination in transcriptionally
active DNA in eukaryotes was first shown in Saccharomyces
cerevisiae during the search for recombination hot spots.
Transcription resulted in a 5- to 10-fold increase in recombination between the nearby duplicated genes in the recombination
hot spot HOT1, which is present in the ribosomal RNA gene
cluster (20). In diploids, transcription driven by RNA polymerase I in the HOT1 region (21,22) stimulates interchromosomal gene conversion (23,24). Several other reports in yeast
also demonstrated that gene conversion is the primary recombination event stimulated by transcription in direct repeats
(22,25).
Later, RNA polymerase II-dependent transcription was also
shown to enhance recombination in yeast indicating that this
effect is not specific for RNA polymerase I-mediated
transcription (25–27). Mitotic intrachromosomal recombination
between non-tandem duplications in S.cerevisiae was enhanced
by transcription mediated by the inducible Gal10 promoter (26).
In addition, meiotic and mitotic interchromosomal recombinations in Schizosaccharomyces pombe were also enhanced by
RNA polymerase II-dependent transcription driven from the
highly active ADH1 promoter (25).
Mating type interconversion is a further example of TAR in
yeast. It involves a specialized gene conversion event initiated
by a HO nuclease-mediated DSB in the MAT locus, which
depends on active transcription. The homologous but transcriptionally silent loci are not cleaved by HO nuclease confirming
the dependence of mating type switching on transcription (28).
Studies in artificial recombination repeats in yeast have
shown that increasing transcription does not further enhance
DSB-induced recombination or affect gene conversion tract
lengths (29). This indicates that once a recombination event is
initiated by a DSB, transcription has no further effect on
recombination levels, suggesting that transcription influences
recombination by initiating it (29).
The yeast THO complex along with Thp1 protein is another
example of TAR in yeast and provides a connection between
transcription elongation and recombination (30,31). THO is
a conserved eukaryotic complex made of four proteins Hpr1,
Tho2, Mft1 and Thp2 (32). This complex plays a role at the
interface between transcription and mRNA export (33–35) and
is recruited only to transcriptionally active chromatin (34,36).
Mutations in any of the genes in this complex lead to defects in
transcription elongation (32,37–39), transcription-dependent
hyper-recombination (30,39,40) and nuclear mRNA accumulation (34). Deletion mutants of THO2 and HPR1 showed 3000fold increase in recombination between direct repeats compared
to the wild-type cells (30) and this hyper-recombination was
dependent on transcription elongation (30) as TAR was abolished
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by terminating transcription elongation by placing a transcription
terminator downstream of one repeat (40). The null mutants of
THO2 and HPR1 were capable of carrying out all types of recombination in the artificial recombination substrates tested,
including both RAD51-independent single-strand annealing
events and Rad51-dependent gene conversion involving of
strand exchanges (39), unlike spontaneous TAR between direct
repeats in yeast which primarily involves gene conversion events
(22–25).
THO together with certain RNA export factors forms a
larger complex called TREX (34). Mutations in these RNA
export factors confer a similar transcription-dependent hyperrecombination phenotype and defect in transcription elongation
as THO mutations (33,41). In addition, yeast mutants in proteins
involved in various processes associated with transcription like
3#-end cleavage/polyadenylation, components of nuclear exosome (multi-subunit RNA processing and degradation machine),
and mRNA export showed a significant transcription-dependent
hyper-recombination phenotype though not to the same extent
as the THO mutants (42). THO–TREX complex and the hyperrecombination observed in yeast mutants in cleavage/polyadenylation factors and nuclear exosome (42) suggest that there is
a strong interdependence between transcription elongation and
hyper-recombination and also between mRNP (export competent ribonucleoprotein particle) biogenesis and genetic stability
in general (33).
TAR in mammalian cells
Nickoloff and Reynolds (43) provided the first direct evidence
of TAR in mammalian cells. Transcription of heteroallelic
neomycin genes was shown to stimulate recombination 6-fold
between transfecting plasmids in Chinese hamster ovary
(CHO) cells. Direct evidence that transcription stimulates
intrachromosomal recombination was provided by studies on
a similar system in CHO cells (44). There was a 2- to 7-fold
increase in intrachromosomal recombination between duplicated neomycin genes stably integrated into the CHO cell lines
in the presence of transcription. Transcription through only one
repeat was enough for stimulating recombination in both direct
and inverted repeats (44). Using a similar recombination
reporter system containing two non-functional copies of the
neomycin gene under the regulation of tetracycline promoter,
we showed a quantitative relation between transcription levels
and recombination levels. About 60-fold increase in transcription through the construct stimulated recombination by
.20-fold (45).
As in yeast, gene conversion was the main spontaneous TAR
event between direct repeats in mammals (44,45) but
recombination between inverted repeats produced a variety of
rearranged products including cross overs and unequal sister
chromatid exchanges, suggesting that transcription does not
influence the mechanism of recombination (44). Studies
performed in our laboratory further revealed that transcription
enhances short-tract and long-tract gene conversion events in
similar frequencies without preference for either (45). Similar
to yeast, transcription in mammalian cells does not seem to
influence DSB-induced recombination any further (45,46) and
has no effect on the DSB-induced gene conversion spectra
(46,47) again confirming that transcription has no influence
once recombination is initiated. However, transcription enhanced the use of a donor allele 2- to 3-fold during DSBinduced gene conversion in human cells, as shown using
Transcription-associated recombination in eukaryotes
a triple neomycin repeat recombination substrate (48). This
suggests that TAR may play a role in maintaining genomic
stability by preventing the use of non-transcribed pseudogenes
as donors during gene conversion (48).
Overall, the available data in mammalian cells suggest that
TAR and DSB-induced HR are mechanistically different
processes with little influence on each other. Our recent data
show that there is a differential genetic requirement for the two
processes (49). It was found that the RAD51 paralogue
XRCC2, which is required for HR induced by a DSB (50), is
dispensable for TAR (49). This is interesting as it proves that
TAR is carried out in absence of XRCC2 and is genetically
distinct from HR induced by a DSB. The XRCC2 protein has
been shown to be critical early in HR for RAD51 foci
formation following ionizing radiation (51) and is also likely
involved in processes late in resolution of HR intermediates,
which has been shown to be the case for other RAD51
paralogues (52). If it is the early role of XRCC2 that is
dispensable for TAR, it would suggest that TAR may be
independent of RAD51-mediated strand invasion. However,
this is not likely to be the case as it was shown in the same
study that TAR is dependent on BRCA2 (53), which is also
involved in the early step of HR, loading RAD51 onto DNA
and required for RAD51 foci formation (54,55). Thus, it is
plausible that TAR requires RAD51-mediated strand invasion,
but is resolved differently than HR induced by a DSB.
TAR may also be linked to the immunoglobulin-diversifying
processes in mammals (56). Immunoglobulin V(D)J recombination (57,58), class switching (59,60) and SHM (60,61) in
mammalian B cells, thought to be mediated by specific
recombination events, were shown to be dependent on
transcription (62–65). During V(D)J recombination, recombination frequencies are higher in transcriptionally active
immunoglobulin genes and this is regulated at the level of
transcription (62,66). In SHM, there is a high frequency of
DSBs in and around the targeted V(D)J region (67) and
mutation frequencies correlate with promoter strength and
transcriptional activity (64). Many reports suggest the involvement of transcription and recombination in class switching (68–70). Therefore, in mammals, TAR seems to play an
important role both in maintaining genomic stability and
generating genetic diversity as in the case of immunoglobulin
diversification.
Mechanism of TAR
In spite of the importance of TAR in all organisms, its
mechanism is still elusive. There are many theories postulated
over the years to explain TAR (7) and there have been some
recent advancements in deciphering its mechanism in both
yeast and mammals. All the proposed models to explain the
stimulation of spontaneous recombination by transcription are
summarized in Figure 1 and can be grouped into one of the two
main non-exclusive categories:
(i) Transcription could lead to increased accessibility of a target
region to recombination proteins or DNA damage thus
stimulating recombination (66)—the accessibility theory.
(ii) TAR could be the consequence of stalled replication forks
formed during transcription due to either collision between
the transcription and replication machineries or transcription-associated DNA:RNA hybrids obstructing the replication fork movement (7)—the collision theory.
The accessibility theory
According to the earliest model proposed to account for TAR
in k phage, transcription leads to strand separation and the
single-stranded DNA (ssDNA) thus formed invade homologous regions, stimulating recombination (71). Alternatively,
this ssDNA is more accessible to recombinogenic DNA
damage (72). This theory was directly tested in yeast by
Aguilera and group (73) by studying the effect of different
DNA-damaging agents in several different recombination
systems that were either transcribed or not transcribed. The
DNA-damaging agents and transcription showed a synergetic
effect on both intermolecular and intramolecular recombination
between direct repeats, suggesting that TAR induced by DNAdamaging agents may be to a large extent due to increased
accessibility of DNA to these agents mediated by transcription.
For example, while transcription alone or treatment with
Fig. 1. Possible mechanisms of TAR. (A) When RNA polymerase opens up the dsDNA during transcription, the nascent ssDNA on the non-template strand is more
susceptible to DNA damage, which may be repaired by HR. The ssDNA may also be more accessible to the recombination machinery, another reason for increase in
HR levels. (B) Transcription results in the negative supercoiling of DNA (underwound state of DNA-facilitating unwinding allowing transcription to occur) behind
the advancing RNA polymerase. This may facilitate the formation of R-loops by the nascent mRNA as the DNA is underwound. R-loops also result in ssDNA
during transcription, which in turn may cause an increase in HR as discussed above. (C) R-loops may form roadblocks to the RNA polymerases behind it or to the
replication machinery working on the same DNA substrate, stalling replication forks, which require recombination to be rescued. (D) Stalled forks may also be
formed when transcription and replication machineries travelling towards each other on the same DNA substrate run into each other and collide. (E) Finally, TAR
may be a parallel pathway to TCR in S phase. When replication is blocked by active transcription, RNA polymerase may recruit recombination machinery, so
replication can bypass transcription.
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P. Gottipati and T. Helleday
4-nitroquinoline-N-oxide (a carcinogen that forms bulky adducts
on DNA) alone increased recombination by 2- and 40-fold,
respectively, both together increased recombination .4000
times. Similarly, all types of recombination events tested in
different recombination systems were greatly increased by
DNA-damaging agents in actively transcribed DNA (73). In
mammals, a similar study evaluated the relationship between
DNA damage and TAR by looking at the effect of transcription
on ultraviolet (UV)-induced intrachromosomal recombination
(74). The study showed that both transcription and UV
irradiation alone stimulated recombination as expected. However, both together did not show any synergistic effect. Elevated
levels of transcription diminished UV-induced recombination,
suggesting that recombination is stimulated by damage and not
by repair. On the same lines, UV repair-deficient cells required
lower UV doses to stimulate intrachromosomal recombination
compared to repair-proficient cells (75). These reports confirm
that ssDNA formed during transcription is more accessible to
recombinogenic DNA damage and this may be one of the
explanations for TAR. Not only does the accessibility of ssDNA
to damage increase upon transcription but also the accessibility
to nucleases increases upon transcription, which seems to be the
case for HO nuclease cleavage of MAT loci in yeast (28) and the
cleavage during V(D)J recombination in vertebrates (62,66).
Transcription could alter the repair of already existing
damage by recruiting the recombination proteins to damaged
regions. This is supported by the fact that there are several
mechanistic similarities between transcription and recombination including DNA unwinding and protein recruitment (76).
Furthermore, some transcription factors play a dual role in
transcription and DNA repair (77,78). This theory suggests that
the mechanism of TAR is perhaps parallel to TCR of other
types of DNA damage (2) and is further supported by the
available biochemical evidence (53). HR protein RAD52
[reviewed in (79)] physically associates with different subunits
of transcription factor TFIIH and RNA polymerase II and can
either activate or repress transcription (53). In addition, TAR is
shown to be dependent on rad52 gene product in yeast (80).
Similarly, another recombination protein BRCA1 associates
and forms a part of the RNA polymerase II holoenzyme (81).
In yeast, Hpr1 component of the THO complex also associates
with RNA polymerase II holoenzyme (82) and is shown to
participate in transcriptional elongation (30).
Further, there are reports suggesting that proteins required
for HR are also involved in TCR. HR proteins BRCA1 and
BRCA2 are suggested to be involved in TCR of oxidative
DNA damage (83), though this is unconfirmed. Recently, it
has been shown that HR is also involved in TCR of UV
damage (84). In S.cerevisiae, HR proteins RAD51 and
RAD54 were shown to play an important role in the repair
of UV damage on transcribed strand only during G2/M phase
of the cell cycle, providing evidence for the involvement of
HR in the repair of transcribed strands of active genes (84).
However, this repair was shown to be transcription dependent
but unrelated to replication lesions (84). These reports suggest
that the processes of TAR and TCR could be interlinked.
Other topological changes in DNA during transcription may
also facilitate the increase in recombination manifesting in
TAR. For example, transcription-induced supercoiling of DNA
may stimulate recombination by bringing the homologous
regions closer together and facilitating strand exchange (85).
Similarly, transcription-associated (86,87) or repair-associated
(88) remodelling of chromatin could result in increased
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interactions between transcriptionally active DNA during HR
repair of DSBs resulting in TAR. This is supported by the
observation that transcription enhances the use of a donor allele
during DSB repair (48). Furthermore, transcription elongation
results in the accumulation of negatively supercoiled DNA
behind the advancing RNA polymerase II. DNA in this
underwound state favours for the local unwinding of DNA and
this allows the formation of DNA:RNA hybrids or R-loops
with the nascent mRNA synthesized during transcription
(discussed below in more detail).The non-transcribing ssDNA
formed in the process may be more susceptible to DNAdamaging agents, thus forming recombinogenic lesions (7).
The collision theory: dependence of TAR on replication
Many of the theories proposed to explain the mechanism of
TAR link TAR to replication-associated lesions and we discuss
these theories in the following section.
As transcription and replication can take place on the same
DNA substrate at the same time, it is possible that they obstruct
each other in more than one way, resulting in stalled or
collapsed replication forks, which may be possible substrates
for TAR. Transcription may obstruct replication in many
different ways. Secondary structures or DNA adducts formed
during transcription elongation are one possible form of
obstruction to replication. As discussed previously, lesions
obstructing replication forks could cause the stalling and
collapse of replication forks creating DSBs. Hence, transcription elongation might contribute to the formation of lesions that
could be subsequently converted into one-ended DSBs and are
then repaired by HR (89). When DSB-induced and spontaneous recombination events were compared in yeast mutants of
different recombination proteins under low and high transcription conditions, it was found that the effect of respective
mutations was the same in DSB-induced recombination and
TAR, suggesting that the initiation events stimulated by
transcription might be DSBs or lesions leading to DSBs (89).
Our work in mammalian cells further suggests that TAR is not
associated with a classical two-ended DSB. We showed that
HR repair of DSBs involves short-tract gene conversion in all
the phases of the cell cycle in mammalian cells (90) where as
transcription induces similar levels of short-tract and long-tract
gene conversion events (45); the same pattern of recombinants
is formed following thymidine treatment (13,14). Hence, it
seems more likely that transcription results in lesions that occur
in absence of DSBs.
As explained in the previous section, negative supercoiling
of DNA behind the advancing RNA polymerase during
transcription may result in the formation of DNA:RNA hybrids
or R-loops. These DNA:RNA hybrids might also obstruct
replication, stalling replication forks. This in turn might be
resolved by recombination (7) resulting in TAR. Cotranscriptional accumulation of DNA:RNA hybrids or R-loops behind
the elongating RNA Polymerase II is the major cause of TAR
and impairment of transcriptional elongation in the THO
mutants (91) and also in mutants of a RNA-splicing factor
ASF/SF2 in chicken DT40 cells and human HeLa cells (92).
Depletion of ASF/SF2 in these cell lines resulted in high levels
of DNA rearrangements due to the formation of R-loops. In
addition, DSBs were found around the R-loop region in these
mutant cells that led to a G2 arrest. Over-expression of human
RNAse H-1 in these mutants eliminated the R-loops, DSBinduced G2 arrest and the accompanying genomic instability (92). Similarly, there was a significant reduction in
Transcription-associated recombination in eukaryotes
hyper-recombination in the THO mutant cells when the
resulting transcript during transcription of the direct repeats
under study was self-cleaved by an active artificially
engineered hammerhead ribozyme or degraded by overexpression of RNase H1 (91). These studies provide a clear
mechanistic link between R-loops and hyper-recombination. Rloops could also explain the mechanism of TAR in CSR,
though there is no in vivo evidence. In vitro experiments have
shown that the transcript of the switch region where the
recombination event takes place hybridizes with the template
DNA-forming R-loop structures during transcription (93–96),
suggesting that R-loops play an important role in TAR.
Another possible criterion where transcription might obstruct
replication is when transcription and replication machineries
collide with each other. Replication fork pausing (RFP) by
RNA polymerases I and III during transcription is a known
phenomenon (97,98) and the collision between replication and
transcription in the ribosomal DNA in yeast (99) increases
recombination (97,100). The link between RFPs and recombination is well established in fission yeast. The specific
recombination reaction involved in mating type switching in
fission yeast is promoted by a RFP (101) caused by a replication
fork barrier (RFB) {a region where particular proteins bind
tightly to DNA obstructing replication [see ref. (102) for
a review on RFBs]}. When this specific RFB is transported to
other loci in fission yeast, this led to a stimulation of
recombination in the adjacent sequences (103,104) confirming
the link between RFPs and recombination. Recently, it has
emerged that RFPs also occur during RNA Polymerase II
transcription. RFP-led increase in recombination was observed
in RNA Polymerase II transcribed leu2 genes (105) and also in
the genes highly transcribed by RNA Polymerase II in THO
mutants (discussed below) (106), suggesting that hyperrecombination led by RFP is a phenomenon that possibly
occurs during transcription of all genes. In S.cerevisiae, TAR
in the leu2 direct repeat constructs required head-on oncoming
replication (105). TAR occurred only when there was
replication fork progression opposite to transcription and it
was associated with the appearance of a RFP in the region of
homology. Further, the appearance of transcription-associated
RFP correlates with TAR (105), indicating that TAR in this
construct is a result of the collision between the machineries of
transcription and replication causing replication blockage.
Recent work in THO mutants in yeast shed more light on the
mechanism of TAR and provided further evidence for the
association of TAR with replication impairment. As explained
before, mutations in the THO complex in yeast result in
transcription-dependent hyper-recombination (30,39) dependent on transcription elongation and also on the type of DNA
segment through which transcription elongation took place
(40). Long and GC-rich regions are found to be particularly
difficult to transcribe in hpr1D mutants (30) and the hyperrecombination was shown to be higher in GC-rich regions (40).
These THO mutants also showed an impairment in the
replication fork progression due to obstruction by DNA:RNA
hybrids at the exact same regions which were previously
shown to have decreased transcription (106). Ribozymemediated cleavage of the nascent mRNA partially suppressed
the replication defect and TAR (106), confirming the role of
DNA:RNA hybrids in TAR. Moreover, the transcriptiondependent hyper-recombination in these mutants occurred only
in the S phase of the cell cycle. This further confirmed that
TAR in these mutants is associated with replication. One of the
mutations in the yeast hpr1 that results in transcription
elongation impairment and hyper-recombination phenotype
also increases chromosome (107) and plasmid loss (32) in the
presence of transcription, also consistent with the notion that
replication blockage is associated with TAR. Stalling and
collapse of replication forks by transcription can result in DNA
breaks and HR-mediated repair or bypassing of these lesions
may result in the observed chromosome and plasmid loss.
Interestingly, one particular point mutant of the hpr1 gene
showed no hyper-recombination phenotype though it showed
a strong defect in transcription and a general defect in mRNA
export like the other mutants of the THO complex (108).
This was due to the absence of replication fork blockage in
this mutant, suggesting that stalled replication is a prerequisite
for hyper-recombination. In addition, transcription-dependent
hyper-recombination observed in mutants with aberrant mRNA
processing (42) further supports this theory as incomplete
processing of mRNA may result in the DNA:RNA hybrids due
to accumulation of nascent mRNA.
Another possibility for stalled replication and transcription
to result in TAR is for RNA polymerase to simply push the
stalled replication fork backwards, thus forming a Holliday
junction, which then requires HR in order to be resolved (7).
Hence, the data in yeast suggest that stalled replication
forks formed either by collision between transcription and
replication machineries or due to RNA:DNA hybrids formed
during transcription elongation in the S phase are the substrates
for TAR.
In our recent study, we have shown that TAR is S phase
associated in mammalian cells also, suggesting that TAR is
probably ubiquitously dependent on replication (45). We studied
recombination between duplicated neomycin repeats under the
regulation of a bi-directional tetracycline promoter. This
regulatable bi-directional promoter also controlled transcription
through the luciferase gene, which allowed us to quantify both
transcription and recombination through the substrate at any
given time. Cells stalled in S phase with transcription showed
much higher frequencies of HR compared to cells stalled to the
same degree in S phase but with no transcription, probably due
to more frequent collisions between transcription and replication
machineries. Further, inhibiting transcription and replication at
the same time abolishes recombination induced by thymidine
[a drug that slows the progression of replication forks (12)],
suggesting that stalled replication forks are involved in TAR in
mammalian cells (45).
In a recent study, we show that both TAR and HR induced at
replication forks by thymidine are dependent on BRCA2
protein and at the same time independent of XRCC2 (53).
XRCC2 has been shown to be required for most HR events
(50,51,109–112), but specifically not for recombination events
induced by thymidine (112). This data suggest that HR induced
by thymidine and transcription have similar requirements and
that a similar HR substrate is formed following thymidine
treatments and transcription. The mechanism of action of
thymidine is unique, in that it slows down the replication forks
through depletion of dCTP levels without causing collapsed
replication forks (12). It is possible that transcription similarly
slows down replication forks and utilizes a similar recombination bypass pathway (53).
A model for bypassing transcription during replication may
involve collision of transcription and replication machineries
causing a DNA polymerase block, which can trigger uncoupling
of the leading and lagging strand synthesis. Template switching
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P. Gottipati and T. Helleday
may then promote replication by using lagging strand as template until the DNA polymerase bypasses RNA polymerase.
Replication may then restart in front of the RNA polymerase
using HR.
A B-cell-specific protein activation-induced cytidine deaminase (AID) that mediates immunoglobulin-diversifying processes SHM and class switching (113) provides further proof of
the link between TAR and replication-associated lesions. Action
of AID is transcription dependent and mutations in THO
complex in yeast result in strong and transcription-dependent
hyper-recombination induced by AID (114). A recently proposed model for SHM in immunoglobulin genes of vertebrates
explains SHM as the result of action of AID and other DNA
repair proteins on DSBs created during collision between the
transcription apparatus and the replication fork (115). As
explained previously, GC-rich S regions form R-loops
(93,94,96) which can in turn obstruct the replication machinery
leading to the stalling of replication forks, which might further
contribute to the high levels of recombination during class
switching. Hence, TAR seems to be the result of replication
impairment by transcription either by physical obstruction to the
replication fork progression or by creating lesions that impair
replication.
Concluding thoughts
All the evidence in both lower and higher eukaryotes suggests
that TAR is a double-edged sword. While TAR in THO–TREX
mutants in yeast suggests that it plays a role in genomic
instability, spontaneous TAR in both yeast and mammals
seems to prevent genetic instability in more than one way. For
example, it appears that TAR prevents genetic instability by
preventing the use of pseudogenes during recombination as
transcription increases preference for particular donor alleles
(48). On the other hand, TAR may help in bypassing
transcription during replication thus preventing genetic instability associated with replication blockage (45,53). In
addition, by taking advantage of the association between
transcription and recombination, immunoglobulin genes seem
to utilize TAR in generating genetic diversity. Considering the
different manifestations of TAR and the widespread nature of
TAR, it is likely that TAR in eukaryotes does not occur by
a single mechanism, but by simultaneous involvement of
several mechanisms as these are not mutually exclusive.
Funding
Medical Research Council; the Swedish Cancer Society;
Swedish Children’s Cancer Foundation; Swedish Research
Council; Swedish Pain Relief Foundation.
Acknowledgements
Conflict of interest statement: None declared.
References
1. Hoeijmakers, J. H. (2001) Genome maintenance mechanisms for preventing cancer. Nature, 411, 366–374.
2. Svejstrup, J. Q. (2002) Mechanisms of transcription-coupled DNA repair.
Nat. Rev. Mol. Cell Biol., 3, 21–29.
3. Hanawalt, P. C. (2001) Controlling the efficiency of excision repair.
Mutat. Res., 485, 3–13.
4. Andreassen, P. R., Ho, G. P. and D’Andrea, A. D. (2006) DNA damage
responses and their many interactions with the replication fork.
Carcinogenesis, 27, 883–892.
208
5. Soulas-Sprauel, P., Rivera-Munoz, P., Malivert, L., Le Guyader, G.,
Abramowski, V., Revy, P. and de Villartay, J. P. (2007) V(D)J and
immunoglobulin class switch recombinations: a paradigm to study the
regulation of DNA end-joining. Oncogene, 26, 7780–7791.
6. Teng, G. and Papavasiliou, F. N. (2007) Immunoglobulin somatic hypermutation. Ann. Rev. Genet., 41, 107–120.
7. Aguilera, A. (2002) The connection between transcription and genomic
instability. EMBO J., 21, 195–201.
8. Helleday, T. (2003) Pathways for mitotic homologous recombination in
mammalian cells. Mutat. Res., 532, 103–115.
9. Helleday, T., Lo, J., van Gent, D. C. and Engelward, B. P. (2007) DNA
double-strand break repair: from mechanistic understanding to cancer
treatment. DNA Repair, 6, 923–935.
10. Pfeiffer, P., Goedecke, W. and Obe, G. (2000) Mechanisms of DNA
double-strand break repair and their potential to induce chromosomal
aberrations. Mutagenesis, 15, 289–302.
11. Michel, B., Flores, M. J., Viguera, E., Grompone, G., Seigneur, M. and
Bidnenko, V. (2001) Rescue of arrested replication forks by homologous
recombination. Proc. Natl Acad. Sci. USA, 98, 8181–8188.
12. Lundin, C., Erixon, K., Arnaudeau, C., Schultz, N., Jenssen, D.,
Meuth, M. and Helleday, T. (2002) Different roles for nonhomologous
end joining and homologous recombination following replication arrest in
mammalian cells. Mol. Cell. Biol., 22, 5869–5878.
13. Arnaudeau, C., Lundin, C. and Helleday, T. (2001) DNA double-strand
breaks associated with replication forks are predominantly repaired by
homologous recombination involving an exchange mechanism in
mammalian cells. J. Mol. Biol., 307, 1235–1245.
14. Saleh-Gohari, N., Bryant, H. E., Schultz, N., Parker, K. M., Cassel, T. N.
and Helleday, T. (2005) Spontaneous homologous recombination is
induced by collapsed replication forks that are caused by endogenous
DNA single-strand breaks. Mol. Cell. Biol., 25, 7158–7169.
15. Cox, M. M., Goodman, M. F., Kreuzer, K. N., Sherratt, D. J.,
Sandler, S. J. and Marians, K. J. (2000) The importance of repairing
stalled replication forks. Nature, 404, 37–41.
16. Sonoda, E., Sasaki, M. S., Buerstedde, J. M., Bezzubova, O., Shinohara, A.,
Ogawa, H., Takata, M., Yamaguchi-Iwai, Y. and Takeda, S. (1998) Rad51deficient vertebrate cells accumulate chromosomal breaks prior to cell
death. EMBO J., 17, 598–608.
17. Kraus, E., Leung, W. Y. and Haber, J. E. (2001) Break-induced
replication: a review and an example in budding yeast. Proc. Natl Acad.
Sci. USA, 98, 8255–8262.
18. Heller, R. C. and Marians, K. J. (2006) Replication fork reactivation
downstream of a blocked nascent leading strand. Nature, 439, 557–562.
19. McGlynn, P. and Lloyd, R. G. (2002) Recombinational repair and restart
of damaged replication forks. Nat. Rev. Mol. Cell Biol., 3, 859–870.
20. Keil, R. L. and Roeder, G. S. (1984) Cis-acting, recombination-stimulating
activity in a fragment of the ribosomal DNA of S. cerevisiae. Cell, 39, 377–386.
21. Stewart, S. E. and Roeder, G. S. (1989) Transcription by RNA polymerase
I stimulates mitotic recombination in Saccharomyces cerevisiae. Mol.
Cell. Biol., 9, 3464–3472.
22. Voelkel-Meiman, K., Keil, R. L. and Roeder, G. S. (1987) Recombinationstimulating sequences in yeast ribosomal DNA correspond to sequences
regulating transcription by RNA polymerase I. Cell, 48, 1071–1079.
23. Voelkel-Meiman, K. and Roeder, G. S. (1990) Gene conversion tracts
stimulated by HOT1-promoted transcription are long and continuous.
Genetics, 126, 851–867.
24. Voelkel-Meiman, K. and Roeder, G. S. (1990) A chromosome containing
HOT1 preferentially receives information during mitotic interchromosomal gene conversion. Genetics, 124, 561–572.
25. Grimm, C., Schaer, P., Munz, P. and Kohli, J. (1991) The strong ADH1
promoter stimulates mitotic and meiotic recombination at the ADE6 gene
of Schizosaccharomyces pombe. Mol. Cell. Biol., 11, 289–298.
26. Thomas, B. J. and Rothstein, R. (1989) Elevated recombination rates in
transcriptionally active DNA. Cell, 56, 619–630.
27. Saxe, D., Datta, A. and Jinks-Robertson, S. (2000) Stimulation of mitotic
recombination events by high levels of RNA polymerase II transcription
in yeast. Mol. Cell. Biol., 20, 5404–5414.
28. Klar, A. J., Strathern, J. N. and Hicks, J. B. (1981) A position-effect
control for gene transposition: state of expression of yeast mating-type
genes affects their ability to switch. Cell, 25, 517–524.
29. Weng, Y. S., Xing, D., Clikeman, J. A. and Nickoloff, J. A. (2000)
Transcriptional effects on double-strand break-induced gene conversion
tracts. Mutat. Res., 461, 119–132.
30. Chavez, S. and Aguilera, A. (1997) The yeast HPR1 gene has a functional
role in transcriptional elongation that uncovers a novel source of genome
instability. Genes Dev., 11, 3459–3470.
Transcription-associated recombination in eukaryotes
31. Gallardo, M. and Aguilera, A. (2001) A new hyperrecombination
mutation identifies a novel yeast gene, THP1, connecting transcription
elongation with mitotic recombination. Genetics, 157, 79–89.
32. Chavez, S., Beilharz, T., Rondon, A. G., Erdjument-Bromage, H.,
Tempst, P., Svejstrup, J. Q., Lithgow, T. and Aguilera, A. (2000)
A protein complex containing Tho2, Hpr1, Mft1 and a novel protein,
Thp2, connects transcription elongation with mitotic recombination in
Saccharomyces cerevisiae. EMBO J., 19, 5824–5834.
33. Jimeno, S., Rondon, A. G., Luna, R. and Aguilera, A. (2002) The yeast
THO complex and mRNA export factors link RNA metabolism with
transcription and genome instability. EMBO J., 21, 3526–3535.
34. Strasser, K., Masuda, S., Mason, P. et al. (2002) TREX is a conserved
complex coupling transcription with messenger RNA export. Nature, 417,
304–308.
35. Aguilera, A. (2005) Cotranscriptional mRNP assembly: from the DNA to
the nuclear pore. Curr. Opin. Cell Biol., 17, 242–250.
36. Zenklusen, D., Vinciguerra, P., Wyss, J. C. and Stutz, F. (2002) Stable
mRNP formation and export require cotranscriptional recruitment of the
mRNA export factors Yra1p and Sub2p by Hpr1p. Mol. Cell. Biol., 22,
8241–8253.
37. Rondon, A. G., Jimeno, S., Garcia-Rubio, M. and Aguilera, A. (2003)
Molecular evidence that the eukaryotic THO/TREX complex is required
for efficient transcription elongation. J. Biol. Chem., 278, 39037–39043.
38. Mason, P. B. and Struhl, K. (2005) Distinction and relationship between
elongation rate and processivity of RNA polymerase II in vivo. Mol.r Cell,
17, 831–840.
39. Garcia-Rubio, M., Chavez, S., Huertas, P., Tous, C., Jimeno, S., Luna, R.
and Aguilera, A. (2008) Different physiological relevance of yeast THO/
TREX subunits in gene expression and genome integrity. Mol. Genet.
Genomics, 279, 123–132.
40. Prado, F., Piruat, J. I. and Aguilera, A. (1997) Recombination between
DNA repeats in yeast hpr1delta cells is linked to transcription elongation.
EMBO J., 16, 2826–2835.
41. Fan, H. Y., Merker, R. J. and Klein, H. L. (2001) High-copy-number
expression of Sub2p, a member of the RNA helicase superfamily, suppresses
hpr1-mediated genomic instability. Mol. Cell. Biol., 21, 5459–5470.
42. Luna, R., Jimeno, S., Marin, M., Huertas, P., Garcia-Rubio, M. and
Aguilera, A. (2005) Interdependence between transcription and mRNP
processing and export, and its impact on genetic stability. Mol. Cell, 18,
711–722.
43. Nickoloff, J. A. and Reynolds, R. J. (1990) Transcription stimulates
homologous recombination in mammalian cells. Mol. Cell. Biol., 10,
4837–4845.
44. Nickoloff, J. A. (1992) Transcription enhances intrachromosomal homologous recombination in mammalian cells. Mol. Cell. Biol., 12, 5311–5318.
45. Gottipati, P., Cassel, T. N., Savolainen, L. and Helleday, T. (2008)
Transcription-associated recombination is dependent on replication in
mammalian cells. Mol. Cell. Biol., 28, 154–164.
46. Taghian, D. G. and Nickoloff, J. A. (1997) Chromosomal double-strand
breaks induce gene conversion at high frequency in mammalian cells.
Mol. Cell. Biol., 17, 6386–6393.
47. Allen, C., Miller, C. A. and Nickoloff, J. A. (2003) The mutagenic potential
of a single DNA double-strand break in a mammalian chromosome is not
influenced by transcription. DNA Repair, 2, 1147–1156.
48. Schildkraut, E., Miller, C. A. and Nickoloff, J. A. (2006) Transcription of
a donor enhances its use during double-strand break-induced gene
conversion in human cells. Mol. Cell. Biol., 26, 3098–3105.
49. Savolainen, L. and Helleday, T. (2008) Transcription-associated recombination is independent of XRCC2 and mechanistically separate from
homology-directed DNA double-strand break repair. Nucleic Acids Res.
doi: 10.1093/nar/gkn971.
50. Johnson, R. D., Liu, N. and Jasin, M. (1999) Mammalian XRCC2
promotes the repair of DNA double-strand breaks by homologous
recombination. Nature, 401, 397–399.
51. Takata, M., Sasaki, M. S., Tachiiri, S., Fukushima, T., Sonoda, E.,
Schild, D., Thompson, L. H. and Takeda, S. (2001) Chromosome
instability and defective recombinational repair in knockout mutants of the
five Rad51 paralogs. Mol. Cell. Biol., 21, 2858–2866.
52. Liu, Y., Masson, J. Y., Shah, R., O’Regan, P. and West, S. C. (2004)
RAD51C is required for Holliday junction processing in mammalian cells.
Science, 303, 243–246.
53. Liu, J., Meng, X. and Shen, Z. (2002) Association of human RAD52
protein with transcription factors. Biochem. Biophys. Res. Commun., 297,
1191–1196.
54. Kraakman-van der Zwet, M., Overkamp, W. J., van Lange, R. E. et al.
(2002) Brca2 (XRCC11) deficiency results in radioresistant DNA
synthesis and a higher frequency of spontaneous deletions. Mol. Cell.
Biol., 22, 669–679.
55. Davies, A. A., Masson, J. Y., McIlwraith, M. J., Stasiak, A. Z.,
Stasiak, A., Venkitaraman, A. R. and West, S. C. (2001) Role of BRCA2
in control of the RAD51 recombination and DNA repair protein. Mol.
Cell, 7, 273–282.
56. Alt, F. W., Blackwell, T. K. and Yancopoulos, G. D. (1987) Development
of the primary antibody repertoire. Science, 238, 1079–1087.
57. Bassing, C. H., Swat, W. and Alt, F. W. (2002) The mechanism and
regulation of chromosomal V(D)J recombination. Cell, 109 (Suppl.),
S45–S55.
58. Gellert, M. (2002) V(D)J recombination: RAG proteins, repair factors, and
regulation. Ann. Rev. Biochem., 71, 101–132.
59. Chaudhuri, J. and Alt, F. W. (2004) Class-switch recombination: interplay
of transcription, DNA deamination and DNA repair. Nat. Rev., 4,
541–552.
60. Li, Z., Woo, C. J., Iglesias-Ussel, M. D., Ronai, D. and Scharff, M. D.
(2004) The generation of antibody diversity through somatic hypermutation and class switch recombination. Genes Dev., 18, 1–11.
61. Neuberger, M. S., Di Noia, J. M., Beale, R. C., Williams, G. T., Yang, Z.
and Rada, C. (2005) Somatic hypermutation at A.T pairs: polymerase
error versus dUTP incorporation. Nat. Rev., 5, 171–178.
62. Blackwell, T. K., Moore, M. W., Yancopoulos, G. D., Suh, H.,
Lutzker, S., Selsing, E. and Alt, F. W. (1986) Recombination between
immunoglobulin variable region gene segments is enhanced by transcription. Nature, 324, 585–589.
63. Jung, S., Rajewsky, K. and Radbruch, A. (1993) Shutdown of class switch
recombination by deletion of a switch region control element. Science,
259, 984–987.
64. Peters, A. and Storb, U. (1996) Somatic hypermutation of immunoglobulin genes is linked to transcription initiation. Immunity, 4, 57–65.
65. Sikes, M. L., Meade, A., Tripathi, R., Krangel, M. S. and Oltz, E. M.
(2002) Regulation of V(D)J recombination: a dominant role for promoter
positioning in gene segment accessibility. Proc. Natl Acad. Sci. USA, 99,
12309–12314.
66. Alt, F. W., Blackwell, T. K., DePinho, R. A., Reth, M. G. and
Yancopoulos, G. D. (1986) Regulation of genome rearrangement events
during lymphocyte differentiation. Immunol. Rev., 89, 5–30.
67. Jacobs, H. and Bross, L. (2001) Towards an understanding of somatic
hypermutation. Curr. Opin. Immunol., 13, 208–218.
68. Bottaro, A., Lansford, R., Xu, L., Zhang, J., Rothman, P. and Alt, F. W.
(1994) S region transcription per se promotes basal IgE class switch
recombination but additional factors regulate the efficiency of the process.
EMBO J., 13, 665–674.
69. Leung, H. and Maizels, N. (1992) Transcriptional regulatory elements
stimulate recombination in extrachromosomal substrates carrying immunoglobulin switch-region sequences. Proc. Natl Acad. Sci. USA, 89,
4154–4158.
70. Xu, L., Gorham, B., Li, S. C., Bottaro, A., Alt, F. W. and Rothman, P.
(1993) Replacement of germ-line epsilon promoter by gene targeting
alters control of immunoglobulin heavy chain class switching. Proc. Natl
Acad. Sci. USA, 90, 3705–3709.
71. Ikeda, H. and Matsumoto, T. (1979) Transcription promotes recAindependent recombination mediated by DNA-dependent RNA polymerase in Escherichia coli. Proc. Natl Acad. Sci USA, 76, 4571–4575.
72. White, M. A., Detloff, P., Strand, M. and Petes, T. D. (1992) A promoter
deletion reduces the rate of mitotic, but not meiotic, recombination at the
HIS4 locus in yeast. Curr. Genet., 21, 109–116.
73. Garcia-Rubio, M., Huertas, P., Gonzalez-Barrera, S. and Aguilera, A.
(2003) Recombinogenic effects of DNA-damaging agents are synergistically increased by transcription in Saccharomyces cerevisiae. New
insights into transcription-associated recombination. Genetics, 165,
457–466.
74. Deng, W. P. and Nickoloff, J. A. (1994) Preferential repair of UV damage
in highly transcribed DNA diminishes UV-induced intrachromosomal
recombination in mammalian cells. Mol. Cell. Biol., 14, 391–399.
75. Bhattacharyya, N. P., Maher, V. M. and McCormick, J. J. (1990) Effect of
nucleotide excision repair in human cells on intrachromosomal homologous recombination induced by UV and 1-nitrosopyrene. Mol. Cell. Biol.,
10, 3945–3951.
76. Kodadek, T. (1998) Mechanistic parallels between DNA replication,
recombination and transcription. Trends Biochem. Sci., 23, 79–83.
77. Svejstrup, J. Q., Vichi, P. and Egly, J. M. (1996) The multiple roles of
transcription/repair factor TFIIH. Trends Biochem. Sci., 21, 346–350.
78. Hoogstraten, D., Nigg, A. L., Heath, H., Mullenders, L. H., van Driel, R.,
Hoeijmakers, J. H., Vermeulen, W. and Houtsmuller, A. B. (2002) Rapid
209
P. Gottipati and T. Helleday
switching of TFIIH between RNA polymerase I and II transcription and
DNA repair in vivo. Mol. Cell, 10, 1163–1174.
79. West, S. C. (2003) Molecular views of recombination proteins and their
control. Nat. Rev. Mol. Cell Biol., 4, 435–445.
80. Thomas, B. J. and Rothstein, R. (1989) The genetic control of directrepeat recombination in Saccharomyces: the effect of rad52 and rad1 on
mitotic recombination at GAL10, a transcriptionally regulated gene.
Genetics, 123, 725–738.
81. Scully, R., Anderson, S. F., Chao, D. M., Wei, W., Ye, L., Young, R. A.,
Livingston, D. M. and Parvin, J. D. (1997) BRCA1 is a component of the
RNA polymerase II holoenzyme. Proc. Natl Acad. Sci. USA, 94, 5605–5610.
82. Chang, M., French-Cornay, D., Fan, H. Y., Klein, H., Denis, C. L. and
Jaehning, J. A. (1999) A complex containing RNA polymerase II, Paf1p,
Cdc73p, Hpr1p, and Ccr4p plays a role in protein kinase C signaling. Mol.
Cell. Biol., 19, 1056–1067.
83. Le Page, F., Randrianarison, V., Marot, D., Cabannes, J., Perricaudet, M.,
Feunteun, J. and Sarasin, A. (2000) BRCA1 and BRCA2 are necessary for
the transcription-coupled repair of the oxidative 8-oxoguanine lesion in
human cells. Cancer Res., 60, 5548–5552.
84. Aboussekhra, A. and Al-Sharif, I. S. (2005) Homologous recombination is
involved in transcription-coupled repair of UV damage in Saccharomyces
cerevisiae. EMBO J., 24, 1999–2010.
85. Wang, J. C. (1985) DNA topoisomerases. Ann. Rev. Biochem., 54,
665–697.
86. Orphanides, G. and Reinberg, D. (2000) RNA polymerase II elongation
through chromatin. Nature, 407, 471–475.
87. Shen, X., Mizuguchi, G., Hamiche, A. and Wu, C. (2000) A chromatin
remodelling complex involved in transcription and DNA processing.
Nature, 406, 541–544.
88. Tsukuda, T., Fleming, A. B., Nickoloff, J. A. and Osley, M. A. (2005)
Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature, 438, 379–383.
89. Gonzalez-Barrera, S., Garcia-Rubio, M. and Aguilera, A. (2002)
Transcription and double-strand breaks induce similar mitotic recombination events in Saccharomyces cerevisiae. Genetics, 162, 603–614.
90. Saleh-Gohari, N. and Helleday, T. (2004) Conservative homologous
recombination preferentially repairs DNA double-strand breaks in the
S phase of the cell cycle in human cells. Nucleic Acids Res., 32, 3683–3688.
91. Huertas, P. and Aguilera, A. (2003) Cotranscriptionally formed
DNA:RNA hybrids mediate transcription elongation impairment and
transcription-associated recombination. Mol. Cell, 12, 711–721.
92. Li, X. and Manley, J. L. (2005) Inactivation of the SR protein splicing
factor ASF/SF2 results in genomic instability. Cell, 122, 365–378.
93. Reaban, M. E. and Griffin, J. A. (1990) Induction of RNA-stabilized DNA
conformers by transcription of an immunoglobulin switch region. Nature,
348, 342–344.
94. Daniels, G. A. and Lieber, M. R. (1995) RNA:DNA complex formation
upon transcription of immunoglobulin switch regions: implications for the
mechanism and regulation of class switch recombination. Nucleic Acids
Res., 23, 5006–5011.
95. Mizuta, R., Iwai, K., Shigeno, M., Mizuta, M., Uemura, T., Ushiki, T. and
Kitamura, D. (2003) Molecular visualization of immunoglobulin switch
region RNA/DNA complex by atomic force microscope. J. Biol. Chem.,
278, 4431–4434.
96. Yu, K., Chedin, F., Hsieh, C. L., Wilson, T. E. and Lieber, M. R. (2003)
R-loops at immunoglobulin class switch regions in the chromosomes of
stimulated B cells. Nat. Immunol., 4, 442–451.
97. Takeuchi, Y., Horiuchi, T. and Kobayashi, T. (2003) Transcriptiondependent recombination and the role of fork collision in yeast rDNA.
Genes Dev., 17, 1497–1506.
98. Deshpande, A. M. and Newlon, C. S. (1996) DNA replication fork pause
sites dependent on transcription. Science, 272, 1030–1033.
99. Brewer, B. J. and Fangman, W. L. (1988) A replication fork barrier at the
3# end of yeast ribosomal RNA genes. Cell, 55, 637–643.
100. Mayan-Santos, M. D., Martinez-Robles, M. L., Hernandez, P.,
Schvartzman, J. B. and Krimer, D. B. (2008) A redundancy of processes
that cause replication fork stalling enhances recombination at two distinct
sites in yeast rDNA. Mol. Microbiol., 69, 361–375.
101. Dalgaard, J. Z. and Klar, A. J. (1999) Orientation of DNA replication
establishes mating-type switching pattern in S. pombe. Nature, 400,
181–184.
102. Labib, K. and Hodgson, B. (2007) Replication fork barriers: pausing for
a break or stalling for time? EMBO Rep., 8, 346–353.
103. Ahn, J. S., Osman, F. and Whitby, M. C. (2005) Replication fork blockage
by RTS1 at an ectopic site promotes recombination in fission yeast.
EMBO J., 24, 2011–2023.
210
104. Lambert, S., Watson, A., Sheedy, D. M., Martin, B. and Carr, A. M.
(2005) Gross chromosomal rearrangements and elevated recombination at
an inducible site-specific replication fork barrier. Cell, 121, 689–702.
105. Prado, F. and Aguilera, A. (2005) Impairment of replication fork
progression mediates RNA polII transcription-associated recombination.
EMBO J., 24, 1267–1276.
106. Wellinger, R. E., Prado, F. and Aguilera, A. (2006) Replication fork
progression is impaired by transcription in hyperrecombinant yeast cells
lacking a functional THO complex. Mol. Cell. Biol., 26, 3327–3334.
107. Santos-Rosa, H. and Aguilera, A. (1994) Increase in incidence of
chromosome instability and non-conservative recombination between
repeats in Saccharomyces cerevisiae hpr1 delta strains. Mol. Gen. Genet.,
245, 224–236.
108. Huertas, P., Garcia-Rubio, M. L., Wellinger, R. E., Luna, R. and
Aguilera, A. (2006) An hpr1 point mutation that impairs transcription and
mRNP biogenesis without increasing recombination. Mol. Cell. Biol., 26,
7451–7465.
109. Liu, N., Lamerdin, J. E., Tebbs, R. S. et al. (1998) XRCC2 and XRCC3,
new human Rad51-family members, promote chromosome stability and
protect against DNA cross-links and other damages. Mol. Cell, 1,
783–793.
110. Deans, B., Griffin, C. S., Maconochie, M. and Thacker, J. (2000) Xrcc2 is
required for genetic stability, embryonic neurogenesis and viability in
mice. EMBO J., 19, 6675–6685.
111. O’Regan, P., Wilson, C., Townsend, S. and Thacker, J. (2001) XRCC2 is
a nuclear RAD51-like protein required for damage-dependent RAD51
focus formation without the need for ATP binding. J. Biol. Chem., 276,
22148–22153.
112. Liu, N. and Lim, C. S. (2005) Differential roles of XRCC2 in homologous
recombinational repair of stalled replication forks. J. Cell. Biochem., 95,
942–954.
113. Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y. and
Honjo, T. (2000) Class switch recombination and hypermutation require
activation-induced cytidine deaminase (AID), a potential RNA editing
enzyme. Cell, 102, 553–563.
114. Gomez-Gonzalez, B. and Aguilera, A. (2007) Activation-induced cytidine
deaminase action is strongly stimulated by mutations of the THO
complex. Proc. Natl Acad. Sci. USA, 104, 8409–8414.
115. Shen, H. M. (2007) Activation-induced cytidine deaminase acts on
double-strand breaks in vitro. Mol. Immunol., 44, 974–983.
Received on September 9, 2008; revised on December 8, 2008;
accepted on December 10, 2008