A Microhomology-Mediated Break-Induced Replication Model for the Origin of Human Copy Number Variation

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DOI: 10.1371/journal.pgen.1000327 · Source: PubMed
Abstract
Chromosome structural changes with nonrecurrent endpoints associated with genomic disorders offer windows into the mechanism of origin of copy number variation (CNV). A recent report of nonrecurrent duplications associated with Pelizaeus-Merzbacher disease identified three distinctive characteristics. First, the majority of events can be seen to be complex, showing discontinuous duplications mixed with deletions, inverted duplications, and triplications. Second, junctions at endpoints show microhomology of 2-5 base pairs (bp). Third, endpoints occur near pre-existing low copy repeats (LCRs). Using these observations and evidence from DNA repair in other organisms, we derive a model of microhomology-mediated break-induced replication (MMBIR) for the origin of CNV and, ultimately, of LCRs. We propose that breakage of replication forks in stressed cells that are deficient in homologous recombination induces an aberrant repair process with features of break-induced replication (BIR). Under these circumstances, single-strand 3' tails from broken replication forks will anneal with microhomology on any single-stranded DNA nearby, priming low-processivity polymerization with multiple template switches generating complex rearrangements, and eventual re-establishment of processive replication.
Review
A Microhomology-Mediated Break-Induced Replication
Model for the Origin of Human Copy Number Variation
P. J. Hastings
1
*, Grzegorz Ira
1
, James R. Lupski
1,2,3
1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America, 2 Department of Pediatrics, Baylor College of
Medicine, Houston, Texas, United States of America, 3 Texas Children’s Hospital, Houston, Texas, United States of America
Abstract: Chromosome structural changes with nonre-
current endpoints associated with genomic disorders
offer windows into the mechanism of origin of copy
number variation (CNV). A recent report of nonrecurrent
duplications associated with Pelizaeus-Merzbacher dis-
ease identified three distinctive characteristics. First, the
majority of events can be seen to be complex, showing
discontinuous duplications mixed with deletions, inverted
duplications, and triplications. Second, junctions at end-
points show microhomology of 2–5 base pairs (bp). Third,
endpoints occur near pre-existing low copy repeats
(LCRs). Using these observations and evidence from
DNA repair in other organisms, we derive a model of
microhomology-mediated break-induced replication
(MMBIR) for the origin of CNV and, ultimately, of LCRs.
We propose that breakage of replication forks in stressed
cells that are deficient in homologous recombination
induces an aberrant repair process with features of break-
induced replication (BIR). Under these circumstances,
single-strand 39 tails from broken replication forks will
anneal with microhomology on any single-stranded DNA
nearby, p riming low-processivity polymerization with
multiple template switches generating complex rear-
rangements, and eventual re-establishment of processive
replication.
Introduction
In the past few years, we have learnt that a major component of
the differences between individuals is variation in the number of
copies of segments of the genome, and of genes included in these
segments (copy number variation or CNV) (for definition of
abbreviations, see Table 1). A considerable portion of the genome
is involved in CNV [1–11]—with estimates of up to 12% [4]—
which can arise meiotically and also somatically as shown by the
finding that identical twins can differ in CNV [12]. CNV has been
a significant component of primate evolution [13–16]. Here we
draw on evidence on the mechanism of DNA transactions in
Escherichia coli, yeast, Drosophila, mammals, and human cancer to
derive a model for the origin of CNV based on the mechanism of
BIR occurring at sites of microhomology (microhomology-
mediated BIR or MMBIR).
Genomic Disorders
Although we can see that considerable variation in copy
number is tolerated or is advantageous to its carrier, some genes
are dosage-sensitive, and duplication or deletion involving these
genes gives rise to human clinical phenotypes collectively referred
to as genomic disorders [17]. This has allowed the ascertainment
of structural changes and thus the study of the origin of CNV. For
recurrent rearrangements, much CNV stems from homologous
recombination between segments that already occur as two or
more copies. When this happens, sequences that lie between the
repeats that recombine will be either duplicated or deleted, thus
changing the copy number. This process is referred to as nonallelic
homologous recombination, or NAHR [18]. The repeated
sequences that recombine might occasionally be highly repetitive
sequences that occur widely in the human genome [19] but are
usually sequences that occur only twice or a few times (i.e., low-
copy repeats, LCRs, or segmental duplications, SDs). The LCRs
tend to occur in clusters in highly complex regions of the genome.
These repeated segments might be short (about 10 kilobases (kb)),
or up to several hundreds of kb in length, and they occur in either
orientation. Some examples of genomic complex regions are
shown in Figure 1.
The endpoints of CNVs that arose by NAHR occur in a few
positions where there is sufficient homology for homologous
recombination. Although many genomic disorders arise by NAHR
[20], some rearrangements have endpoints in many different
positions. These CNVs arose de novo by rearrangements at sites
that lack extensive homology. Recent evidence on the distribution
of nonpathological CNVs in two individuals suggests that most
differences in copy number from the reference sequence arose by
nonrecurrent events [2]. Thus nonrecurrent chromosomal chang-
es arise quite frequently [21]. Because the nonrecurrent events
presumably reflect the origin of most genome complexity, the
study of them is important to the understanding of genomic
disorders, genetic variability due to CNV, and human evolution.
Pelizaeus-Merzbacher disease (PMD; Online Mendelian Inher-
itance in Man (OMIM) accession code 312080; http://www.ncbi.
nlm.nih.gov/omim/) is a recessive X-linked genomic disorder
affecting the central nervous system that arises by nonrecurrent
chromosomal changes. The changes involve duplication, triplica-
tion, or deletion of the PLP1 gene. The clinical phenotype allows
identification of individuals showing nonrecurrent chromosomal
changes in the PLP region. In a study of the structural variation in
the genomes of patients with PMD, Lee et al. [22] describe some
aspects of the fine structure of newly arising CNVs with
nonrecurrent endpoints and report three striking properties of
Citation: Hastings PJ, Ira G, Lupski JR (2009) A Microhomology-Mediated Break-
Induced Replication Model for the Origin of Human Copy Number Variation. PLoS
Genet 5(1): e1000327. doi:10.1371/journal.pgen.1000327
Published January 30, 2009
Copyright: ß 2009 Hastings et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Funding: This work was supported by grants from the National Institute s of
Health; R01 GM64022 to PJH and R01 GM80600 to GI.
* E-mail: hastings@bcm.tmc.edu
Editor: Ivan Matic, Universite
´
Paris Descartes, INSERM U571, France
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their structure that help us to understand the origin of CNVs.
First, the authors report that the novel junctions form at sites of
microhomology, i.e., lengths of homology 2 to 5 nucleotides long
that are too short to support homologous recombination. Such
junctions have been reported previously in cases of nonrecurrent
endpoints of deletions and duplications [19,23,24]. Second, they
observed that the new structures are complex, showing duplication
and deletion interspersed with nonduplicated or with triplicated
lengths, and showing duplicated segments in either orientation.
These characteristics were reported previously [25–31]. Third,
although these events did not arise by NAHR, the novel junctions
tend to occur in close proximity to LCRs [32–34]. Figures 2 and 3
illustrate examples of these complex non-recurrent events.
Nonrecurrent rearrangements had previously been attributed to
a mechanism of nonhomologous end-joining (NHEJ)
[19,20,24,33]. However, the characteristics of microhomology
junctions and structural complexity in these new structures, as
revealed by nucleotide sequencing and high-resolution array
comparative genomic hybridization, led Lee et al. [22] to propose
that the rearrangements arose through a replication-based
mechanism termed FoSTeS (fork stalling and template switching),
a mechanism proposed previously for amplification in E. coli [35].
Replication-based models have also been proposed to explain the
origin of gross chromosomal rearrangements seen in a low
proportion of patients with cystic fibrosis and hemophilia A.
Analysis of deletions of the genes involved reveals complex
structures similar to those described for PLP1 [28,29,31].
Genome Rearrangements in Cancer
The amount of structural variation in cancer cells is sometimes
so extreme [36] that it is not possible to determine which changes
occurred within the same event. However, it can be seen that
duplications are often discontinuous, and junction regions include
insertions of nearby, unlinked, and unknown sequences, and
deletions and inversions [37], showing that rearrangement events
in cancer cells are complex. Many studies report microhomology
at junctions of a large proportion of the structural variation (e. g.,
[37–39]). Studies of translocation endpoints in leukemia and other
cancers find that many junctions have microhomology and are
associated with insertions and deletions of various lengths [40–42].
These observations are compatible with at least some of the
genomic instability seen in tumor formation and progression
having stemmed from the same underlying mechanism as the
formation of nonrecurrent duplications in genomic disorders.
Involvement of Replication in Chromosomal
Structural Change
In the Lac assay system in E. coli [43], amplification of the lac
operon to 20–100 copies occurs in response to the stress of
starvation [44,45]. The novel junctions of the amplified segments
(amplicons) show that endpoints occurred at sites of microhomol-
ogy of 2–15 bp [35,46]. Some of the amplicons are complex,
containing both direct and inverted repeats. Many others cannot
Table 1. Abbreviations Used in the Text.
Abbreviation Meaning
BIR Break-induced replication, a recombination-based mechanism for restarting broken replication forks.
CNV Copy number variation, variation within a population of the number of copies of a gene or length of genome.
DSB Double-strand break, a break in both strands of a DNA molecule.
FoSTeS Fork stalling and template switching, a replicative mechanism for changing chromosome structure.
LCR Low copy repeat, a length of genome that occurs twice or a few times.
MMBIR Microhomology-mediated break-induc ed replication, a replication-based mechanism of recombination between sequences with very little
base identity, proposed here.
NAHR Nonallelic homologous recombination, homologous recombination occurring between low copy repeats.
NHEJ Nonhomologous end joining, a mechanism for repair of DNA double-strand breaks that does not require homology.
SD Segmental duplication, a repetition of a length of genome.
doi:10.1371/journal.pgen.1000327.t001
Figure 1. In silico analyses revealed complex genomic architecture in regions of nonrecurrent rearrangement. (A) The ,3Mb
surrounding the PLP1 gene and (B) the ,4 Mb surrounding the MECP2 gene on the X chromosome contain numerous LCRs in various orientations
[33,106]. LCRs are represented by the colored block arrows, and like LCR copies are designated by color and letter for a given sequence. Orientation is
depicted by the direction of the block arrow.
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be identified by outward-facing polymerase chain reaction (an
observation also encountered frequently for PLP1 duplication
junction analysis [22]), which would reveal the junctions of simple
tandem repeats, and so are presumed to be complex, rather than
simple tandem repeats [35,46,47]. By these criteria, about 25% of
amplicons are complex. Thus, with respect to microhomology and
complexity, the chromosomal structural changes in this system
resemble those found in nonrecurrent events in human genomic
disorders.
Homologous recombination requires RecA protein (Rad51 in
eukaryotes) (reviewed in [48]). Microhomology-mediated deletion
formation in E. coli (less than 25 nucleotides of homology) has long
been known to be RecA-independent [49–52]. RecA-independent
short homology-mediated deletions (25–50 nucleotides) have
previously been attributed to template switching within a
replication fork during DNA replication (reviewed in [53]). The
evidence for this is, first, that mutations in genes encoding
replication functions affect the formation of these events; second,
that mutations affecting post-replicational mismatch repair affect
them, placing the event very near to the replication fork; third,
that mutation of 39 exonucleases has an effect that is consistent
with the ends being used to prime DNA synthesis; and fourth, that
it is very difficult to obtain mutations affecting the process by
transposon mutagenesis, suggesting essential functions.
In the E. coli Lac system, study of genetic requirements of stress-
induced amplification has revealed some details of the mechanism.
First, the events involve 39 DNA ends. This is seen by an increase in
amplification when a 39 exonuclease gene (xonA) is deleted, and a
decrease when the 39 exonuclease is over-expressed. Similar
manipulation of 59-exonuclease has no effect [35]. This suggests
that amplification results from free 39 ends in the cell most of which
are normally removed by exonuclease. As above, the involvement of
39 ends but not 59 ends is consistent with priming of DNA synthesis.
Second, lagging-strand processing at replication forks is
implicated by a requirement for the 59 exonuclease domain of
DNA polymerase I (Pol I) [35,45]. Pol I is involved in lagging-
Figure 2. Complex rearrangements involving
PLP1
detected by junction analysis (A) and oligonucleotide array comparative
genomic hybridization analysis (B) [22]. (A) A complex duplication of the PLP1 region detected by outward facing polymerase chain reaction.
Panel (i) shows the PLP1 region with the positions of the outward facing primers. The structure of the duplicated region is shown in (ii), with an
enlargement of the complex junction region in (iii). Two or three bp of microhomology, shown by the letters A, C, G and T, were found at the
breakpoint junctions (open arrows). (B) Deletion and duplications found in two patients with Pelizaeus-Merzbacher disease and their carrier mother
[24], shown by comparative genomic hybridization. A ,190-kb deletion is followed by a ,9-kb segment with no copy-number change, and an
interrupted ,190-kb duplication was detected (i). Panel (ii) shows enlargement of the array revealing interruption of the ,190-kb duplication. In
each horizontal yellow box above, blue lines represent an average of the data points. Red data points indicate copy-number gains, green data points
indicate losses, and black data points indicate no copy-number change. The y-axes show relative hybridization; genomic position is on the x-axis.
Panel (iii) summarizes the structure based on comparative genomic hybridization where a green box shows the region deleted, red boxes show the
regions duplicated, and black lines show regions of no change.
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strand replication, base excision repair, and nucleotide excision
repair, but these excision repair processes are not involved in
amplification [35], so lagging strands at replication forks are
implicated in amplification.
Third, there is a requirement for the proteins of double-strand
break (DSB) repair by homologous recombination [35] (the
RecBC system, reviewed in [48]). That this is actually a
requirement for DSB repair (not just the proteins) is shown by
the discovery that in vivo double-strand cleavage of DNA near lac
enhances amplification rates [54].
Taken together, these observations suggest a model for
amplification in the Lac system in E. coli in which replication is
restarted at sites of repair of DNA double-strand ends [35]. The
hypothesis proposed was that template switching occurs during
replication restart at stalled replication forks. Because the distances
involved exceed the lengths that are expected to be exposed as
single-stranded at a single replication fork, it was proposed that the
switches occurred between different replication forks [35].
The idea that chromosomal structural changes originate from
DNA replication has received support from a study of micro-
homology-mediated SD formation in yeast [55]. These authors
support the idea that the mechanism of SD formation involves
replication by showing that its frequency is enhanced by treatment
with camptothecin and is dependent on Pol32, a component of
Pold (discussed below). Camptothecin is a topoisomerase I
inhibitor that leaves nicks in DNA. These nicks are believed to
become collapsed forks when a replication fork reaches them.
Thus, increasing the frequency of fork collapse increases the
frequency of duplication formation. These authors also report that
situations that lead to fork stalling rather than collapse have little
effect on the frequency of duplication formation [55]. Thus, it
appears that the substrate for duplication is a single double-strand
end at a collapsed replication fork.
This long-distance template-switch model was also used by Lee
et al. [22] to explain the observations of nonrecurrent chromo-
somal changes seen in Pelizaeus-Merzbacher disease discussed
above and the juxtaposition of multiple genomic sequences
normally separated by large genomic distances [22,56]. Experi-
ments on the integration of nonhomologous DNA into mamma-
lian cells revealed microhomology junctions and insertion of
sequence from other parts of the genome at the junctions. These
observations were interpreted in terms of a similar model of
repeated copying and switching to another template [57].
Break-Induced Replication
A more specific model for restarting replication at collapsed
(broken) replication forks, BIR [58], has been developed for yeast,
and a similar mechanism was proposed to explain telomere
maintenance in yeast and human cell lines that have lost
telomerase activity (reviewed in [59]). Recent evidence [60,61]
suggests that the BIR mechanism can be modified to explain the
complexity of chromosomal structural changes described above for
human and E. coli. Figure 4 illustrates the mechanism of BIR.
When the replicative helicase encounters a nick on the template
strand (Figure 4A), one arm of a replication fork breaks off
(Figure 4B). There is no second end to be involved in the
mechanisms of DSB repair that are available at a DSB consisting
of two double-strand ends: homologous recombination or
nonhomologous end-joining. The 59 end of the broken arm is
resected by an exonuclease to leave a 39 overhang (Figure 4C).
This 39 tail invades a homologous sequence, normally the sister
chromatid from which it came. This invasion is mediated by
RecA/Rad51 protein (Figure 4D). The 39 end primes DNA
synthesis and establishes a replication fork consisting of both
leading and lagging strand synthesis [61] (Figure 4E). This
replication is of low processivity, and the extended arm is
separated from the sister chromatid (Figure 4E). Such separation
might be achieved by migration of the Holliday junction shown in
Figure 4D and 4E. The 39 end reinvades and the process is
repeated (Figure 4G and 4H). After a few cycles of invasion,
extension, and separation, the replication fork becomes more
processive, and replication continues to the end of the chromo-
some arm or to the end of the replicon. The change from low
processivity to highly processive replication can be attributed to a
switch in the DNA polymerases involved [61]. Initial extension
from a double-strand end was shown to require the primase
Figure 3. Complex genomic rearrangements at
PLP1
seen in patients with Pelizaeus-Merzbacher disease, illustrating long-range as
well as short-range complexity. Duplications are shown in red, deletions in green, triplications in blue, and no copy number change in black. The
figure is not drawn to scale. Approximate positions are given relative to PLP1.
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complex and Pold, notably the nonessential Pol32 subunit,
whereas the more processive Pole was required for the 30-kb
extension to the telomere. Figure 4I shows the completed pair of
chromatids with the new material segregating conservatively as
suggested for E. coli [62]. This would result if the Holliday junction
followed the replication fork. Another possibility is that the
Holliday junction is resolved so that there will be semi-
conservative segregation of old and new DNA strands [60],
(reviewed in [63]). Evidence for conservative segregation of new
DNA strands in BIR, suggesting that the Holliday junction was not
resolved, was reported for E. coli [62].
The repeated extension and separation have been interpreted as
repeated attempts to find the other side of a break consisting of two
double-strand ends. When, eventually, none is found because this
is a collapsed fork rather than a two ended DSB, the remainder of
the chromosome is replaced by replication [60,63]. The pattern of
repeated rounds of template switching followed by a long length of
replication is supported by observations of BIR in yeast. BIR can
be induced experimentally by transforming a chromosomal
fragment into a yeast cell [64]. Using such a system, Smith et al.
[60] placed a chromosomal fragment with a centromere and one
telomere-forming sequence into a diploid yeast cell. The fragment
had homology to both homologues of chromosome III. These
homologues were differentially marked. Selection for a marker on
the fragment selected for cells in which the fragment had acquired
a second telomere. These authors found that most fragments had
completed the replication of 50 kb to the end of the chromosome
to which the fragment had homology. The striking result was that
many of the chromosomes recovered had switched from one
homologue to the other. In some cases, more than one switch was
seen. The switches were confined to the first 10 kb, after which a
single homologue was copied. In a few percent of cases, the switch
was to a different chromosome at sites of repeated homology
consisting of the long terminal repeat of a retrotransposon. Thus,
BIR was demonstrated to produce complexity of the sorts reported
above for E. coli amplification and for nonrecurrent end-points in
human genomic disorders.
BIR has been suggested as the mechanism that underlies SD
and other structural changes in yeast, e.g., [55,65,66], and human,
e.g., [31,67]. As discussed below, BIR is strongly RecA/Rad51-
dependent and homology-dependent, and so cannot account for
the observations of microhomology associated with complex
rearrangements without substantial change.
Microhomology-Mediated BIR (MMBIR)
BIR, as described above, is usually an accurate process, because
the repeated invasions are RecA/Rad51-mediated and involve long
lengths of homology between DNA sequences. Invasion catalyzed
by RecA/Rad51 requires extensive homology of about 50 bp in E.
coli [68] and more in eukaryotes [69,70]. This does not fit with the
microhomology junctions described above. We therefore suggest
that in these systems, replication forks are reestablished in a RecA/
Rad51-independent manner. Rad51-independent BIR occurs in
yeast at a much lower efficiency than the Rad51-dependent BIR
[71,72], though its frequency is very much enhanced, at the expense
of fidelity, by the presence of unusual structures such as an inverted
repeat [71]. However, telomere recombination in the absence of
telomerase is proficient in the absence of Rad51 and is mediated by
very short homologies [73,74] (reviewed in [59]). The fact that
telomere recombination occurs by BIR is supported by the finding
that it requires the same set of enzymes as BIR that is initiated in the
middle of a chromosome [61]. Absence or shortage of RecA/Rad51
might arise because the cells are stressed, as described below. That
microhomology-mediated SD formation occurs in yeast by a BIR
mechanism is supported by the finding that, like homology-
mediated BIR [61], it requires Pol32 [55].
In mammalian cells, there is a surprisingly efficient micro-
homology-mediated DSB repair pathway. Most, if not all,
experimental research on microhomology-mediated DSB repair
has been performed with nuclease-induced breaks. This recently
Figure 4. Repair of a collapsed replication fork by BIR. When a replication fork encounters a nick in a template strand (A) (arrowhead), one arm
of the fork breaks off (red), producing a collapsed fork (B). At the single double-strand end, the 59 strand is resected, giving a 39 overhang (C). The 39
single-strand end invades the sister molecule (blue), forming a D-loop (D), which subsequently becomes a replication fork with both leading and
lagging strand replication (E). There is a Holliday junction at the site of the D-loop. Migration of the Holliday junction, or some other helicase activity,
separates the extended double-strand end from its templates (F). The separated end is again processed to give a 39 single-strand end, which again
invades the sister, and forms a replication fork (G). Eventually the replication fork becomes fully processive, and continues replication to the
chromosome end (H and I). This process is shown here with the Holliday junction following the fork so that newly formed strands are segregated
together (conservative segregation) (H). Each line represents a DNA nucleotide chain (strand). Polarity is indicated by half arrows on 39 end. New DNA
synthesis is shown by dashed lines. The publications on which this model is based are cited in the text.
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described pathway was characterized in recombination events
induced by I-SceI or RAG1/RAG2 nucleases in cells deficient in
classical NHEJ and in cancer cells [75,76]. Nucleases generate
two-ended breaks at random with respect to ongoing replication
forks. However, BIR acts under circumstances when DSB repair,
including NHEJ, is not an option, because after replication fork
breakage, there is only a single end with no second end to which
the one end can be annealed or ligated. Spontaneous damage to
DNA occurs predominantly during replication [77–79], so that
mechanisms that repair single DNA ends are more appropriately
invoked for spontaneous damage than are mechanisms that act on
two-ended DSBs. We suggest that a novel pathway, microhomol-
ogy-mediated BIR (MMBIR), is used to repair single double-
strand ends when stretches of single-stranded DNA are available
and share microhomology with the 39 single-strand end from the
collapsed fork.
Single-stranded DNA might be expected to occur in replication
forks, from stalled transcription complexes, at excision repair
tracts, or at secondary structures in DNA such as cruciforms or
hairpins caused by complex genomic architecture, and possibly in
other situations such as in promoter regions and replication
origins. The dimensions of most of the template switches discussed
here (tens to hundreds of kb distant, i.e., the length of a duplication
or deletion) preclude mechanisms of replication slippage within a
single replication fork. An ability of any single-stranded DNA
region that shares microhomology with the single-stranded 39 end
to take part in the events would explain why MMBIR is inexact
and liable to lead to chromosomal structural changes. Very short
homology should not be a barrier to replication fork restart
because polymerase eta, used in DSB repair in vertebrates [80,81],
is efficient in initiating new DNA synthesis from mismatched
primers, and even primers as short as 2–3 bp [82].
The presence of inverted repeats could generate hairpin loops
that expose single-stranded sequence [22,32]. In addition, hairpin
structures might increase the likelihood of replication fork stalling,
which might then initiate BIR. Such major roles for secondary
DNA structures in the generation of chromosomal structural
changes offers an explanation for the clustering of structural
changes, producing complex chromosomal regions such as that
illustrated in Figure 1. The model of MMBIR is presented in
Figure 5.
The clear distinction between NHEJ and BIR mediated by
microhomology is that, in the second instance, microhomology
junctions are followed by shorter or longer stretches of DNA
sequence derived from elsewhere. Ten to 20% of nonhomologous
junctions in mammalian cells have sequence inserted at the
junction [83]. Some events that had previously been interpreted as
occurring by an NHEJ mechanism might have occurred by
MMBIR with a single template switch. In addition, events that
appeared to be simple end-joining events might have had
complexity that was not revealed by the techniques in use.
Control of BIR and MMBIR
A major question remains—why do cells use microhomology-
and not homology-driven repair? The likely answer is that Rad51
is not available or is in short supply. This might be caused by stress
responses. Evidence supporting this comes from cancer research.
Hypoxia in the tumor microenvironment is correlated with genetic
instability [84,85] (reviewed in [86]). It has been shown that
hypoxia leads to repression of RAD51 and BRCA1 [87,88] and to
reduced homologous recombination [87,89] (reviewed in [89,90]).
This has been interpreted as a switch from high-fidelity
homologous recombination to lower fidelity NHEJ caused by
stress [87,88]. At collapsed replication forks, where NHEJ is not
possible, we suggest that down-regulation of RAD51 prevents BIR
from following the Rad51- homology-dependent BIR route but
still allows a Rad51-independent BIR route that requires very
much less homology, as observed in telomere recombination in
budding yeast [73,74]. If Rad51 is down-regulated but not absent,
a condition might prevail in which some homologous invasion is
allowed, but not enough to prevent some illegitimate events
occurring, as was witnessed in Drosophila with reduced gene dosage
of Rad51 [91]. We do not know whether the error-prone nature of
this repair is aided by down-regulation of mismatch repair, which
has also been reported for stressed cancer cells [92,93]. There
might be other changes in gene expression under stress that
promote genomic instability (e.g., [94]).
A similar switch from high fidelity to low-fidelity DSB repair is
seen in E. coli in response to the stress of starvation [54]. Similarly
the microhomology-mediated amplification seen in the Lac system
in E. coli discussed above is induced by stress, as evidenced by the
observation that the event occurred after the beginning of
starvation [44], and by the finding that adaptive amplification in
this system requires the starvation and general stress response
transcriptional activator RpoS [95].
The mechanism of MMBIR, as described above, features
annealing of single-stranded DNA with minimal homology. Hence
the enzyme responsible for this has a central role in the proposed
mechanism. We suggest that annealing is catalyzed by Rad52.
Rad52 is essential for the single-strand annealing reaction that
Figure 5. MMBIR. The figure shows successive switches to different
genomic positions (distinguished by color) forming microhomology
junctions (arrows). For clarity, the nature of the single-stranded regions
of annealing is not defined (see text). (A) shows the broken arm of a
collapsed replication fork, which forms a new low-processivity fork as
shown at (B). The extended end dissociates repeatedly ((C and E) shown
with 59-ends resected) and reforms the fork on different templates (D
and F). In (F), the switch returns to the original sister chromatid (blue),
forming a processive replication fork that completes replication. (G)
shows the final product containing sequence from different genomic
regions. Each line represents a DNA nucleotide chain (strand). Polarity is
indicated by half arrows on 39 end. Whether the return to the sister
chromatid occurs in front of or behind the position of the original
collapse determines whether there is a deletion or duplication (see
Table 2).
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PLoS Genetics | www.plosgenetics.org 6 January 2009 | Volume 5 | Issue 1 | e1000327
deletes sequence between direct repeats [96], and it anneals single
strands in vitro [97]. Chromosomal rearrangements in yeast that
have microhomology at the junctions have been seen to occur in
the absence of Rad51, but they require Rad52 [42,66,98]. In one
of these cases, frequent switches were associated with micro-
homology junctions in a Rad51-independent, Rad52-dependent
process that produced translocations and inversions at sites of
highly diverged genes [66]. These authors proposed that these
events occurred by template switching during BIR [66]. In vitro,
Rad51 inhibits the single-strand annealing activity of Rad52 [99],
suggesting that the absence of Rad51 might exercise tight control
of the switch from strand invasion to annealing of single strands.
However, the formation of microhomology-mediated Rad51-
independent SDs in yeast was found to be Rad52-independent
[55]. Rad52 is also not required for microhomology-mediated
end-joining [100]. These observations show that microhomology
junction formation can be mediated by a different protein in yeast,
as well as by Rad52.
In summary, we are suggesting that, because stress induces a
reduction in the amount of Rad51 available, while leaving Rad52
unchanged, the amount of homologous interaction that is used for
repair is reduced, leaving annealing of single DNA strands as the
main mechanism available for the repair of collapsed replication
forks. Thus, classical BIR will be reduced, and MMBIR will be
substituted.
Long-Range Discontinuities in Duplications
The idea that there is a cell-wide physiological condition that
favors nonhomologous interactions has further implications. If a
condition prevails that allows one such event, it is possible that
further nonhomologous events will occur in the same cell. The
possibility of multiple rounds of events was suggested for a yeast
system to correct for an inversion that would produce a dicentric
chromosome [66]. We also note that, in human duplications, there
are discontinuities (short regions that are not duplicated) and
triplicated regions within duplications on a scale of hundreds of kb
or Mb apart (Figures 2 and 3). These long-distance interruptions
are not readily explained by template switching during the early
stages of a single BIR event, where switching occurs after one
template is copied for hundreds of bp to a few kb (Figure 2 and
[60]), but rather suggest that more than one BIR event occurred
along the same chromosome. MMBIR requires, in addition to a
cell-wide stress response, a specific DNA structure: a single double-
strand end. To explain why single double-strand ends should
occur serially along the same chromosome, we propose that the
Holliday junction formed during BIR follows the replication fork,
as we have suggested above as the mechanism of separation of the
extended broken end. If the replication fork formed by BIR stalls
for any reason, the Holliday junction might then process through
the fork, separating the newly synthesized DNA from its template,
and so generating a collapsed fork anew (as in Figure 4E and 4F)
and leading to the long range discontinuities seen in duplicated
segments, as illustrated in Figure 3.
Chromosome Structural Consequences of MMBI R
The ways in which MMBIR would lead to the various
chromosomal structural changes are summarized in Table 2.
Translocations would be formed by a switch to a different
chromosome. Duplication would occur when the switch was to
either the sister or the homologue behind the position at which the
fork collapsed (with respect to the direction of movement of the
fork). Deletion happens when there is a switch to a position ahead of
the fork collapse. A switch to a sequence that has already been
duplicated, behind the end of the duplicated sequence, would
produce a triplication. Switching to the same molecule behind the
position of fork collapse has the potential to initiate rolling-circle
replication and consequent amplification. Switching to either the
sister molecule or the homologue in inverted orientation would give
an inverted chromosomal segment. If long-distance replication
follows, this might form a dicentric chromosome, so that this would
have to be followed by a second inversion to allow a cell to be viable.
This need for a second switch has led to the idea that there might be
more than one round of switching events involved in the formation
of some structural changes [66] as discussed above. Alternatively, a
second inverted template switch within a single series of switches
would restore a viable chromosomal structure.
Implications of the Model
We suggest that the replicative mechanism described here
contributes to genomic disorders that show nonrecurrent end-
points, contributes to much of the chromosomal structural
instability that occurs somatically in cancer formation and tumor
progression and also to the origin of the genomic constitutional
structural complexity that underlies NAHR genomic disorders,
and is a driving force in evolution. We offer evidence from diverse
organisms that such a mechanism exists, and suggest that the
model offers directions for future research that will further
elucidate the molecular details.
The mechanism of MMBIR affects human biology at many
levels. First, at the cellular level, the mechanism might apply to the
events underlying much cancer formation and progression. Second,
at the organismal level, we propose that MMBIR acting in the
germline will give rise to CNV, and the accompanying genomic
disorders and chromosomal syndromes. At the same time MMBIR
could create LCRs that provides the homology required for NAHR,
leading to genomic disorders in future generations. Third, at the
species level, we suggest that complex genomic regions generate
secondary structures that increase the likelihood of MMBIR, so that
complex architecture becomes more complex on an evolutionary
timescale, as has been documented for primate evolution [13,16].
We suggest that MMBIR might underlie genomic rearrangements
and CNV associated with the emergence of primate-specific traits
[10,13,101]. Furthermore, MMBIR provides material on which
natural selection and evolution operate: variation in copy number
might change the expression levels of included genes and also
provide redundant copies of genes that could then be mutated and
changed to encode new functions [102–104]. Further, the formation
of nonhomologous junctions might shuffle exons of different genes
to attain new functions (F. Zhang and J. Lupski, unpublished
observations). Indeed, these regions of complex genomic architec-
Table 2. Chromosomal Consequences of Template Switches
during MMBIR.
When Switch Is to: Consequence:
Sister or homologue behind position of fork breakage Duplication
Sister or homologue ahead of position of fork breakage Deletion
Sister or homologue in wrong orientation Inversion
Nonhomologous sequence Translocation
Sequence already duplicated Triplication
On same molecule behind the break Rolling circle
doi:10.1371/journal.pgen.1000327.t002
PLoS Genetics | www.plosgenetics.org 7 January 2009 | Volume 5 | Issue 1 | e1000327
ture have been referred to as gene nurseries, i.e., regions in which
new genes are formed [13,14].
The MMBIR model predicts that complex genomic rearrange-
ments will often be accompanied by extensive loss of heterozy-
gosity and, in some cases, by loss of imprinting, because the
chromosome that is copied might be either the sister or the
homologue. Such loss of heterozygosity could lead to regional
uniparental disomy [105] as a potential novel mechanism for
disease. We also predict that the events described here will be seen
in model systems under conditions where the cells are stressed, and
study of DNA repair activities in stressed cells might be a fertile
field for investigation.
Acknowledgements
We are grateful to Drs. J.A. Lee, S.M. Rosenberg, and J.H. Wilson for
assistance and comment.
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PLoS Genetics | www.plosgenetics.org 9 January 2009 | Volume 5 | Issue 1 | e1000327
    • "The number of rearrangements induced by microhomologydriven pathways is likely to be higher than is currently thought [54] . The outcomes of these currently underappreciated repair pathways could include an increased copy number of the sequences being repaired [56] . Consequently , these mechanisms may be a significant contributor to the formation of heterochromatic blocks. "
    [Show abstract] [Hide abstract] ABSTRACT: Background A prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained. Results Using a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences. Conclusion The large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2667-5) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "In this model, microhomology-induced template switching occurs where nearby single-stranded DNA is used as template to repair DNA breaks. Depending on the template , this results in the formation of deletions, duplications, triplications inversions or translocations that are flanked by minimal sequence homology of 2–6 bp at the breakpoints [45]. Further complexity at the genomic rearrangement breakpoints, involving small deletions and/ or small insertions of unlinked or unknown sequences, are also commonly observed and is likely due to multiple template-switching events occurring during the repair process [49]. "
    [Show abstract] [Hide abstract] ABSTRACT: With the advent of whole exome sequencing, cases where no pathogenic coding mutations can be found are increasingly being observed in many diseases. In two large, distantly-related families that mapped to the Charcot-Marie-Tooth neuropathy CMTX3 locus at chromosome Xq26.3-q27.3, all coding mutations were excluded. Using whole genome sequencing we found a large DNA interchromosomal insertion within the CMTX3 locus. The 78 kb insertion originates from chromosome 8q24.3, segregates fully with the disease in the two families, and is absent from the general population as well as 627 neurologically normal chromosomes from in-house controls. Large insertions into chromosome Xq27.1 are known to cause a range of diseases and this is the first neuropathy phenotype caused by an interchromosomal insertion at this locus. The CMTX3 insertion represents an understudied pathogenic structural variation mechanism for inherited peripheral neuropathies. Our finding highlights the importance of considering all structural variation types when studying unsolved inherited peripheral neuropathy cases with no pathogenic coding mutations.
    Full-text · Article · Jul 2016
    • "As a result, BRCA1/ 2-deficient cells are hypersensitive to chemotherapeutic agents, such as PARP inhibitors (PARPis) (Bryant et al., 2005; Farmer et al., 2005; Konstantinopoulos et al., 2015), and to replication stress-inducing poisons (Howlett et al., 2005). In BRCA1/2-deficient cells, unstable replication forks lead to chromosomal translocation and copy number variation (Hastings et al., 2009). Although genomic instability is critical to tumor progression, its excess can limit cell survival (Bartkova et al., 2005; Negrini et al., 2010 ). "
    [Show abstract] [Hide abstract] ABSTRACT: BRCA1/2 proteins function in homologous recombination (HR)-mediated DNA repair and cooperate with Fanconi anemia (FA) proteins to maintain genomic integrity through replication fork stabilization. Loss of BRCA1/2 proteins results in DNA repair deficiency and replicative stress, leading to genomic instability and enhanced sensitivity to DNA-damaging agents. Recent studies have shown that BRCA1/2-deficient tumors upregulate Polθ-mediated alternative end-joining (alt-EJ) repair as a survival mechanism. Whether other mechanisms maintain genomic integrity upon loss of BRCA1/2 proteins is currently unknown. Here we show that BRCA1/2-deficient tumors also upregulate FANCD2 activity. FANCD2 is required for fork protection and fork restart in BRCA1/2-deficient tumors. Moreover, FANCD2 promotes Polθ recruitment at sites of damage and alt-EJ repair. Finally, loss of FANCD2 in BRCA1/2-deficient tumors enhances cell death. These results reveal a synthetic lethal relationship between FANCD2 and BRCA1/2, and they identify FANCD2 as a central player orchestrating DNA repair pathway choice at the replication fork.
    Full-text · Article · Jun 2016
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