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Reviews
Bacterial artificial chromosomes and herpesvirus genomics
20 Eggleston, A.K. and West, S.C. (1996) Exchanging partners:
recombination in E. coli. Trends Genet. 12, 20–26
21 O’Connor, M. et al. (1989) Construction of large DNA segments
in Escherichia coli. Science 244, 1307–1312
22 Kempkes, B. et al. (1995) Immortalization of human B
lymphocytes by a plasmid containing 71 kilobase pairs of
Epstein–Barr virus DNA. J. Virol. 69, 231–238
23 Yang, X.W. et al. (1997) Homologous recombination based
modification in Escherichia coli and germline transmission in
transgenic mice of a bacterial artificial chromosome. Nat.
Biotechnol. 15, 859–865
24 Brune, W. et al. (1999) Rapid identification of essential and
nonessential herpesvirus genes by direct transposon
mutagenesis. Nat. Biotechnol. 17, 360–364
25 Zhang, Y. et al. (1998) A new logic for DNA engineering using
recombination in Escherichia coli. Nat. Genet. 20, 123–128
26 Muyrers, J.P. et al. (1999) Rapid modification of bacterial
artificial chromosomes by ET-recombination. Nucleic Acids
Res. 27, 1555–1557
27 Narayanan, K. et al. (1999) Efficient and precise engineering
of a 200 kb beta-globin human/bacterial artificial
chromosome in E. coli DH10B using an inducible homologous
recombination system. Gene Ther. 6, 442–447
28 Berg, C.M. et al. (1989) Transposable elements and the
genetic engineering of bacteria. In Mobile DNA (Berg, D.E. and
Howe, M., eds), pp. 879–925, American Society for
Microbiology, Washington, DC, USA
29 Chatterjee, P.K. and Coren, J.S. (1997) Isolating large nested
deletions in bacterial and P1 artificial chromosomes by in vivo
P1 packaging of products of Cre-catalysed recombination
between the endogenous and a transposed loxP site. Nucleic
Acids Res. 25, 2205–2212
30 Chatterjee, P.K. et al. (1999) Direct sequencing of bacterial
and P1 artificial chromosome-nested deletions for identifying
position-specific single-nucleotide polymorphisms. Proc. Natl.
Acad. Sci. U. S. A. 96, 13276–13281
31 Luckow, V.A. et al. (1993) Efficient generation of infectious
recombinant baculoviruses by site-specific transposonmediated insertion of foreign genes into a baculovirus genome
propagated in Escherichia coli. J. Virol. 67, 4566–4579
32 Fink, D.J. and Glorioso, J.C. (1997) Engineering herpes simplex
virus vectors for gene transfer to neurons. Nat. Med. 3, 357–359
33 Sclimenti, C.R. and Calos, M.P. (1998) Epstein–Barr virus
vectors for gene expression and transfer. Curr. Opin.
Biotechnol. 9, 476–479
34 Messerle, M. et al. Cytomegalovirus BACs – a new herpesvirus
vector approach. Adv. Virus Res. (in press)
35 Banerjee, S. et al. (1995) Therapeutic gene delivery in human
B-lymphoblastoid cells by engineered non-transforming
infectious Epstein–Barr virus. Nat. Med. 1, 1303–1308
36 Sun, T.Q. et al. (1996) Human artificial episomal
chromosomes for cloning large DNA fragments in human cells.
Nat. Genet. 8, 33–41
37 Sun, T.Q. et al. (1996) Engineering a mini-herpesvirus as a
general strategy to transduce up to 180 kb of functional selfreplicating human mini-chromosomes. Gene Ther. 3,
1081–1088
38 Wang, S. and Vos, J.M. (1996) A hybrid herpesvirus infectious
vector based on Epstein–Barr virus and herpes simplex virus
type 1 for gene transfer into human cells in vitro and in vivo.
J. Virol. 70, 8422–8430
39 Suter, M. et al. (1999) BAC-VAC, a novel generation of (DNA)
vaccines: a bacterial artificial chromosome (BAC) containing
a replication-competent, packaging-defective virus genome
induces protective immunity against herpes simplex virus 1.
Proc. Natl. Acad. Sci. U. S. A. 96, 12697–12702
Partners and pathways
repairing a double-strand break
Double-strand chromosome breaks can arise in a number of ways, by ionizing radiation, by spontaneous
chromosome breaks during DNA replication, or by the programmed action of endonucleases, such as in
meiosis. Broken chromosomes can be repaired either by one of several homologous recombination
mechanisms, or by a number of nonhomologous repair processes. Many of these pathways compete actively for
the repair of a double-strand break. Which of these repair pathways is used appears to be regulated
developmentally, genetically and during the cell cycle.
roken chromosomes pose a serious threat to cell survival. The presence of an unrepaired double-strand
break (DSB) will trigger the DNA-damage response systems of a cell to arrest its progression through the cell
cycle and, sometimes, to cause apoptotic cell death. But
even if a cell with an unrepaired DSB continues to divide,
the broken chromosome fragments will mis-segregate and
be degraded, producing aneuploidy.
In response to this threat, cells have elaborated an
impressive arsenal of DNA-repair pathways. There are
two general types of repair: homologous recombination
(HR) and nonhomologous end-joining (NHEJ). These two
processes are in competition with each other and one
focus of this review is to examine the way that this competition is regulated. But the cell’s options are far more complex than simply electing to employ HR or NHEJ. There
are several types of homologous repair: gene conversion,
break-induced replication and single-strand annealing
(reviewed in Ref. 1). Similarly, there are also several alternative end-joining mechanisms2,3. Moreover, even once a
process such as gene conversion is initiated, there are
additional genetically regulated decisions in choosing
among alternative homologous templates to carry out
repair. How does the cell decide to use a template on a sister
B
0168-9525/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0168-9525(00)02022-9
chromatid, on a homologous chromosome or at an ectopic
site? Moreover, how are these choices tied to the cell’s
DNA damage-sensing checkpoints?
This review surveys the impressive recent progress in
delineating the different mechanisms of homologous and
nonhomologous repair and the way in which they all compete in repairing DSBs. The emphasis will be on what has
been learned in the best-studied organism, Saccharomyces
cerevisiae, but I also discuss studies in other model eukaryotic systems and in mammalian cells.
Homologous recombination mechanisms
The three major types of HR all begin in the same way, as
the ends of the DSB are resected by 59 to 39 exonucleases
or by a helicase coupled to an endonuclease, to produce
long, 39-ended single-stranded DNA tails (Fig. 1).
James E. Haber
[email protected]
Single-strand annealing
In the simplest process (Fig. 1d), resection exposes complementary regions of homologous sequences originally
flanking the DSB, creating a deletion by single-strand
annealing (SSA). SSA will occur with as little as 30 bp of
homology, although it is much more efficient with
200–400 bp (Ref. 44).
TIG June 2000, volume 16, No. 6
Rosentiel Basic Medical
Sciences Research
Center, MS 029 Brandeis
University, 415 South
Street, Waltham, MA
02454-9110, USA.
259
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DNA double-stranded break repair
FIGURE 1. Alternative outcomes of the homologous recombinational repair of a DSB
5′ to 3′ resection
Strand invasion
New DNA synthesis
(a)
Replication fork
capture
(c)
Continued
DNA synthesis
Holliday junction
resolution
Break-induced replication
No crossing-over
(b)
Crossing-over
Conservative
inheritance of
DNA strands
(d)
Continued
5′ to 3′ resection
Strand annealing
No crossing-over
Deletion
trends in Genetics
After a double-strand break (DSB) is created, the ends are resected and one of the 39 single-stranded ends can invade an intact template. Strand invasion requires
the participation of the Rad51p strand exchange protein and a number of associated proteins, including Rad52p, Rad54p and Tid1p, discussed in this review. During
meiosis, these recombination proteins are joined by a second strand-exchange protein, Dmc1p. Strand invasion is believed to establish a modified replication fork, in
which both leading and lagging-strand DNA synthesis occurs43. As new DNA synthesis proceeds, branch migration displaces the two newly synthesized strands. (a) If
the replication fork encounters the other end of the DSB, an intermediate containing two Holliday junctions can be formed, allowing gene conversions to be resolved
both with and without crossing-over. (b) If the strands are completely displaced or if the leading strand pairs with the second end of the DSB, a simple synthesisdependent strand annealing (SDSA) will occur, producing gene conversions without crossing-over. (c) If the second end of the DSB fails to engage, replication can
proceed all the way to the end of the chromosome (or until it encounters a converging replication fork). This process is known as break-induced replication (BIR). (d)
If resection proceeds far enough to expose complementary strands of homologous sequences flanking a DSB (shown as black boxes), repair can occur by single-strand
annealing (SSA), leading to a deletion of all intervening sequences.
Gene conversion
SSA is in competition with gene conversion (Fig. 1a), during which the two resected ends of the DSB invade and
copy sequences from a homologous template located on a
sister chromatid, a homologous chromosome, or at an
ectopic location. The molecular mechanism by which gene
conversion occurs is not precisely known; recent evidence
has prompted several suggested revisions of the DSB gaprepair mechanism proposed by Szostak et al.4 (these vari260
TIG June 2000, volume 16, No. 6
ations are reviewed in Ref. 1). The mechanism shown in
Fig. 1 is termed synthesis-dependent strand annealing
(SDSA).
It would seem that gene conversion is the most conservative and non-mutagenic process of DNA repair, but surprisingly, in S. cerevisiae, SSA out-competes gene conversion in mitotic cells even when the donor and recipient are
intrachromosomal5,6, apparently because the resection of
DNA ends is not restrained. It is possible that one reason
Reviews
DNA double-stranded break repair
why SSA is less evident in meiotic cells is that 59 to 39
resection is down-regulated or impaired.
Break-induced replication
Under certain circumstances only one end of a DSB might
be able to engage in homologous recombination, for
example in haploid or hemizygous chromosomes of G1
diploids, where there is no homologous chromosome.
Sequences near the centromere-proximal side of the DSB
might be able to find homologous sequences elsewhere in
the genome and create a nonreciprocal translocation by a
process known as recombination-dependent DNA replication or break-induced replication (BIR; Fig. 1c)7. This
same circumstance appears to occur when chromosomes
lack telomerase, the enzyme that normally maintains the
short, repeated sequences at telomeres that protect ends
from fusions and other types of recombination8. Even
when there is homology on both sides of a DSB, BIR
(using only one of the ends to initiate recombination)
appears to be in competition with gene conversion9. This
mechanism also accounts for extensive DNA replication
found in many gene targeting events10.
Nonhomologous recombination
DSB ends can be repaired by several nonhomologous
repair mechanisms in which the DNA ends are joined with
little or no base-pairing at the junction2,3. End joining in
yeast and in mammals requires the same core set of proteins: the DNA end-binding proteins Ku70p and Ku80p,
as well as DNA ligase IV and its associated Xrcc4 protein.
Vertebrate cells also require DNA-PKcs, for which no
homologue has been demonstrated in fungi. In budding
yeast there is also a requirement for Rad50p, Mre11p and
Xrs2p, three proteins that have endo- and exonuclease
activity (reviewed in Ref. 11) although there is also a
Mre11-independent pathway of chromosome rearrangements12; but in fission yeast13 and vertebrate cells14 the
absence of Mre11p has little effect.
When the ends of a DSB are created by a nuclease that
leaves 4-bp overhanging, complementary ends, yeast cells
readily promote their religation by NHEJ, effectively competing with homologous recombinational alternatives15
(Fig. 2). However, when the ends of DNA are not complementary, NHEJ in budding yeast is much less efficient,
succeeding in only ~2 in 1000 cells. This is one of the distinctive differences between yeast and mammalian cells,
which can efficiently join ends of all types by NHEJ
(Ref. 3). The basis of this important difference is not
known, but it seems to account in part for why mammalian gene targeting is much more likely to result in mistargeted insertions. There is also likely to be a Ku- and
Rad52-independent end-joining process as well, as some
deletions are recovered after DSBs are created in yku70D
and rad52D cells. No genes have yet been identified to
define this pathway.
Competition between homologous and
nonhomologous recombination: inferences from
gene targeting and other types of repair
Based on gene-targeting experiments, it appears that mammalian cells are much less adept in accomplishing accurate
integration of homologous sequences than is budding
yeast. However, several recent observations argue that S.
cerevisiae is much more capable of nonhomologous
recombination than we had suspected, and that vertebrate
FIGURE 2. Nonhomologous end-joining
Religation
AACA
TTGT
AACA
TTGT
A
T
AACAACA
TTGTTGT
AACACA
TTGTGT
Misalignment
Fill-in
Deletion
trends in Genetics
Nonhomologous end-joining requires the participation of the DNA end-binding
proteins Ku70p and Ku80p and a specialized DNA ligase IV with its associated
Xrcc4 protein. In mammalian cells, NHEJ also requires the Ku-associated DNAPKCS, but no homologue of this protein has been found in yeast. In Saccharomyces
cerevisiae, most NHEJ processes also require the Mre11p–Rad50p–Xrs2p
complex, but their homologues in Schizosaccharomyces pombe or in vertebrate
cells do not seem to have a key role. An example is shown in the precise ligation
and imprecise end-joining of DNA cleaved by the S. cerevisiae site-specific HO
endonuclease. S. cerevisiae efficiently joins sequences with complementary 59 or
39 overhanging ends, but only inefficiently joins unmatched DNA ends. The
junctions of deletions and insertions usually have one or a few bp of
microhomology. New DNA synthesis is shown in grey. In budding yeast, the
insertion pathway is seen only in S and/or G2 cells, whereas the much less
efficient deletion pathway (which also does not seem to require
Mre11p–Rad50p–Xrs2p) is found at all stages of the cell cycle. Mammalian cells
join any DNA ends together efficiently, often creating junctions with 1–5 bp of
base pairing (with the removal of the un-paired flaps of the single-stranded
ends).
cells are far more accomplished in carrying out homologous recombination. In mammalian cells, approximately 30–50% of breaks created by the site-specific
I-SceI endonuclease can be repaired by homologous
recombination, with the rest repaired by NHEJ (Ref. 16).
Similarly, in yeast, when a DSB is made on a chromosome
with HO endonuclease, about 30% of the cells simply religate the DSB while the remaining cells engage in a homologous gene conversion to repair the break15.
Very little is known in general about the way cells elect
homologous or nonhomologous recombination. Such
knowledge might provide a way to improve mammalian
gene targeting. Two DNA end-binding complexes, Ku70pKu80p and a multimer of Rad52p, might act as ‘gatekeepers’ to control access to NHEJ or HR, respectively17. A
remarkable discovery that supports this idea is that the
level of the Ku proteins is dramatically lower in mouse
cells that are undergoing meiosis18. It is presumed that
mouse, like yeast, initiates recombination with hundreds
of DSBs created by the Spo11 protein, and it would be
highly disadvantageous if these DSBs were repaired by
imprecise NHEJ.
Yeast has provided additional insights into choice
between HR and NHEJ. The largest effects are associated
with expression of yeast’s mating-type locus. Haploid
cells express either MATa or MATa alleles, whereas
TIG June 2000, volume 16, No. 6
261
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DNA double-stranded break repair
FIGURE 3. Alternative homologous partners with which
homologous recombination can occur
Allelic
Sister
Ectopic
trends in Genetics
In meiosis, sister-chromatid recombination is apparently suppressed by the presence of protein
structures (axial elements of the synaptonemal complex) and by the action of a meiosis-specific
recombination complex (Dmc1p–Tid1p) that direct recombination to non-sister homologous sequences.
Partner choice is also changed by mutations in genes that are involved in the DNA-damage checkpoint
pathway, leading to an increased use of ectopic and sister sequences over allelic sites.
diploids are normally MATa/MATa. MATa/MATa
diploids are more radiation-resistant and are significantly
more adept in repairing an HO-induced DSB than
diploids expressing only one MAT allele15,19. NHEJ is also
under mating-type control15,20; both haploid and diploid
cells expressing one MAT allele are much more proficient
in NHEJ than cells expressing both MATa and MATa.
These differences make sense: haploid strains in G1 can
only repair DSBs by NHEJ, whereas diploids always have a
homologous partner with which to repair a DSB and hence
might be expected to down-regulate NHEJ. The search is
on for the genes controlled by mating type that carry out
this regulation.
Another key question is whether a cell irrevocably commits the DNA ends towards one repair pathway. For example, when a haploid G1 cell suffers a DSB, the ends do
not have homologous sequences with which to repair the
damage, and hence repair can only occur by NHEJ. But as
DNA ends are resected, flanking homologous sequences
can become exposed, allowing SSA to occur, sometimes as
long as several hours later5. By that time, has the cell given
up trying to complete NHEJ, or are all solutions being
tried out at the same time?
Choosing a template for gene conversion in
mitotic cells: a sister chromatid or a homologue?
As noted above, gene conversion, BIR and SSA all compete with each other; but even if we consider only the
process of gene conversion, the cell still confronts important choices. In a diploid cell, after DNA replication, a
DSB can be repaired either from a sister chromatid or
from a homologous sequence located either in an allelic or
ectopic location (Fig. 3). There is increasing evidence that
262
TIG June 2000, volume 16, No. 6
the choice between inter-homologue recombination and
sister-chromatid exchange is strongly regulated.
The selection of a partner for HR is particularly crucial
during meiosis. Recombination is initiated by DSBs, but if
repair occurs between sister chromatids, there will be no
inter-homologue crossing-over, which is essential for
proper chromosome segregation. Thus, meiotic cells have
evolved mechanisms to ensure that most recombination
events will occur between non-sister chromatids21, apparently the opposite of what occurs in mitotic cells, where
most DSBs are likely to occur during replication and the
most conservative choice of a partner is the sister chromatid22. There seem to be two major components involved
in this process, the induction of a meiosis-specific recombination protein and the meiosis-specific formation of
proteinaceous axial elements between sister chromatids
(reviewed in Ref. 23).
The new recombination protein, Dmc1p, is found in all
meiotic cells, from yeast to humans, in addition to its
homologue, the Rad51p strand-exchange protein, which
is also present in mitotic cells. Two-dimensional gel analysis shows that a dmc1 mutant strain fails to produce the
joint molecule (JM) intermediates either formed between
homologous sister- or between non-sister chromatids,
whereas the absence of Rad51p particularly reduces interhomologue exchanges21. Interestingly, the defects caused
by dmc1 are partially suppressed by a red1 deletion that
prevents normal axial element formation21. red1 dmc1
double mutants can form JMs with normal kinetics, but
they are almost exclusively derived from sister-chromatid
exchanges, apparently by allowing Rad51p to work on
substrates from which it is somehow normally excluded.
Recently, Thompson et al.24 showed that red1, mek1
and hop1 mutations that prevent normal axial element
formation all increase sister-chromatid recombination.
Another pair of proteins involved in this decision are
Rad54p and its homologue Tid1p (Rdh54p). Tid1p interacts strongly with Dmc1p and less so with Rad51p; conversely, Rad54p strongly interacts with Rad51p but apparently not with Dmc1p (Ref. 25). These proteins also play
important roles in mitotic cells. Rad54p seems to be critical
in sister-chromatid repair, whereas the absence of Tid1p has
no effect26. Conversely, the absence of Tid1p reduces interhomologue mitotic recombination but does not affect intrachromosomal or inter-sister recombination27,28. Taken
together, these data argue that normally Dmc1p and Tid1p
direct DSB repair towards non-sisters, whereas Red1p
prevents sister-chromatid exchange that would be mediated
by Rad51p and Rad54p. The absence both of Red1p and of
Dmc1p permits DSB repair from sisters (presumably carried
out by Rad51p and Rad54p) and improves spore viability.
Allelic and ectopic partners in mitosis
In some instances, the cell must make a further decision,
whether to recombine with an allelic partner or with homologous sequences located at ectopic sites. Some sequences
are found in multiple copies, dispersed throughout the
genome and crossovers between such non-allelic sites can
lead to chromosome rearrangements. One way to restrict
ectopic interactions would be to make the lengths of
homology needed for a successful encounter greater than
the size of the dispersed elements. This might be the case
for small sequences such as Alu sequences in humans or
delta sequences in yeast, but recombination between
larger transposable elements do not appear to be limited
Reviews
DNA double-stranded break repair
by this constraint29,30. Recently Inbar and Kupiec31 presented evidence that yeast will forgo recombining with a
homologous partner that shares a few hundred bp of
homology located at the very ends of a DSB in favor of
recombining with a second, competing locus that shares
greater homology, but further away from the DSB ends.
This could explain how recombination between short, dispersed sequences might be avoided and why allelic events
(where the homology stretches out to the ends of the chromosome) would be favored over ectopic ones.
Burgess and colleagues32,33 have used fluorescent in situ
hybridization and site-specific recombination to examine
allelic and ectopic recombination in mitotic yeast cells.
Their results reveal several layers of interaction. First,
homologous chromosomes show frequent, but transient
pairing at most times in the cell cycle. Second, because
centromeres cluster, two sequences equally distant from
their respective centromeres are more likely to recombine
than those at different distances. Finally, for three of the
four sites analysed there was a fourfold advantage to being
in allelic, rather than ectopic, positions.
Studies of HR initiated by endogenous nucleases have
not found much constraint on the ability of broken chromosomes to search the entire genome for homologous
partners. When cells were given a choice between repairing simultaneous DSBs on two different chromosomes,
either by a pair of intrachromosomal deletions or by two
interchromosomal translocations, the two outcomes
occurred at equal frequencies34. Even though mammalian
chromosomes are much larger and homology searches
must be more difficult, quite similar results have been
found in mouse cells. Expressing the site-specific I-SceI
endonuclease to create DSBs in mouse cells, Jasin and her
colleagues have shown that ectopic recombination is, at
best , eightfold less frequent than allelic events35. It is possible that in these cases, the presence of DSB damage
induces a checkpoint gene-mediated response that causes
the disruption of the normal localization of sequences and
promotes a search of the entire genome for partners.
In meiosis, allelic recombination is about five times
more frequent than ectopic events36. Moreover, when the
ectopic events are between different locations on two
homologous chromosomes, the advantage of allelic over
ectopic recombination increases as the two sequences are
moved further apart, leading to the suggestion that homologous chromosomes are already loosely aligned before
the initiation of recombination that will cause their true
synapsis. Homologues are associated even in cells that
cannot recombine37. The advantage of allelic over ectopic
events can also be explained if the recombination event
under study occurs later than another recombination
event on the same chromosome that would bring the rest
of the sequences closer together.
Efficient meiotic ectopic recombination is not simply a
feature of fungi, whose small chromosomes might not be
as organized as those in mammals. For example, in meiosis of male mice, Schimenti and his colleagues30 have
shown that ectopic recombination is surprisingly robust.
One set of DNA sequences seems to be particularly
restricted in ectopic interactions, both in meiosis and
mitosis. Sequences near telomeres can recombine freely
with homologous sequences at other chromosome
ends38,39, but these subtelomeric regions are somehow
insulated from recombining with similar homologous
regions that are located away from telomeres36.
It is important to know if ectopic meiotic recombination
is carried out by the same machinery that performs allelic
events. Recent results from Grushcow et al.40 argue that
meiotic, ectopic and allelic recombination might, indeed,
use somewhat different recombination enzymes. A dmc1
mutant enhances the frequency with which ectopic sites are
used, suggesting that Dmc1p (and Tid1p, perhaps) are
especially focused on interallelic events. A similar finding
has been made by Thompson and Stahl24, who observed
that a dmc1 mutation (as well as mutations in a number of
other genes) increases unequal sister-chromatid recombination at the expense of normal allelic recombination.
Effect of checkpoint genes on DSB repair in
mitosis and meiosis
One critical function of the DNA-damage checkpoint
response is to give cells enough time to complete recombination. Consequently, deletion of S. cerevisiae checkpoint
genes MEC1, RAD17 or RAD24 produces a loss of spore
viability. The mutations also suppress the arrest of cells at
the pachytene stage of meiosis in dmc1 recombinationdefective mutants41. Recently, Grushcow et al.40 have discovered that checkpoint genes also regulate both the choice
of homologous partners and the frequency of crossing-over.
There is an increase both in inter- and intra-chromosomal
ectopic recombination that is independent of the increase
seen with dmc1 mutants, discussed above. Moreover the
proportion of ectopic gene convertants undergoing
crossovers increased from 65% to 90%. It would be fascinating to know if checkpoint mutations also affect the low
level of crossing-over in msh4 or zip1 mutants, as it is not
clear that ectopic recombination between short sequences
will involve the synaptonemal complex.
Another recent study by Thompson and Stahl24 found
that mutations in some checkpoint genes also altered the
choice between an allelic partner or a sister chromatid
during yeast meiosis, including RAD17, RAD24 and
MEC3. So it looks like checkpoint genes are important to
ensure inter-homologue allelic recombination in competition both with ectopic and with (unequal) sister partners.
Checkpoint defects also affect ectopic recombination in
mitotic cells. A rad9 deletion increased translocations five- to
tenfold, but this was suppressed if cells were arrested in
G2/M before induction42, suggesting that cells that are capable of G2/M arrest can repair lesions by sister-chromatid
exchange, so that the possibility of translocations is
reduced. But Fasullo et al. also note that translocations
might arise from repair of the damaged chromosome following mitosis42; indeed, half of selected events carry nonreciprocal translocations, in addition to intact copies of the
two chromosomes involved in these rearrangements. This
can be explained if a broken chromosome is segregated
during mitosis, in the absence of checkpoint arrest, and can
then initiate BIR to create a nonreciprocal translocation.
Conclusions
We are beginning to see how the cell can achieve a balance
between competing mechanisms to repair a chromosomal
DSB. Insights from these studies may help provide new
approaches to ensuring more accurate gene targeting in
mammalian cealls, as well as in other organisms, where this
has proven difficult. We are still left with many questions
about how homology searches are carried out and about
the precise mechanisms of any of these pathways, but the
rate of information is growing at an ever more rapid pace.
TIG June 2000, volume 16, No. 6
263
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Acknowledgements
The author wishes to thank S. Lovett and members of the
Haber lab for generously providing comments. Work from
References
1 Pâques, F. and Haber, J.E. (1999) Multiple pathways of
recombination induced by double-strand breaks in
Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349–404
2 Moore, J.K. and Haber, J.E. (1996) Cell cycle and genetic
requirements of two pathways of nonhomologous end-joining
repair of double-strand breaks in Saccharomyces cerevisiae.
Mol. Cell. Biol. 16, 2164–2173
3 Jeggo, P.A. (1998) DNA breakage and repair. Adv. Genet. 38,
185–218
4 Szostak, J.W. et al. (1983) The double-strand-break repair
model for recombination. Cell 33, 25–35
5 Fishman-Lobell, J. et al. (1992) Two alternative pathways of
double-strand break repair that are kinetically separable and
independently modulated. Mol. Cell. Biol. 12, 1292–1303
6 Wu, X. et al. (1997) Rules of donor preference in
Saccharomyces mating-type gene switching revealed by a
competition assay involving two types of recombination.
Genetics 147, 399–407
7 Bosco, G. and Haber, J.E. (1998) Chromosome break-induced
DNA replication leads to non-reciprocal translocations and
telomere capture. Genetics 150, 1037–1047
8 Le, S. et al. (1999) RAD50 and RAD51 define two different
pathways that collaborate to maintain telomeres in the
absence of telomerase. Genetics 152, 143–152
9 Voelkel-Meiman, K. and Roeder, G.S. (1990) Gene conversion
tracts stimulated by HOT1-promoted transcription are long
and continuous. Genetics 126, 851–867
10 Morrow, D.M. et al. (1997) ‘Break copy’ duplication: a model
for chromosome fragment formation in Saccharomyces
cerevisiae. Genetics 147, 371–382
11 Haber, J.E. (1998) The many interfaces of Mre11. Cell 95,
583–586
12 Chen, C. and Kolodner, R.D. (1999) Gross chromosomal
rearrangements in Saccharomyces cerevisiae replication and
recombination defective mutants. Nat. Genet. 23, 81–85
13 Wilson, S. et al. (1999) The role of Schizosaccharomyces
pombe Rad32, the Mre11 homologue, and other DNA damage
response proteins in non-homologous end joining and
telomere length maintenance. Nucleic Acids Res. 27,
2655–2661
14 Yamaguchi-Iwai, Y. et al. (1999) Mre11 is essential for the
maintenance of chromosomal DNA in vertebrate cells. EMBO J.
18, 6619–6629
15 Lee, S.E. et al. (1999) Role of yeast SIR genes and mating type
in channeling double-strand breaks to homologous and
nonhomologous recombination pathways. Curr. Biol. 9,
the author’s laboratory has been supported by grants from
the National Institutes of Health, the Department of
Energy and the National Science Foundation.
767–770
16 Liang, F. et al. (1998) Homology-directed repair is a major
double-strand break repair pathway in mammalian cells.
Proc. Natl. Acad. Sci. U. S. A. 95, 5172–5177
17 Van Dyck, E. et al. (1999) Binding of double-strand breaks in
DNA by human Rad52 protein. Nature 398, 728–731
18 Goedecke, W. et al. (1999) Mre11 and Ku70 interact in
somatic cells, but are differentially expressed in early meiosis.
Nat. Genet. 23, 194–198
19 Fasullo, M. et al. (1999) Expression of Saccharomyces
cerevisiae MATa and MATa enhances the HO endonucleasestimulation of chromosomal rearrangements directed by his3
recombinational substrates. Mutat. Res. 433, 33–44
20 Åström, S.U. et al. (1999) Yeast cell-type regulation of DNA
repair. Nature 397, 310
21 Schwacha, A. and Kleckner, N. (1997) Interhomolog bias
during meiotic recombination: meiotic functions promote a
highly differentiated interhomolog-only pathway. Cell 90,
1123–1135
22 Kadyk, L.C. and Hartwell, L.H. (1992) Sister chromatids are
preferred over homologs as substrates for recombinational
repair in Saccharomyces cerevisiae. Genetics 132, 387–402
23 Zickler, D. and Kleckner, N. (1999) Meiotic chromosomes:
integrating structure and function. Annu. Rev. Genet. 33,
603–754
24 Thompson, D.A. and Stahl, F.W. (1999) Genetic control of
recombination partner preference in yeast meiosis. Isolation
and characterization of mutants elevated for meiotic unequal
sister-chromatid recombination. Genetics 153, 621–641
25 Dresser, M.E. et al. (1997) DMC1 functions in a
Saccharomyces cerevisiae meiotic pathway that is largely
independent of the RAD51 pathway. Genetics 147, 533–544
26 Arbel, A. et al. (1999) Sister chromatid-based DNA repair is
mediated by RAD54, not by DMC1 or TID1. EMBO J. 18,
2648–2658
27 Shinohara, M. et al. (1997) Characterization of the roles of the
Saccharomyces cerevisiae RAD54 gene and a homologue of
RAD54, RDH54/TID1, in mitosis and meiosis. Genetics 147,
1545–1456
28 Klein, H.L. (1997) RDH54, a RAD54 homologue in
Saccharomyces cerevisiae, is required for mitotic diploidspecific recombination and repair and for meiosis. Genetics
147, 1533–1543
29 Kupiec, M. and Petes, T.D. (1988) Allelic and ectopic
recombination between Ty elements in yeast. Genetics 119,
549–559
30 Cooper, D.M. et al. (1998) Factors affecting ectopic gene
conversion in mice. Mamm. Genome 9, 355–360
31 Inbar, O. and Kupiec, M. (1999) Homology search and choice
of homologous partner during mitotic recombination. Mol.
Cell. Biol. 19, 4134–4142
32 Burgess, S.M. and Kleckner, N. (1999) Collisions between
yeast chromosomal loci in vivo are governed by three layers of
organization. Genes Dev. 13, 1871–1883
33 Burgess, S.M. et al. (1999) Somatic pairing of homologs in
budding yeast: existence and modulation. Genes Dev. 13,
1627–1641
34 Haber, J.E. and Leung, W.Y. (1996) Lack of chromosome
territoriality in yeast: promiscuous rejoining of broken
chromosome ends. Proc. Natl. Acad. Sci. U. S. A. 93,
13949–13954
35 Richardson, C. et al. (1998) Double-strand break repair by
interchromosomal recombination: suppression of
chromosomal translocations. Genes Dev. 12, 3831–3842
36 Goldman, A.S. and Lichten, M. (1996) The efficiency of meiotic
recombination between dispersed sequences in
Saccharomyces cerevisiae depends upon their chromosomal
location. Genetics 144, 43–55
37 Weiner, B.M. and Kleckner, N. (1994) Chromosome pairing via
multiple interstitial interactions before and during meiosis in
yeast. Cell 77, 977–991
38 Horowitz, H. et al. (1984) Rearrangements of highly
polymorphic regions near telomeres of Saccharomyces
cerevisiae. Mol. Cell. Biol. 4, 2509–2517
39 Louis, E.J. and Haber, J.E. (1990) Mitotic recombination
among subtelomeric Y’ repeats in Saccharomyces cerevisiae.
Genetics 124, 547–559
40 Grushcow, J.M. et al. (1999) Saccharomyces cerevisiae
checkpoint genes MEC1, RAD17 and RAD24 are required for
normal meiotic recombination partner choice. Genetics 153,
607–620
41 Lydall, D. et al. (1996) A meiotic recombination checkpoint
controlled by mitotic checkpoint genes. Nature 383, 840–843
42 Fasullo, M. et al. (1998) The Saccharomyces cerevisiae RAD9
checkpoint reduces the DNA damage-associated stimulation
of directed translocations. Mol. Cell. Biol. 18, 1190–1200
43 Holmes, A. and Haber, J.E. (1999) Double-strand break repair
in yeast requires both leading and lagging strand DNA
polymerases. Cell 96, 415–424
Reference added in proof
44 Sugawara, N. et al. DNA length dependence of the singlestrand annealing pathway and the role of Saccharomyces
cerevisiae RAD59 in double-strand break repair. Mol. Cell.
Biol. (in press)
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