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Journal of Bacteriology, December 1998, p. 6269-6275, Vol. 180, No. 23
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Sister Chromatid Exchange Frequencies in
Escherichia coli Analyzed by Recombination at the
dif Resolvase Site
Walter W.
Steiner
and
Peter L.
Kuempel*
Department of Molecular, Cellular, and
Developmental Biology, University of Colorado, Boulder, Colorado
80309
Received 8 June 1998/Accepted 29 September 1998
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ABSTRACT |
Sister chromatid exchange (SCE) in Escherichia coli
results in the formation of circular dimer chromosomes, which are
converted back to monomers by a compensating exchange at the
dif resolvase site. Recombination at dif is
site specific and can be monitored by utilizing a density label assay
that we recently described. To characterize factors affecting SCE
frequency, we analyzed dimer resolution at the dif site in
a variety of genetic backgrounds and conditions. Recombination at
dif was increased by known hyperrecombinogenic mutations
such as polA, dut, and uvrD. It was
also increased by a fur mutation, which increased oxidative
DNA damage. Recombination at dif was eliminated by a
recA mutation, reflecting the role of RecA in SCE and
virtually all homologous recombination in E. coli.
Interestingly, recombination at dif was reduced to
approximately half of the wild-type levels by single mutations in
either recB or recF, and it was virtually
eliminated when both mutations were present. This result demonstrates
the importance of both RecBCD and RecF to chromosomal recombination
events in wild-type cells.
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INTRODUCTION |
Linear and circular chromosomes both
present unique problems for replication. Due to the enzymatic
limitations of DNA polymerases, for example, a problem is encountered
when the ends of linear chromosomes are replicated. Eukaryotes have
solved this problem by capping the ends of their chromosomes with
specialized structures called telomeres, which can then be replicated
by the telomerase enzyme (18). Because circular chromosomes
do not have ends, no difficulty is encountered in replicating the
entire molecule with conventional DNA polymerases. However,
circular chromosomes are faced with a new problem when
recombination occurs between the growing daughter chromosomes, a
process we refer to as sister chromatid exchange (SCE). One exchange or
any odd number of exchanges results in the formation of circular dimer
chromosomes (24). Dimers must be resolved back to monomers
prior to cell division in order to properly partition daughter
chromosomes to daughter cells. This resolution process requires a
compensating exchange, which occurs by site-specific recombination at
the dif locus in the terminus of the Escherichia
coli chromosome (39).
SCE is difficult to analyze in haploid organisms, such as
Escherichia coli, due to the absence of genetically
distinguishable homologous chromosomes. Previously, the only direct
attempts to analyze SCE in E. coli used autoradiographic
analyses to determine how often daughter cells each received parts of
3H-labeled chromosomes (14, 43). The difficulty
and lack of sensitivity of those assays have limited their use, and no
attempts have been made to extend these earlier studies in order to
characterize the various mutations that might affect SCE. Efforts to
identify factors contributing to SCE have generally involved genetic
analyses of interactions between chromosomal regions present as direct or inverted repeats and which are either adjacent to or at some distance from each other (3, 9, 16, 29, 30, 31, 48, 49). The
main problem with genetic assays of this kind is that recombination
between chromosomal repeats can occur both intramolecularly as well as
intermolecularly (true SCE), and it is not generally possible to
distinguish which of these types of interactions predominates.
Important concepts have certainly emerged from genetic analyses of SCE,
but it is difficult to estimate whether it is valid to extrapolate
these findings to events that occur between growing daughter
chromosomes during their replication and partition.
Because dimer chromosomes can form only as a consequence of
intermolecular recombination between daughter chromosomes, we reasoned that analysis of chromosome dimer resolution at the
dif site provides a completely different method to study
SCE. Previously, we reported that the frequency of recombination
occurring at dif was increased above wild-type levels by a
polA mutation, which is known by other assays to increase
the frequency of chromosomal recombination events (22, 49).
This result suggested that the frequency of SCE was higher in a
polA mutant, that is, more dimers were formed and
subsequently resolved at dif. Here we extend our analysis of
chromosome dimer resolution at dif to other
hyperrecombinogenic (hyper-rec) mutations and show that the
dif recombination frequency is increased by these mutations
as well.
We also analyzed the effects on SCE of mutations in some of the major
genes associated with recombination. Recombination in E. coli has traditionally been thought of in terms of
recombination pathways. The three pathways originally defined by Clark
(7) are the RecBCD, RecE, and RecF pathways. The RecBCD
pathway predominates when one of the recombination substrates is a
linear DNA molecule (23, 38). Since most recombination
assays in E. coli (e.g., conjugation and transduction)
involve linear molecules, the RecBCD pathway has generally been
considered the major pathway of recombination in wild-type cells. In
conjugation or transduction assays, the RecE and RecF pathways of
recombination normally contribute very little to the overall frequency
of recombination events (6). These pathways become active,
however, and restore recombination proficiency to recB or
recC cells when suppressor mutations are introduced. For
example, sbcA mutations activate the RecE pathway of
recombination, and sbcB sbcC mutations activate the RecF
pathway (27).
The recE gene forms part of the cryptic Rac prophage located
in the terminus of the E. coli chromosome (5).
Since recE is absent in some strains and is not expressed in
sbcA+ cells, it can be considered a minor, or
cryptic, recombination pathway. RecF is not encoded by a cryptic gene,
and although it is not normally involved in conjugational or
transductional recombination, recF mutants are as sensitive
to UV irradiation as recB cells are (20).
However, the reason for this sensitivity is not completely clear. It
has recently been suggested that the primary role of RecF may not be in
recombination at all but rather in the reassembly of a replication
holoenzyme at the site of a stalled replication fork (10).
Although this might be one function of RecF, our data demonstrate a
significant role for RecF in recombination between sister chromosomes.
This conclusion is consistent with that recently reported by Galitski
and Roth (16), who used a genetic assay to analyze SCE in
Salmonella typhimurium.
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MATERIALS AND METHODS |
Strains.
All strains used in this study were E. coli K-12 strains and are listed in Table
1. PK3772 is C600 thi-1 thr-1 leuB6
thyA deoB or deoC. PK3872 is PK3772
leu+ thr+. All mutations
were introduced into PK3772 or PK3872 by bacteriophage P1 transduction.
Media.
Glucose-minimal medium was M-9 salts (2)
containing 0.2% glucose, 2 µg of thymine ml
1, 1 µg of thiamine ml1, and 2 µCi of
[3H]thymine ml1 (Sigma). Rich medium was
identical to glucose-minimal medium but contained in addition 0.2%
Isogro (39). Acetate-minimal medium was identical to
glucose-minimal medium except that sodium acetate was substituted for
glucose at a final concentration of 40 mM. Heavy minimal medium for the
initial growth of cultures contained the appropriate
13C-labeled carbon compound and
15NH4Cl at 99% atom purity. Heavy rich medium
contained [13C]glucose, 15NH4Cl,
and [13C-15N]Isogro, all at 99% atom purity.
Heavy isotopes were obtained from Isotec, Inc., Miamisburg, Ohio.
Density label assay.
The density label assay was performed
as previously described (39). Net percentages of
semihybrid density DNA for a given experiment (Table
2) were determined by subtracting the
average percentage of semihybrid density DNA found in six independent density assays of xerC and xerD mutants, in which
site-specific recombination at dif is prevented
(39). For each experiment, the percentage of semihybrid
density DNA in three, four, or five fractions was determined, and the
average percentage of semihybrid density DNA in the same number of
fractions from the experiments with xerC and xerD
mutants was subtracted from this value. The number of fractions which
gave the highest net value was then used to determine the average
percentage of semihybrid density DNA for a given genotype. The use of
either three, four, or five fractions was always found to give the
highest net value in any given experiment. The background percentages
of semihybrid density DNA from the xerC and xerD
experiments were 7.0, 10.6, and 15.2% for three, four, and five
fractions, respectively.
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RESULTS |
Density label assay for dif recombination.
The
results reported here were obtained by using the density label assay
that we recently described (39) and which is shown in a
simplified form in Fig. 1. Exponentially
growing cells uniformly labeled with heavy isotopes (15N
and 13C) were shifted to light medium and grown for two
more generations before DNA purification. All DNA was hybrid density
after the first generation in light medium, and recombination between
hybrid-density dif sites produces hybrid-density DNA in
which the density of the strands changes at dif. Cell
division is required for this recombination, and dimer chromosomes are
resolved to monomers (39). An additional generation of
growth and replication converts the hybrid-density dif sites
into semihybrid-density sites (i.e., one-fourth heavy density),
which are detected by the assay.
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FIG. 1.
Density label assay for site-specific recombination at
dif. Cells are grown in heavy medium, so that the DNA is
uniformly labeled with 13C and 15N isotopes.
Following a shift to light medium, the cells are allowed to grow for
two more generations. Heavy DNA is indicated by solid lines, and light
DNA is indicated by dashed lines. The dif site is in the
middle of the molecule. If no recombination occurs at dif,
equal amounts of hybrid and light DNA at dif are produced
after two generations. If recombination occurs between dif
sites in sister chromosomes after the first generation, recombinant
hybrid DNA is produced, in which the density of both single strands
switches from heavy to light at dif. After growth for a
second generation in light medium, recombinant hybrid density DNA is
converted to semihybrid density DNA, in which one-fourth of each duplex
molecule is heavy density DNA. Semihybrid density DNA can be separated
from hybrid and light density DNA in CsCl density gradients. See the
text and reference 39 for further details. Rep.,
replication.
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Recombination between
dif sites can also occur in some cells
after the second replication of the
dif site. Two DNA
doublings
produce cells in which one
dif site is hybrid
density and the
other is light density. Recombination between these
sites at the
time of cell division also produces semihybrid-density
DNA. In
the two generations of exponential growth that we used for the
analysis, recombination of this type can occur only in cells that
have
not yet entered the D period (the interval between termination
and cell
division) at the time of the density shift (
39).
To monitor the extent of DNA replication and the distribution of
genomic DNA within the gradients, the cells were continuously
labeled with [
3H]thymine. The distribution of a
9.9-kb
BglI
dif fragment in the
gradients was
then determined by hybridization of the gradient
fractions with a
32P-labeled
dif probe. Figure
2 shows a typical result obtained
when
the density assay was performed with our wild-type strain.
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FIG. 2.
Recombination at dif in wild-type (WT) cells.
The density assay was performed with PK3872 grown in rich medium. (A)
Analysis of 3H-genomic DNA shows equal amounts of hybrid
and light DNA, indicating that two generations of growth and DNA
replication had occurred. Since fractions are collected from the bottom
of each gradient, higher fractions indicate lower densities. kdpm,
1,000 decays per minute. (B) Probing the same fractions shown in panel
A with a 32P-labeled dif probe reveals a
distinct semihybrid density DNA peak, which results from site-specific
recombination at dif (39). y-axis
units are arbitrary units from the phosphorimager.
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Mutations increasing SCE.
A prediction from our previous
results was that hyper-rec mutations should increase the frequency of
SCE. The increased frequency of SCE should produce more dimer
chromosomes, which in turn can be detected by the increased frequency
of dimer resolution at dif. To test this correlation,
we initially examined a polA mutant. polA
codes for DNA polymerase I, and mutations in this gene were among the
original hyper-rec mutations identified by E. B. Konrad (22). An increased frequency of SCE in a polA
mutant background was also suggested by our earlier observation that
polA dif double mutants exhibit increased filamentation and
SOS induction, presumably due to the increased number of
unresolvable dimer chromosomes (24). By use of the
density assay, we observed that the level of recombination
occurring at the dif locus was substantially increased in a
polA mutant relative to that in its
polA+ parent (Table 2).
To test further the correlation between dimer formation and dimer
resolution at the
dif site, we analyzed additional mutations
originally identified by Konrad (
22) as being hyper-rec.
dut (also called
dnaS) codes for dUTPase, and
mutations in this gene
result in increased incorporation of uracil into
DNA. Uracil incorporated
into DNA is rapidly removed by excision repair
functions within
the cell, and as a consequence,
dut
mutants tend to accumulate
nicks in nascent DNA (
21,
42).
Similar to
polA, the frequency
of recombination at
dif was also increased by a
dut mutation (Fig.
3A; Table
2).
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FIG. 3.
Recombination frequency at dif is sensitive
to changes in SCE frequency. The density assay was performed with
several different strains carrying mutations known to increase
(dut and uvrD) or decrease (recA)
genetic recombination. Only the distribution of DNA at dif
in CsCl density gradients is shown for each experiment. (A) PK4001
(dut); (B) PK3984 (uvrD); (C) PK3802
(recA). The density assay of PK4001 (A) was performed in
glucose-minimal medium; the other two experiments (B and C) were
performed in rich medium. y-axis units in panel C indicate
net counts obtained with an Ambis Systems imager.
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Konrad also observed that a
uvrD mutant (originally called
mutU) was hyper-rec. Mutations in
uvrD affect DNA
helicase II,
which functions in both excision and mismatch repair.
Consistent
with the results obtained with other hyper-rec mutants, a
uvrD mutant also showed increased recombination at
dif in the density
assay (Fig.
3B; Table
2). The basis of
the hyper-rec phenotype
caused by
uvrD mutations is not
entirely clear, but in an assay
based on recombination between tandem
repeats, it appears to require
RecF and SOS induction (
3).
SOS induction has been shown previously to stimulate recombination in a
different assay of SCE based on duplication formation
due to unequal
crossing over between direct repeats (
13,
35).
Because it
was also involved in
uvrD-stimulated recombination
(
3), we tested the possibility that some level of SOS
induction
was a general requirement of SCE. For this experiment,
we analyzed
a
lexA3 mutant by the density label assay. Since
lexA3 codes for
a noncleavable repressor of the SOS regulon,
cells carrying this
mutation cannot be induced for SOS. Recombination
at
dif, however,
was not significantly affected by
lexA3 (Table
2).
It was also possible that the hyperrecombination seen in
uvrD cells was a general property of cells affected in
excision repair.
Therefore, we tested the effect of another
excision-deficient
mutation,
uvrA. SCE did not appear to
be affected by this mutation
(Table
2).
We also tested whether the frequency of SCE was influenced by oxidative
DNA damage, as suggested by Galitski and Roth (
16).
For this experiment, we analyzed a
fur mutant, which
lacks a protein
regulating the uptake of iron into the cell.
fur mutants incorporate
iron constitutively, and the excess
iron in the cell stimulates
the production of hydroxyl radicals and
other DNA-damaging agents
(
41). The
fur mutant we
tested showed substantially increased
recombination at the
dif site (Table
2), which is consistent
with DNA damage
being a cause of SCE. That the increased levels
of SCE occur as a
result of repair in
fur mutants is indicated
by the fact
that
fur recA or
fur recB double mutants are not
viable
under aerobic conditions (
41).
Recombination pathways involved in SCE.
As mentioned above,
the recombination-related genes involved in SCE have previously been
studied by genetic assays based on recombination between repeated
regions (9, 16, 48). Since our experiments demonstrated a
correlation between recombination at dif and SCE elsewhere
in the chromosome, we have also utilized the density assay as an
independent test to identify recombination functions required for SCE.
We initially tested a recA mutant, which would be expected
to block all recombination and undergo no SCE. Consistent with this
expectation, no recombination was observed at dif (Fig. 3C).
Since recombination at dif itself is RecA independent
(24), this result demonstrates that SCE does not occur in a
recA mutant. It also is consistent with the ability of
recA mutations to suppress the Dif phenotype: in the absence of SCE, dimer chromosomes are not formed, and resolution of dimers at
dif is not required (24).
Since the RecBCD pathway is usually considered the major route for
recombination and chromosome interactions in wild-type
E. coli, we tested whether resolution at
dif was affected
in a
recB mutant. Figure
4A
shows a typical result obtained with such
strains. Although
recombination was reduced by the
recB mutation
(51% of
wild-type levels), it still clearly occurred. This was
a somewhat
surprising result, because
recB mutations, as well
as
recA mutations, had previously been shown to suppress the
Dif
phenotype (
24). We originally proposed that the
suppression
of the Dif phenotype seen in
recB mutants was
due to a lack of
dimer chromosome formation caused by this mutation.
Since SCE
obviously still occurred in
recB mutants, the
ability of
recB to suppress the Dif phenotype was likely due
to another effect
of this mutation. We have subsequently observed that
the lack
of filamentation of
dif recB mutants is due to the
inability to
induce the SOS response (
39a).
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FIG. 4.
RecBCD and RecF provide alternate routes to SCE. The
density assay was performed with strains carrying mutations in
recB, recF, or both of these genes. The
recombination frequency at dif was reduced to approximately
the same extent by either single mutation, and it was virtually absent
when both mutations were present. (A) PK3798 (recB); (B)
PK3874 (recF); (C) PK3882 (recB recF).
y-axis units in panel A indicate net counts obtained with an
Ambis Systems imager.
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Since recombination at
dif was still observed in a
recB mutant, it suggested that SCE could also occur by a
pathway other
than the RecBCD pathway. Therefore, we tested a
recF mutant, and
the result was similar to that observed in
the
recB strain (Fig.
4B; Table
2). When the
recB
recF double mutant was tested in
the density assay, recombination
at
dif was reduced even further
and only a small, residual
amount of resolution still occurred
at
dif (Fig.
4C). These
results demonstrate that RecBCD and RecF
both contribute to SCE and the
formation of dimer
chromosomes.
Frequency of SCE for cells grown in different media.
Most of the experiments described above were performed on
cells grown in rich medium, in which the generation time was 33 min. In
some cases, however, it was necessary to grow the cells in
glucose-minimal medium. For example, PK4001 (dut) grew very poorly in rich medium, and minimal medium was used for the experiment shown in Fig. 3A. To determine whether the growth medium itself affected SCE frequency, we also conducted experiments on our wild-type strain (PK3872) grown in glucose-minimal or acetate-minimal medium. In
glucose-minimal medium, the generation time was increased to approximately 63 min (39). At this growth rate, the
frequency of recombination observed in cells grown for two
generations in light medium was essentially unchanged (16%) (Table
2). When PK3872 was grown in acetate-minimal medium, the
generation time was increased to approximately 130 min. Under these
conditions, a slight decrease in recombination was observed at
dif (10.5%) (Table 2). These data suggest that the SCE
frequency may decrease slightly with a decreasing growth rate, but the
effect is not substantial.
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DISCUSSION |
Assay for SCE.
The density assay that we have used here to
analyze SCE differs considerably from the genetic assays previously
used for this purpose. The dif locus reduces chromosome
dimers, which are the products of SCE, to monomers by providing a
compensating exchange. Therefore, the frequency of recombination at
dif should reflect the frequency of recombination that
occurs throughout the rest of the chromosome, and our results with
known hyper-rec and hyporecombinogenic mutations are consistent with
this interpretation (Fig. 3; Table 2). In contrast to genetic assays,
an advantage of the density assay is that it uses the entire bacterial
chromosome as the target for SCE. Furthermore, all regions of the
chromosome are at their normal locations and undergo the normal
interactions inherent in replication, repair, and recombination.
The density assay does, of course, have some disadvantages. One
disadvantage is that the assay itself is labor-intensive.
Also,
differences of orders of magnitude cannot be detected. The
assay works
well when the frequency of SCE falls between 10 and
15% per
generation, as with the wild-type strain we used (
39),
but
very low levels of SCE would be difficult to reliably detect.
Increases
in SCE can be readily observed (Fig.
3), but the maximum
level of
recombination and semihybrid density DNA that can be
detected is
50%. This arises since even numbers of SCE events
will not
produce dimer chromosomes, and at very high frequencies
of SCE, even
and odd numbers of exchanges will become equally
frequent.
Consequently, the density assay rapidly loses sensitivity
as the
frequency of recombination approaches 50%.
Since our assay is quite different from those previously used to
analyze SCE, it is interesting to compare our results for
the frequency
of SCE (~16% for wild-type cells) with those recently
reported by
Galitski and Roth (
16). Their assay was based on
recombination between tandem repeats and consequently used a much
smaller target (~40 kb). They estimated a frequency of recombination
events per generation of 0.49%, and when this value is extrapolated
to
the size of the entire chromosome, an SCE frequency of 58%
results.
Considering the differences between the two procedures
(recombination
detected by physical rejoining versus sectored
colonies) as well as the
species difference (
E. coli versus
S. typhimurium), the results are rather similar. A possible source
of
the higher estimate by their assay is that in addition to scoring
events due to unequal crossing over, which is similar to SCE,
their
assay might also score events due to intrachromosomal recombination
and
looping out of the scored loci. It should be noted that if
the
difference is due to looping out, this implies that SCE and
looping out
have similar genetic
requirements.
Two pathways of SCE.
Perhaps the most interesting result that
we report here is that both RecBCD and RecF are involved in producing
SCE. The data presented in Fig. 4 and Table 2 suggest that each
contributes more or less equally to SCE, and each operates
independently of the other. Although this might seem consistent with
current interpretations of recombination pathways, it should be
stressed that sbcB sbcC mutations were not required to
observe RecF-dependent recombination in our assay. Consequently, SCE
occurred in the absence of any mutations that stabilize 3' ends of DNA,
so it did not involve the classical recF recombination
pathway. Except for some possible unknown effect of the recB
mutation that was necessary to conduct the assay, the RecF
recombination should be occurring at wild-type levels. These results
are consistent with those recently reported by Galitski and Roth
(16), who also observed that RecF contributed substantially
to chromosomal recombination events in wild-type cells.
Given that both RecBCD and RecF contribute to SCE, what does this tell
us about the nature of the recombination substrates
present within the
cell?
recB mutant cells show substantially
reduced viability
in comparison to
recB+ cells (
4), so
the RecBCD nuclease obviously plays an important
role in the cell
apart from its role in conjugational and transductional
recombination. Since RecBCD is a very potent double-stranded
DNA
exonuclease, degradation of DNA by this enzyme could be quite
damaging to a cell unless it contains other copies of the degraded
genes. Keeping this in mind, a current model for the normal role
of
RecBCD is in the repair of broken replication forks (
1,
26). When a replication fork breaks and releases the end of
one
of the growing daughter chromosomes, it presents a double-strand
end,
which is required for the loading of the RecBCD nuclease
(
40). Degradation would proceed backwards, towards the
origin
of replication (
oriC), and the other nascent daughter
chromosome
would contain copies of the degraded genes. The
combined actions
of RecBCD and RecA proteins on the broken arm of
the chromosome
would facilitate its invasion into the intact daughter
chromosome.
This establishes a Holliday junction, which is
thought to promote
restoration of the collapsed replication fork.
Since the Holliday
junction can be resolved in either of two
orientations, a dimeric
chromosome should be produced in half of these
events (for example,
see Fig. 8 and 9 in reference
1).
The source of the broken replication forks that are repaired by
RecBCD could be spontaneous breaks in the template strands.
If a
replication fork proceeds through a region containing such
a nick, a
double-strand break would be generated. It can be estimated
that chromosomes contain about 10 single-strand breaks per
template
(
17). If only 1.5% of these single-strand breaks
resulted in
a broken replication fork, about 15% SCE per
generation would
be
produced.
What is the role of RecF in SCE? Breakage and reformation of forks by a
RecF-mediated process seem unlikely by current understanding
of these
mutants, especially in a strain that is
recBC+
sbcBCD+. RecF, therefore, presumably operates on
substrates that do not
involve double strand-breaks. RecF has been
shown to be of major
importance in the repair of daughter strand gaps
left on chromosomes
following UV irradiation of cells (
44,
45). When a replication
fork encounters a lesion in the template,
such as a thymine dimer,
it can reinitiate replication downstream in a
process known as
translesion synthesis (
36). This leaves a
damaged single-stranded
region in one chromosome that can be repaired
only with information
from the undamaged sister chromosome.
Invasion of the single-stranded
region of the damaged
chromosome into the undamaged chromosome
establishes a Holliday
junction which can migrate across the lesion.
The damaged DNA can then
be excised by repair enzymes within the
cell and resynthesized from
information in the undamaged strand
(
28). Resolution of the
Holliday junction, again, can occur
in either of two orientations, and
a dimer chromosome will result
half of the time. Resumption of
synthesis at the stalled replication
fork could require RecF, as
proposed by Courcelle et al. (
10).
Sources of SCE.
As mentioned above, one possible source of
SCE is single-strand breaks in the template strand. These breaks
could arise from the replication process itself. This is
suggested by the increased level of SCE in polA and
dut mutants, which increase the frequency of single-strand
breaks in newly synthesized DNA (21, 25, 34, 42). If some of
these breaks are not closed before they appear as template DNA, a
double-strand break would occur. The increased frequency of SCE in
mutants affected in sealing breaks indicates that at least some of the
spontaneous SCE arises from a background level of breaks of this type.
It can be argued that the distribution of SCE events might not be
uniform around the chromosome. Louarn and coworkers have
observed that
hyperrecombination occurs in the terminus region
of the chromosome and
that the frequency of recombination in that
region increases more than
3 orders of magnitude (
29). The hyperrecombination
is RecBCD
dependent, which indicates that it is derived from double-strand
breaks or from nicks leading to double-strand breaks
(
9). Louarn
and coworkers have demonstrated that this
terminal hyperrecombination
is not related to the meeting of
replication forks in this region
(
29), and they suggest that
it is due to the postreplication
reconstruction of nucleoid
organization that occurs prior to cell
division. Decatenation and
other events associated with this reconstruction
are proposed to
lead to double-strand breaks, which are the basis
of the
hyperrecombination.
Another source of SCE is oxidative damage to DNA, which occurs
spontaneously in cells grown under aerobic conditions (
12,
16). As a test of this hypothesis, we examined a
fur
mutant,
which shows increased amounts of oxidative DNA damage
(
41).
Table
2 shows that SCE was substantially increased by
this mutation.
Oxygen by-products can affect DNA in a variety of ways,
including
the generation of single-strand breaks and damage to DNA
bases
(
15). As discussed above, the breaks would ultimately
be repaired
by RecBCD nuclease, while repair of any gaps left by
translesion
synthesis would involve the action of RecF. As mentioned by
Galitski
and Roth (
16), oxidative DNA damage could
contribute to a large
part of the spontaneous level of SCE observed in
wild-type
cells.
| |
ACKNOWLEDGMENTS |
The research was supported by National Institutes of Health grant GM32968.
We thank Heather Szerlong and Andrias Hojgaard for assistance with
experiments and helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Molecular, Cellular, and Developmental Biology, University of Colorado,
Boulder, CO 80309. Phone: (303) 492-7952. Fax: (303) 492-7744. E-mail: Peter.Kuempel{at}colorado.edu.
Present address: Fred Hutchinson Cancer Research Center, Seattle,
WA 98104-2092.
| |
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Abstract
Introduction
