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Journal of Bacteriology, November 1998, p. 5639-5645, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Interaction of RecBCD Enzyme with DNA at
Double-Strand Breaks Produced in UV-Irradiated Escherichia
coli: Requirement for DNA End Processing
Brigitte
Thoms and
Wilfried
Wackernagel*
Genetik, Fachbereich Biologie,
Universität Oldenburg, D-26111 Oldenburg, Germany
Received 22 June 1998/Accepted 2 September 1998
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ABSTRACT |
The RecBCD enzyme has a powerful duplex DNA exonuclease activity in
vivo. We found that this activity decreased strongly when cells were
irradiated with UV light (135 J/m2). The activity decrease
was seen by an increase in survival of phage T4
2
of about 200-fold (phage T4
2 has defective duplex DNA end-protecting
gene 2 protein). The activity decrease depended on excision
repair proficiency of the cells and a postirradiation incubation.
During this time, chromosome fragmentation occurred as demonstrated by
pulsed-field gel electrophoresis. In accord with previous observations,
it was concluded that the RecBCD enzyme is silenced during interaction
with duplex DNA fragments containing Chi nucleotide sequences. The
silencing was suppressed by induction or permanent derepression of the
SOS system or by the overproduction of single-strand DNA binding
protein (from a plasmid with ssb+) which is
known to inhibit degradation of chromosomal DNA by cellular DNases.
Further, mutations in xonA, recJ, and
sbcCD, particularly in the recJ sbcCD and
xonA recJ sbcCD combinations, impeded RecBCD silencing. The
findings suggest that the DNA fragments had single-stranded tails of a
length which prevents loading of RecBCD. It is concluded that in
wild-type cells the tails are effectively removed by
single-strand-specific DNases including exonuclease I, RecJ DNase, and
SbcCD DNase. By this, tailed DNA ends are processed to entry sites for
RecBCD. It is proposed that end blunting functions to direct DNA ends
into the RecABCD pathway. This pathway specifically activates
Chi-containing regions for recombination and recombinational repair.
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INTRODUCTION |
Mutations in the recB and
recC genes greatly reduce chromosomal recombination in
Escherichia coli and render cells sensitive to UV
irradiation, gamma rays, and other DNA-damaging agents, which suggests
that these genes function in recombinational repair of DNA damage,
particularly of DNA double-strand breaks (8, 44). Together
with recD, these genes encode the three protein subunits of
the RecBCD enzyme. In vitro, the enzyme has exonuclease activity for
duplex DNA (therefore also termed exonuclease V), exo- and
endonucleolytic activities for single-stranded DNA, and a DNA helicase
activity, all of which require ATP (for references, see reference
18). From early studies, it is known that the rapid
degradation of linear duplex DNA in E. coli cells is mainly caused by the RecBCD enzyme, irrespective of whether the DNA has entered the cell during phage infection (43), transfection
(51), or transformation (33, 52). Accordingly,
the gene 2 mutant of phage T4 which lacks the protective
protein attached to the ends of the T4 genome has a very low chance of
surviving in wild-type cells as a result of genome degradation
(34) but multiplies unimpaired in recB or
recC mutants. Therefore, the efficiency of plating (EOP) of
T4 2 has been used to monitor the level of
RecBCD enzyme in a variety of experiments (1, 7, 11, 17,
40).
Although in vivo the RecBCD enzyme effectively degrades linear
double-stranded DNA, it is nevertheless essential for the
recombinational repair of DNA double-strand breaks (41).
These apparently conflicting observations have been explained by the
findings that the octanucleotide sequence 5'-GCTGGTGG-3'
termed Chi which is present in the E. coli genome
about once per 5 kb can block the nucleolytic activity of the RecBCD
enzyme and stimulate recombination in their vicinity (11, 14, 32,
44). How the switch at Chi from the DNA-degrading activity of
RecBCD to a recombination-initiating function is achieved in vivo is
not yet known in detail. It has been proposed (32, 46) and
some evidence provided by in vitro and in vivo experiments (13,
17, 31) that the RecD subunit may play a key role in the switch
process. Before RecBCD can encounter a properly oriented Chi sequence
in a duplex DNA molecule, the enzyme must bind to the end of that DNA.
In vitro, effective binding requires that the DNA is blunt ended or has
single-stranded tails of only a few nucleotides (37, 45).
We have previously observed that the EOP of the T4
2 mutant strongly increased when
E. coli wild-type cells were irradiated with UV prior to
phage infection (48). This indicated a decrease of
RecBCD enzyme activity. Here we report experiments which led us to
conclude that the activity decrease occurs in the UV-irradiated E. coli cells as a result of the interaction of RecBCD
with duplex DNA fragments. These fragments are produced in an excision
repair-dependent process at high UV doses. Results of further
experiments suggest that the ends of the DNA fragments require
processing by other DNases to make them a substrate for RecBCD
enzyme. The possibility that processing functions direct tailed linear
DNA into the RecA- and RecBCD-dependent recombination repair process is
discussed.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The E. coli
strains used are listed in Table 1.
Except for WA485, all strains are AB1157 and its derivatives obtained
by P1 transduction or plasmid transformation using standard procedures. Strain WA235 was used for propagation of T4 2.
Table 1 lists the EOP of T4 2 on all strains
employed. Details of strain constructions can be provided upon request.
Plasmids employed were the cloning vectors pBR322 (
3), pUC19
(
57), and pJF118EH (
15). Plasmid pDW1 is pBR322
with a
17-kbp fragment (obtained after partial
Sau3AI
digestion of
E. coli DNA) cloned into the
BamHI
site spanning the
recC+,
recB+, and
recD+ gene
region of
E. coli (
40). Plasmid pSK1 is pJF118EH
with
a 3.8-kbp
PstI fragment covering the
recD+ gene of
E. coli
(
17). Plasmid pJA40 is pUC9 with a 664-bp
HaeIII
fragment covering
ssb+ of
E. coli
(obtained by J. Brandsma, Leiden, The Netherlands).
Plasmid pUV1 is the
circularized large
HindIII fragment of pJA501
(
6) containing the vector pBR322 and the
uvrA+ gene with its promoter. Plasmids and their
relevant genotypes
are included in the genotypes of strains in Table
1.
Media and growth conditions.
Bacterial cultures were grown
in TBY (10 g of Bacto Tryptone, 5 g of Bacto yeast extract, 5 g of NaCl per 1,000 ml) at 30°C. Plates contained TBY agar (TBY
solidified by 1.5% Bacto Agar). The soft agar consisted of TBY with
0.5% Bacto Agar. Phosphate buffer consisted of 0.04 M
Na2HPO4, 0.02 M KH2PO4,
0.07 M NaCl, and 0.002 M MgSO4.
Determination of the EOP of T4 2.
Phages
(in 0.1 ml of phosphate buffer) were added to 0.2 ml of log-phase
culture (2 × 108 cells/ml). After 15 min at 30°C
for phage adsorption, the suspension was poured together with 3 ml of
soft agar on a plate. Plaques were counted after 24 h at 30°C.
The titer of the T4 2 lysate was determined
with the recB mutant strain WA632. The EOP is the plaque
count determined on a given strain divided by the plaque count on
WA632. The relative EOP is the EOP on irradiated cells divided by the
EOP on unirradiated cells of the same strain determined in the same
experiment. As a control of the cellular capacity for phage
propagation, the EOP of T4+ was determined in parallel.
When the EOPs of T4 2 and T4+ were
determined with irradiated cells, 0.01 ml of a culture grown overnight
(AB1157) was added after phage adsorption and before plating. With some
strains, a decline of the T4+ plating upon irradiation with
the highest doses was observed (up to 0.3 compared to the unirradiated
cells). The T4 2 EOP was corrected
accordingly. Phage adsorption was always more than 95%.
UV irradiation.
The cells of 5 ml of a log-phase culture
were sedimented and resuspended in 4.5 ml of phosphate buffer. Cells
were irradiated in a glass petri dish at room temperature with stirring
as described previously (47). After irradiation, 0.5 ml of
10-fold concentrated TBY was added and the suspension was incubated at
30°C in a 100-ml Erlenmeyer flask on a shaker for a time period given
for each experiment.
Measurement of T4 2 DNA
degradation.
Phage T4 2 labelled with
[3H]thymidine was prepared by standard procedures. The
phages had a specific radioactivity of 2 × 105 cpm
per infective center. The quantification of intracellular DNA
degradation was performed as described previously (47) and corrected for the efficiency of phage adsorption (39).
Pulsed-field gel electrophoresis.
Cells were washed in 10 mM
Tris-HCl (pH 7.5)-1 mM EDTA and resuspended in 100 mM NaCl-10 mM
Tris-HCl (pH 7.5)-25 mM EDTA. The cells were concentrated, embedded in
1% agarose (type III; Biometra, Göttingen, Germany) lysed, and
treated with proteinase K essentially as described previously
(24). Electrophoresis was performed in 1% agarose at 13°C
for 16 h at 180 V with a Rotaphor R22 (Biometra) and using a pulse
time of 3 to 30 s (linear) and an angle of 120 to 95° (log). The
electrophoresis markers were 
DNA concatemers and
HindIII-digested DNA. The gel was stained with
ethidium bromide and photographed.
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RESULTS |
Increased EOP of phage T4 2 on
UV-irradiated cells.
Compared to recB mutant cells
(WA632), the EOP of T4 2 on wild-type cells
(AB1157) is only about 5 × 104 (Table 1) due to
RecBCD-dependent exonucleolytic phage genome destruction (34,
39). When wild-type cells were irradiated with UV and aerated in
broth at 30°C for various time periods before infection with T4
2, a time-dependent increase of the EOP of T4
2 was observed, reaching a maximum after about
90 to 120 min. This phenomenon was termed increase of T4
2 EOP (ITE). Its dependence on the UV dose
showed a typically concave-shaped curve (Fig.
1), with an increase in the EOP of about
200-fold at a UV dose of 135 J/m2. The level of cell
survival at the various UV doses of the wild-type cells (and of other
strains in later experiments) is not relevant since the capacity to
propagate T4 was largely maintained even at the highest doses (see
Materials and Methods).
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FIG. 1.
Increase in the EOP of T4 2
with different UV doses applied to various E. coli strains.
Postirradiation aeration in broth was for 120 min at 30°C. The
relative EOPs were determined as described in Materials and Methods.
The data are means from two to three independent experiments. For
clarity, only some points are given with standard deviations or with
the range (strain WA692). The strains were wild type (AB1157;  ),
lexA3 (BT136;  ), wild type with pDW1 carrying the
recB+C+D+ genes (WA692;
 ), and wild type with vector pBR322 (WA691;  ).
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The degradation of [
3H]thymidine-labelled T4
2 DNA to acid-soluble material decreased from
about 50% acid-soluble material
in nonirradiated cells to 33% in
irradiated cells (135 J/m
2) (Table
2), suggesting that the
EOP increase was related to
lower duplex DNA-degrading activity in
irradiated cells. This
was supported by the finding that in cells with
an about 15-fold-higher
RecBCD enzyme activity due to the multicopy
plasmid pDW1 (with
recB+ recC+
recD+ [
40]), induction of ITE was
weak (Fig.
1). Apparently, in strongly
UV-irradiated cells, less RecBCD
enzyme is available for attacking
T4
2
genomes. A possible reason could be (i) that the enzyme is
inhibited
by an UV-induced protein (ExoV inhibitor
[
56]) or (ii) that
the enzyme interacts with
UV-damaged DNA or repair intermediates
which sequester the enzyme. The
facts that in a
lexA3 mutant (in
which induction of SOS
proteins is blocked) [
53]), full induction
of ITE was
observed at high doses and hyperinduction was observed
at lower doses
(Fig.
1) and the data in the next section are all
in favor of the
second possibility.
The increase of T4 2 EOP depends on
excision repair.
Mutations in uvrA block the
incision step of the excision repair pathway of UV photoproducts and
other DNA damages (50). Compared to wild type, in a
uvrA mutant induction of ITE was strongly reduced at low and
high UV doses (Fig. 2) and after
postirradiation incubation periods between 0.5 and 4 h (data not
shown). Results similar to those with the uvrA mutant were
also obtained with uvrC (Fig. 2) and uvrB (not
shown) mutants. A plasmid expressing uvrA+
restored inducible ITE in the uvrA mutant (Fig. 2). In
contrast to UV-irradiated wild-type cells, a decrease of T4
2 DNA degradation was not observed in
UV-irradiated uvrA cells (Table
2). In polA1 cells, in which
incision at photoproducts is normal but closing of excision repair gaps
is defective (50), UV-induced ITE was seen (data not shown)
and the degradation of T4 2 DNA was reduced
(Table 2). Crossing of a uvrC279::Tn10
mutation into the polA1 strain abolished UV-induced ITE
(data not shown). From these observations, it was concluded that
incision of DNA at photoproducts is required for ITE and that a higher
survival of T4 2 phages is correlated with
less degradation of phage DNA. It should be pointed out that the
increase in T4 2 survival of about 200-fold at
135 J/m2 (Fig. 1) represents the rescue of only about 10%
of the infecting phage. Correspondingly, the production of acid-soluble
material from phage DNA was only partially lowered upon UV irradiation (Table 2).
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FIG. 2.
Increase in the EOP of T4 2
with UV doses applied to a uvrA strain (WA618; ), a
uvrC strain (WA778; ), and a uvrA strain with
plasmid pUV1 carrying the uvrA+ gene (BT241;
). For details, see the legend to Fig. 1. The data are means from
two independent experiments, the range is shown only for the points
after the UV dose of 135 J/m2.
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The notion that ITE became effective only in
uvr+ cells and particularly following high UV
doses (Fig.
1) suggested that excision
repair of closely spaced
photoproducts on opposite strands leads
to the formation of DNA
double-strand breaks (
4,
42). In
excision-proficient
E. coli, Bonura and Smith (
4) observed
20 to 30 double-strand breaks per genome after 100 J/m
2 and
postirradiation incubation of 80 min. Using pulsed-field
gel
electrophoresis, the UV dose-dependent fragmentation of chromosomal
DNA
was detected in wild-type cells, which became very apparent
after 135 J/m
2 and 90 min of postirradiation incubation (Fig.
3). The time dependence
of DNA
fragmentation could indicate that breakage is also a result
of incision
plus replication. Chromosome fragmentation was not
found in the
uvrA mutant (Fig.
3). It was, however, seen in the
polA strain (not shown), showing that ITE depends on
incising
but not on gap closure.
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FIG. 3.
Pulsed-field gel electrophoresis of chromosomal DNA from
UV-irradiated cells of the wild type (AB1157) and a uvrA
mutant (WA618). The two leftmost lanes contain a DNA multimer
ladder and HindIII-digested DNA fragments. The sizes
of the fragments are indicated as kilobase pairs (kb) to the left of
the gel. The other lanes were loaded with agar blocks, with each
containing about 3 × 108 cells. The UV doses applied
to cells and the time spans of postirradiation incubation at 30°C
(min) are indicated at the top.
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Influence of SOS derepression on UV-induced ITE.
Since several
components of the excision repair system are coded by genes under SOS
regulation (see reference 50), we asked whether
derepression of the SOS regulon would stimulate the subsequent UV
induction of ITE. This was not the case. Rather, induction of
ITE was suppressed when wild-type cells had received an SOS-inducing UV
dose (54 J/m2) 90 min before the ITE-inducing irradiation
(135 J/m2) and further incubation (Table
3). In a
lexA71::Tn5 mutant in which the SOS system is
permanently and fully derepressed, ITE was hardly inducible with UV
(without or with prior SOS-inducing treatment [Table 3]), although
chromosomal DNA fragmentation occurred, as in UV-irradiated wild-type
cells (Fig. 3 and data not shown). In contrast, pretreatment of a
lexA3 mutant with UV (54 J/m2) did not prevent
subsequent UV induction of ITE (Table 3). These findings show that (i)
a gene under the control of the lexA repressor can
counteract UV induction of ITE and (ii) that the chromosome fragmentation following high UV doses is necessary but not sufficient to trigger ITE.
Overexpression of
recA in a
lexA3 mutant caused
by the
recAo298 operator mutation (
9) did not
prevent induction of ITE
(Table
3), suggesting that
recA is
not the SOS gene counteracting
induction of ITE. In an attempt to find
the counteracting SOS
gene, defective alleles of SOS genes were crossed
into the
lexA71::Tn
5 (plus
sulA) genetic background including
(
recA-srl)::Tn
10,
dinA::Mud,
dinB::Mud,
dinD::Mud,
umuC::Mud,
recN::Tn
5,
ruvA::Tn
10, and
recQ::Tn
3.
None of these crosses restored the
inducibility of ITE (data not
shown). However, when an
ssb-1
allele was crossed into the
lexA71::Tn
5 (plus
sulA) background (giving strain BT330), induction of ITE
by
UV was partially restored (the EOP of T4
2
increased after 135 J/m
2 and 90 min at 30°C by a factor
of 25.3 ± 5.5;
n = 3). This was
not observed with
the
ssb-113 allele (strain BT335; 2.9 ± 0.8;
n = 4).
The single-strand binding protein (SSB) coded by
ssb-1 has a
1,000-fold-lower single-stranded DNA affinity than the wild-type
protein, whereas the
ssb-113 protein is not impaired in DNA
binding
(
28,
54,
55). The
ssb gene has a
lexA-regulated promoter
(
5), and an increased
rate of SSB synthesis was observed in
cells treated with nalidixic acid
(
35). Meyer and Laine (
28)
concluded that the
ssb gene of
E. coli is inducible by conditions
derepressing the SOS operon. Since in the
lexA71::Tn
5 background,
only the weakly
DNA-binding SSB-1 protein allowed induction of
ITE whereas the
wild-type SSB and the SSB-113 did not, it was
hypothesized that
increased amounts of SSB with normal DNA-binding
activity would prevent
induction of ITE.
Effect of ssb+ overexpression.
In
cells with the multicopy plasmid pJA40 overproducing SSB
(6), induction of ITE was absent (Table
4). A similar result was obtained using
the ssb+ gene cloned in pSY343 (58),
a runaway replicon (data not shown) and also with pJA40 in
lexA3 cells (Table 4). These observations suggest that
overexpression of ssb+ suppresses the induction
of ITE by UV. The results also imply that single-stranded DNA
influences the induction of ITE. Since chromosomal DNA fragmentation in
the SSB-overproducing cells was as strong as in wild-type cells (Fig. 3
and data not shown), excess SSB probably prevents induction of ITE by
interfering with a step occurring subsequent to chromosome breakage.
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TABLE 4.
Influence of SSB overproduction from the multicopy
plasmid pJA40 (with ssb+) on the UV-induced
increase of T4 2 EOP
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Gene functions required for full induction of ITE.
We assumed
that the chromosomal fragments produced after high UV doses contain
single-stranded tails of various length and of 5' and 3' polarity.
Tails of both polarities longer than about 25 nucleotides are known to
prevent loading of RecBCD enzyme on the DNA fragment (37,
45). Thus, long tails would have to be removed or shortened by
single-strand-specific exo- and endonucleases to make the duplex DNA
ends available to RecBCD. To address this point, UV-induced ITE was
determined at different times after irradiation of isogenic strains
defective for various DNases (Fig. 4). A
xonA2 mutation abolishing exonuclease I (36) had
only a minor, if any, effect. A stronger reduction was observed in a
recJ::Tn10 mutation eliminating RecJ DNase. A
xonA recJ double mutant and the recJ single
mutant gave similar results (not shown). We also examined an
sbcCD deletion mutant which lacks the two subunit genes of
an enzyme that has besides duplex DNA exonuclease activity a
single-strand endonuclease activity (10). As shown in Fig.
4, lack of the SbcCD endonuclease strongly decreased induction of ITE
up to 60 min after irradiation, but the effect was almost lost 120 to
180 min after irradiation. An sbcCD xonA double mutant showed a further decrease of ITE, particularly 120 to 180 min after
irradiation. The lowest UV-induced ITE values were obtained in the
sbcCD recJ double mutant and the sbcCD recJ
xonA triple mutant. In these strains, induced ITE after 2 h
of incubation was about 5% of that observed in wild-type cells.
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FIG. 4.
Effect of postincubation time after UV irradiation (135 J/m2) in broth at 30°C on the EOP of T4
2 on strains deficient for various DNases. The
strains were wild type (AB1157;  ), xonA (WA818; ),
sbcCD (BT384; ), recJ (BT122;  ),
sbcCD xonA (BT421;  ), sbcCD recJ (BT386;
×), and sbcCD xonA recJ (BT422; +). The data are means
from three to four independent experiments. For clarity, standard
deviations are given only for the 120-min incubation values.
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Influence of recD+ overexpression.
Two
mechanisms have been proposed to explain how chromosomal DNA fragments
could lower the RecBCD enzyme activity in cells: (i) the interaction of
RecBCD with the DNA (i.e., binding followed by DNA degradation) may
temporarily sequester the enzyme on the substrate DNA (7) or
(ii) the duplex DNA-degrading activity is silenced irreversibly during
DNA degradation at properly oriented Chi sites (17).
Previously it was demonstrated that overexpression of
recD+ partially alleviated the silencing
(17). As shown in Fig. 5, derepression of recD+ located on an expression
plasmid also measurably alleviated induction of ITE by UV.
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FIG. 5.
Influence of recD+ overexpression
from pSK1 (recD+) on the increase of the EOP of
T4 2 in UV-irradiated (135 J/m2)
cells. Strains were wild type with vector plasmid pJF118EH (BT305; )
and wild type with pSK1 (BT306; ). The cells were grown with IPTG
(isopropyl- -D-thiogalactopyranoside) (1 mM). The data
are means from three independent experiments. The standard deviations
are given only for the 150-min point.
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DISCUSSION |
Irradiation with relatively high UV doses (producing thousands of
pyrimidine dimers per chromosome) leads to the fragmentation of the
E. coli chromosomal DNA in an excision repair-dependent process (4) (Fig. 3). Here we have shown that with this DNA fragmentation the EOP of T4 2 increased about
200-fold. This finding is reminiscent of the observation that the
EOP of T4 2 increased strongly on E. coli cells treated with agents producing DNA double-strand
breaks, such as gamma ray (7) or bleomycin (17). Brcic-Kostic et al. (7) concluded that the
concentration of free RecBCD enzyme in the cytoplasm was depleted
because the enzyme was loaded on the duplex DNA ends and that this
helped infecting T4 2 genomes to survive. In
the studies with gamma ray or bleomycin, ITE was seen immediately after
treatment of cells. In contrast, in the UV-experiments reported here,
ITE required a postirradiation incubation of about 1 to 2 h at
30°C. The time span appears to be required for the excision
repair-dependent and possibly replication-dependent chromosome
fragmentation following UV irradiation. The necessity of relatively
high UV doses and the use of stationary-phase cells in earlier
experiments may be the reasons why an effect of UV on the T4
2 survival was not detected in a previous
study (7).
Several conditions besides overexpression of recB+
recC+ recD+ were identified which strongly
suppressed induction of ITE. Two were related to the SOS system. These
conditions were the prior induction of the SOS response by a small UV
dose or the permanent SOS derepression by a
lexA71::Tn5 mutation. Among several defective SOS genes screened for no longer causing suppression of ITE in a
lexA71 background, only the ssb-1 allele coding
for an SSB protein with a low DNA single-strand affinity turned out to
partially alleviate the suppression. The ssb-113 allele
coding for an SSB with normal DNA binding had no such effect. The third
condition preventing ITE, in accord with the previous finding, was the
overexpression of ssb+ from a plasmid. These
observations pointed to a role of single-stranded DNA in the response
of the RecBCD enzyme level to high UV doses. Since excess SSB did not
interfere with the chromosome fragmentation following UV irradiation,
we assumed that SSB associated with single-stranded tails present on
the DNA fragments. In vitro covering of single strands with SSB makes
the DNA refractory to the single-strand-specific endo- and
exonucleolytic activities of the RecBCD enzyme (27). The
lengths of the tails on the fragments in the UV-irradiated cells are
not known. It is conceivable that excision repair gaps (12.4 nucleotides [50]) in close proximity on opposite
strands could result in chromosome breakage leaving tails of well over 25 nucleotides. Tails of a few nucleotides covered with SSB and tails
longer than 25 nucleotides with or without SSB all prevent loading of
RecBCD on the DNA in vitro (27, 37, 45). Thus, if the DNA
fragments in UV-irradiated cells have tails and SSB is overproduced,
then the RecBCD enzyme could not bind to DNA and the level of free
enzyme would remain high. This was observed. In accord with these
considerations is the notion that at low UV doses in lexA3
cells, in which the SOS system is not inducible and therefore the level
of SSB cannot increase, higher levels of induced ITE were observed than
in the wild type (Fig. 1 and Table 3). If induction of the SOS response
occurred in the wild type after application of ITE-inducing UV doses,
it occurred apparently too late or was too weak (as a consequence of
the high UV doses) to suppress ITE.
The conclusion that single-stranded tails are initially present on the
DNA fragments produced in UV-irradiated cells and prevent direct
loading of RecBCD is also supported by the finding that mutational
elimination of single-strand-specific DNases is a further condition
which counteracted induction of ITE (Fig. 4). Apparently, the
activities of exonuclease I (specific for 3' single strands [22]), RecJ DNase (specific for 5' single strands
[26]) and SbcCD DNase (single-stranded DNA
endonuclease [10]) all contribute to high ITE by
acting in the rapid removal of tails from duplex DNA fragments. Perhaps
the long tails are particularly sensitive to shortening by
endonucleolytic cutting, whereas the exonucleases act in the further
trimming. Apparently, when sufficient time is provided, exonucleases
can achieve extensive blunting of DNA ends in the sbcCD
mutant, leading to full induction of ITE (Fig. 4). Since overproduction
of SSB apparently blocks tail removal, the responsible nucleases must
be inhibited by SSB. It is not known whether SSB affects SbcCD and
RecJ. In vitro exonuclease I is not inhibited, whereas protection
of DNA by SSB against various other DNases has been described
(29). In vivo, the ssb-1 mutation was shown to
eliminate the protection of DNA against RecBCD and one or more other
DNases that is conferred by wild-type SSB (23). The function
of single-strand-specific DNases such as RecJ in recombination is
probably not limited to the early step of producing entry sites for
RecBCD but is also necessary for later steps and for RecBCD-independent
recombination (18, 26).
What happens to RecBCD when it has gained access to a chromosomal
duplex DNA fragment after removal of tails? We believe that the short
time sequestration of RecBCD as a result of interacting with DNA is not
the main cause of ITE. Rather the exposure of RecBCD during its
tracking along DNA to properly oriented Chi sites will silence the
exonucleolytic activity for duplex DNA as observed in vitro
(14) and in vivo (11, 17, 21, 31). In vivo, the
silencing appears to be irreversible since RecBCD activity (determined
as ATP-dependent duplex DNA exonuclease) was no longer detectable in
extracts made from bleomycin-treated cells (17). The
silencing of RecBCD is partially alleviated by overexpression of
recD+ (17, 31). Overexpression
of recD+ also partially alleviated UV-induced
ITE (Fig. 5), suggesting that the mechanism of RecBCD silencing
in heavily UV-irradiated cells is the same as that in cells in which
the chromosome is fragmented by gamma rays (7) or bleomycin
(17) or in cells in which linear duplex plasmid DNA with Chi
sites is presented (31). The detailed mechanism of RecBCD
silencing is not yet known. The heavy UV irradiation in our experiments
was necessary to obtain an excess of presumably tailed DNA ends and an
almost synchronous silencing of a large part of RecBCD in the cell
population in order to study factors that lead to silencing or prevent
it. We believe that our conclusions drawn for the bulk of RecBCD in UV-irradiated cells are also true for single enzyme molecules interacting with DNA broken as a result of other impacts.
Recent in vitro experiments show that the 3' single strand that is
exposed at Chi by RecBCD is simultaneously recognized by RecA with high
efficiency in an apparently coordinated reaction and transferred to a
homologous duplex (2). This suggested a physical cooperation
of RecA with RecBCD in Chi-specific recombination (2). It
fits with the observation that in vivo the highest recombination
frequencies and the most effective DNA repair were achieved by
RecA and RecBCD components from the same species instead of
interspecific combinations (12). The necessity of blunt ends for the loading of RecBCD on DNA would make
single-strand-specific DNases important enzymes because their
activity would direct tailed DNA ends at a double-strand break into the
RecABCD recombination repair process. In this, RecA protein is
specifically targeted to the "recombination island" sequences
surrounding the Chi sites which strongly stimulate strand transfer
(2, 49). Random single-stranded tails at DNA ends would not
specifically expose such sequences. The overlapping activities of
the various single-strand-specific DNases in processing tailed
DNA into blunt ends would give mutants defective in one or two of the
DNases, probably not a recombination- or repair-deficient phenotype. In
contrast, absence of the single-strand-degrading activities exonuclease
I and SbcCD DNase restores recombination and repair to recB
or recC mutants (20, 25). In these mutants, stabilization, not removal, of single-stranded tails by the
sbcB and sbcCD mutations, perhaps supported by
increased production of SSB, appears to make recombination possible
which is no longer focused to Chi sites.
| |
ACKNOWLEDGMENTS |
We thank Jourica Brandsma, Rudi Eichenlaub, David Leach, Bob
Lloyd, and Ralph Meyer for providing bacterial strains and plasmids and
Regina Rinken and Johann de Vries for help with some of the experiments.
This work was supported by the Fonds der Chemischen Industrie and the
Volkswagen-Stiftung (Lower Saxony-Israel joint project).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Genetik,
Fachbereich Biologie, Universität Oldenburg, Postfach 2503, D-26111 Oldenburg, Germany. Phone: 49-441-798 3298. Fax: 49-441-798 3298 or 49-441-798 3250. E-mail:
genetics{at}biologie.uni-oldenburg.de.
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Abstract
Introduction
