(Received for publication, December 5, 1994; and in revised form, April 3, 1995)
From the
Genetic recombination occurring in wild type Escherichia
coli is stimulated at DNA sequences known as sites,
5`-GCTGGTGG-3`. In vitro, homologous pairing between duplex
DNA substrates dependent upon the RecA, RecBCD, and SSB proteins is
stimulated by the presence of a
sequence in the donor linear
double-stranded DNA. We show that this stimulation is due to two
factors: 1) the enhanced production of
-specific single-stranded
DNA fragments and 2) their preferential use in the RecA
protein-promoted pairing step. Furthermore, under conditions of
limiting Mg
concentration, joint molecule formation
does not occur, even though DNA unwinding and
-specific
single-stranded DNA fragment production are observed. Also, under these
conditions,
-specific fragments derived from both the upstream
and downstream regions of the DNA strand containing
and from
cleavage of the non-
-containing DNA strand are detected. Finally,
the behavior of mutant RecBCD enzymes (RecBC*D and RecBCD
)
in this in vitro reaction is shown to parallel their in
vivo phenotypes with respect to
stimulation of
recombination. Thus we suggest that, in addition to its ability to
regulate the degradative activities of RecBCD enzyme,
itself may
be a preferred site for initiation of homologous pairing in this
concerted process.
Homologous recombination in Escherichia coli occurs
primarily through the RecBCD pathway of generalized recombination (for
reviews see Smith(1988, 1989), Clark and Sandler(1994), and
Kowalczykowski, et al.(1994)). Genetic analysis demonstrates
that recombination occurring through this pathway is dependent on the
RecA, RecBCD, and single-stranded DNA binding (SSB)(
)<
/a>proteins (Clark and Margulies, 1965; Glassberg et
al., 1979; Emmerson and Howard-Flanders, 1967; Howard-Flanders and
Theriot, 1966). Mutations in the recA gene decrease levels of
recombination by as much as 6 orders of magnitude (Clark and Margulies,
1965), mutations in the recB or recC genes reduce
recombination by 99% (Emmerson and Howard-Flanders, 1967;
Howard-Flanders and Theriot, 1966), and defects in the ssb gene can reduce recombination by 80% (Glassberg et al.,
1979). In addition to these protein components, recombination in the
RecBCD pathway is enhanced by recombination hotspots, called
sites (5`-GCTGGTGG-3`), which increase the frequency of genetic
exchange 5- to 10-fold in their vicinity (Lam et al., 1974;
McMilin et al., 1974; Smith et al., 1981; Smith,
1983; Stahl et al., 1975).
The biochemical roles of RecA and SSB proteins in recombination reactions in vitro are well established (for reviews, see Kowalczykowski (1991a, 1991b), Radding(1991), West(1992), Cox(1993), and Kowalczykowski and Eggleston, 1994). RecA protein promotes both the exchange of DNA strands between complementary single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) and the renaturation of homologous ssDNA. The ability of RecA protein to exchange complementary DNA strands is stimulated by SSB protein in vitro, and studies using mutant RecA proteins suggest that the process of DNA strand exchange is likely to represent recombinational events occurring in vivo (see Kowalczykowski (1991a) and Kowalczykowski et al.(1994)).
The RecBCD enzyme
(exonuclease V) is multifunctional and consists of three nonidentical
subunits, the RecB, RecC, and RecD polypeptides. In vitro, it
has the following biochemical activities: DNA-dependent ATPase, ssDNA
and dsDNA exonuclease, ssDNA endonuclease, and ATP-dependent DNA
helicase (for reviews, see Taylor(1988), Smith(1990),
Kowalczykowski(1994), and Kowalczykowski et al.(1994)). In
addition to its nonspecific nuclease activities, RecBCD enzyme can
generate single-stranded DNA fragments whose 3`-end terminates
4-6 nucleotides to the 3`-side of the recombination
hotspot (Ponticelli et al., 1985; Taylor et al.,
1985) (see Smith(1994)). This specific interaction is
orientation-dependent, since RecBCD enzyme must approach from the
3`-side of the
sequence for recognition to occur (Taylor et
al., 1985).
The ability of to stimulate recombination in
a polar fashion stems from the fact that the
site is a
regulatory DNA sequence, which acts to attenuate the nonspecific
nuclease activity, but not the helicase activity, of the RecBCD enzyme;
this results in the creation and preservation of a ssDNA fragment
containing the
sequence (Dixon and Kowalczykowski, 1991; Dixon
and Kowalczykowski, 1993) (see Kowalczykowski(1994)). This attenuation
of nuclease activity is a result of the loss or functional inactivation
of the RecD subunit through interaction with
(Dixon, et
al., 1994).
Certain mutations in the recB, recC, or recD genes yield altered RecBCD enzymes that
display differential effects with regard to -stimulation of
recombination. The recC* class of mutations was isolated as
pseudorevertants of a presumed missense mutation in the recC gene (Schultz et al., 1983). While being moderately
recombination-proficient and maintaining wild type levels of dsDNA
exonuclease activity, the RecBC*D enzyme is, however, unable to
stimulate recombination at
sites (Schultz et al.,
1983). The recBCD
mutations appear to be nonsense
mutations that map primarily to the recD gene and thus are
devoid of a full-length recD gene product (Amundsen et
al., 1986; Chaudhury and Smith, 1984). These mutants are similar
to wild type cells in that they are fully viable and are
recombination-proficient; in fact, they display elevated levels of
conjugal, plasmid, and phage
recombination (Chaudhury and Smith,
1984; Lovett et al., 1988; Thaler et al., 1989).
However, despite the elevated recombination levels, the recD
mutants do not display
-stimulation of
recombination (Chaudhury and Smith, 1984). The most noticeable
difference between double dagger mutants and wild type RecBCD enzyme is
that the mutant RecBCD
enzymes are devoid of any detectable
nuclease activities in vivo and in cell extracts (Amundsen et al., 1986; Chaudhury and Smith, 1984; Taylor, 1988), but
they retain helicase activity (Rinken et al., 1992).
Previously, the formation of joint molecules in vitro between dsDNA substrates was shown to depend on the RecA, RecBCD,
and SSB proteins in a coordinated process referred to as the
``RecABCD-dependent reaction'' (Roman et al., 1991).
Formation of joint molecules requires creation of ssDNA through
unwinding of linear dsDNA by RecBCD enzyme, trapping of the ssDNA
strands by RecA and SSB proteins, and DNA strand invasion of homologous
supercoiled DNA molecule promoted by RecA protein (Kowalczykowski and
Roman, 1990). enhances the RecABCD-dependent reaction in
vitro with a polarity identical to that expected from in vivo observations (Dixon and Kowalczykowski, 1991). The role of
was to down-regulate the destructive nuclease activity of RecBCD
enzyme, while allowing the recombination-promoting helicase activity to
persist.
In this report, we expand on our previous findings to
demonstrate that -dependent joint molecule formation occurs more
rapidly and at a higher frequency than
-independent pairing
events. At reduced magnesium ion concentrations, the formation of
-specific ssDNA fragments is enhanced, and fragments derived from
both the 5`- and 3`-sides of
are detected; however, under these
conditions, homologous pairing is limited by the inability of the RecA
protein to promote DNA strand invasion. Furthermore, mutant RecBCD
enzymes that do not recognize
in vivo fail to stimulate
-dependent joint molecule formation to the same extent as the
wild type RecBCD enzyme does.
RecA protein was purified using a procedure
based on spermidine precipitation (Griffith and Shores,
1985).(
)Protein concentration was det
ermined
using an extinction coefficient of 2.7
10
M
cm
at 280 nm.
SSB protein was isolated from strain RLM727 and purified according
to LeBowitz(1985). Protein concentration was determined using an
extinction coefficient of 3.0 10
M
c
m
at 280 nm
(Ruyechan and Wetmur, 1975).
All restriction enzymes and DNA modifying enzymes were purchased from New England Biolabs, Pharmacia LKB, or Life Technologies, Inc. The enzymes were used as described by Sambrook et al.(1989) or as indicated by the specific vendor.
For the agarose gel
assay, standard RecABCD reaction conditions were used, except all DNA
and protein components were increased by a factor of 4 unless otherwise
indicated, and the donor linear dsDNA was radiolabeled at the 5`-end.
Aliquots of the reaction mixture (40 µl) were taken at the
indicated time points and were added to 10 µl of stop buffer (0.1 M EDTA, 2.5% SDS, 40% glycerol, 0.125% bromphenol blue, and
0.125 xylene cyanol) to deproteinize the sample. Electrophoresis was
performed for 10 h at 2.1 V/cm using 0.75% agarose gels in TAE buffer
(40 mM Tris acetate (pH 8.0), 2 mM EDTA). The gels
were dried, and autoradiography was at -20 °C with Kodak
XAR-5 film and using an intensifying screen. The autoradiogram was
analyzed using a Bio Image system (Millipore). The reported values are
expressed as the percentage of the total input linear dsDNA. The
-dependent and
-independent joint molecules are defined as
containing either the ssDNA fragment downstream of the
sequence
or a full-length ssDNA strand, respectively, as the invasive ssDNA
strand (Dixon andKowalczykowski, 1991).
Figure 1:
RecABCD-dependent joint molecule
formation at various concentrations of Mg.
RecABCD-dependent reactions were performed using standard
RecABCD-reaction conditions at the indicated concentrations of
magnesium acetate (labeled [Mg Acetate] (mM)). The
ATP concentration was 1 mM, and DNA and protein concentrations
were at the standard levels (i.e. they were not increased
4-fold). The linear dsDNA substrate NdeI-pBR322
FH was radioactively labeled at the 5`-ends.
Reactions were initiated with the addition of 0.31 nM wild
type RecBCD enzyme (0.025 functional enzyme/dsDNA end). All values
reported are a percentage of the total input linear dsDNA contained in
a reaction (Std lane).
-dependent joint molecules (
-dep. JMs) containing either the 3512- or
3057-nucleotide
-specific ssDNA fragment (
-ssDNA)
are indicated as a doublet; the
-independent joint molecule (
-indep. JM) contains a full-length ssDNA strand. A
control reaction containing no Mg
is shown in the far right lane.
The data in Fig. 1also
highlight a common feature of -stimulated, RecABCD-dependent
pairing reactions. Typically, the extent of
-dependent joint
molecule formation reaches a maximum at about 5 min, with 16 ±
5% (based on multiple replicate experiments) of the input linear
-containing dsDNA (after conversion to ssDNA) used in the
formation of this joint molecule species; in contrast,
-independent joint molecule formation (using either
or
° dsDNA) achieved a maximum of at
most 5 and 3% of the input
and
°
(linear duplex) DNA, respectively (data not shown). Significantly, the
limited amount of
-independent joint molecules formed (compared
to the amount of
-dependent joint molecules) is not due to the
lack of full-length ssDNA generated in these reactions, since
approximately 10-20% of the input dsDNA is converted to
full-length ssDNA (all of the dsDNA is unwound, and the remainder of
the ssDNA produced is shorter than full-length), but is not used to
form stable joint molecules. This suggests that
enhances overall
formation of joint molecules because
-specific fragments are used
preferentially for pairing.
Interestingly, at 1 and 2 mM Mg concentrations, two novel discrete ssDNA
species are present. Their sizes (approximately 800 and 1250
nucleotides) are consistent with ssDNA fragments expected from cutting
the DNA strand opposite the
sequence (i.e. the strand
containing the complement of
) in the vicinity of
. Studies
to be reported elsewhere
(
)confirm the
se
fragments to be both
-specific and derived from the
non-
-containing strand. Similar observations regarding cleavage
of the non-
-containing strand near
have been noted by
Taylor and Smith.
(
)
This pattern of product formation is
evident in Fig. 2at Mg concentrations above 1
mM; using 5`-end labeled dsDNA, the predominant product is
half-length ssDNA (panelA), whereas using 3`-end
labeled dsDNA under identical conditions, essentially no discrete
length ssDNA is detected, consistent with the asymmetric pattern of
degradation described above. In addition, as described previously
(Eggleston and Kowalczykowski, 1993a), when the Mg
concentration is increased, degradation increases, resulting in
decreased production of half-length ssDNA. The enhanced degradation of
DNA coincides with reaction conditions in which the Mg
concentrations exceed the ATP concentration (i.e. 2.0-12 mM Mg
) (Egglest
on and
Kowalczykowski, 1993a).
Figure 2:
Unwinding and degradation of °
dsDNA at various concentrations of Mg
. Unwinding
reactions were carried out under standard DNA unwinding reaction
conditions. The dsDNA substrate was pBR322 dsDNA (4363 base pairs)
linearized with the restriction enzyme EcoRI and radioactively
end-labeled at either the 5`- (A) or 3`-ends (B). The
concentration of magnesium acetate used in each reaction is indicated.
The reactions contained 15 nM RecBCD enzyme (1.2 functional
RecBCD enzyme molecules/dsDNA end), and the time for each unwinding
reaction is 1 min. The main ssDNA species produced at low
Mg
concentrations has a size similar to that of a
full-length ssDNA standard (Std) of 4363 nucleotides (A and B, 4363ssDNA); whereas at high
Mg
concentrations, the size of the major ssDNA
species produced is approximately one-half the length of a DNA strand
as compared with the ssDNA standards (2431 and 1932
ssDNA).
In contrast, when the Mg concentration is decreased (to below 1.0 mM) the
decreased frequency of degradation, combined with unwinding, results in
the production of ssDNA whose size is full-length (4363 nucleotides; Fig. 2A). At the lowest Mg
concentration examined (0.125 mM), the amount of
full-length ssDNA produced is 80 ± 10% of the input dsDNA. When
3`-end-labeled dsDNA is unwound under equivalent conditions (Fig. 2B), at low Mg
concentrations,
the major ssDNA product is also full-length, but the yield is somewhat
lower (62 ± 8% of the unwound dsDNA is full-length at 0.125
mM Mg
). When the Mg
concentration is elevated, the 3`-terminal DNA strand is rapidly
degraded. To ensure that the production of full-length ssDNA seen at
the low Mg
ion concentrations is not due to the
inhibition of the ability of RecBCD enzyme to bind and unwind from both
ends of the dsDNA simultaneously, both dsDNA filter binding and
titration of helicase activity control experiments were performed to
confirm that the dsDNA ends are saturated with enzyme at the indicated
Mg
ion concentrations (Taylor and Smith, 1985; data
not shown). These results demonstrate that the enhanced degradation of
the DNA coincides with reaction conditions in which the Mg
concentration exceeds the ATP concentration (2.0-12.0
mM Mg
) (Eggleston and Kowalczykowski,
1993a). The simplest interpretation of these results is that
degradation is asymmetric, and the frequency of nucleolytic cutting by
RecBCD enzyme increases with increased free Mg
concentration (Dixon and Kowalczykowski, 1993; Eggleston and
Kowalczykowski, 1993a; Kowalczykowski, 1994).
Figure 3:
Unwinding and degradation of
dsDNA at various concentrations of
Mg
. Linear
-containing dsDNA substrate was
created by cutting the plasmid pBR322
FH (4316
base pairs), which contains two
sites, with the restriction
enzyme NdeI. The DNA was labeled at either the 5`-end (A) or 3`-end (B) with
P. The
full-length ssDNA fragment produced at low Mg
concentrations is a ssDNA band with a mobility similar to the
ssDNA standard (Std) of 4363 nucleotides (4363ssDNA).
-dependent ssDNA fragments (
-fragments) ha
ve calculated lengths near the predicted
sizes of 3512 and 3057 nucleotides (A) and 1259 and 804
nucleotides (B) compared with the ssDNA standards of 2768 and
1595 nucleotides.
DNA labeled at the 3`-end yields
qualitatively similar results as those obtained with 5`-end-labeled DNA (Fig. 3B). The optimum Mg concentration for the appearance of the upstream
-dependent
ssDNA fragment is 0.25 mM, with 5 ± 1% and 3 ±
1% of input dsDNA being cleaved to create the 1259- and 804- nucleotide
upstream
-dependent fragments, respectively. This optimum is
identical to that observed in Fig. 3A, but the yield of
-specific fragments derived from the 3`-side of
is reduced
by 5-10-fold, despite the higher probability of recovering (due
to the lower probability of a single random cleavage event) a ssDNA
fragment that is at least 5-fold shorter than the downstream fragment.
A reduction of
-specific fragment production can again be seen at
the Mg
concentration of 0.125 mM (3 ±
1% and 2 ± 1% of input dsDNA for the 1259- and 804-nucleotide
fragments, respectively). This decrease coincides with a decrease in
nonspecific degradation; an increase in full-length ssDNA is observed
at 0.125 mM Mg
compared with 0.25 mM Mg
ion (37 ± 4% versus 14
± 2% for Mg
concentrations of 0.125 mM and 0.25 mM, respectively). When elevated Mg
concentrations are used, the ssDNA containi
ng the 3`-end is
degraded into small oligonucleotide fragments (Fig. 3B),
as demonstrated previously (Dixon and Kowalczykowski, 1993). Ponticelli et al.(1985) demonstrated the production of a specific
-dependent ssDNA fragment derived from the 3`-terminal end at the
enzyme's entry site; their reaction conditions employed a low
free Mg
concentration. Our results clearly
demonstrate that the formation of
-dependent ssDNA fragments is
sensitive to Mg
concentration, thus providing an
explanation for the seemingly different results reported (Ponticelli et al., 1985; Dixon and Kowalczykowski, 1991, 1993). The yield
of
-specific fragments is obviously governed by a compromise
between the reduced overall level of cleavage (at both nonspecific and
sites) at low Mg
concentrations versus the enhanced DNA cleavage at all sites seen at high Mg
concentrations; at the higher Mg
concentrations,
-specific ssDNA fragments are being
produced, but their detection is completely obscured by the higher
level of nucleolytic activity displayed by the attenuated enzyme
downstream of
. Regardless of experimental conditions, the DNA
strand 3` at the entry site is always cleaved more often than the
5`-strand, and a productive interaction with
always results in
an attenuation of degradation.
Both mutant enzymes
were examined in RecABCD-dependent pairing reactions using
FH dsDNA (
d
sDNA;
see Fig. 1) to maximize
-dependent stimulation of joint
molecule formation and using subsaturating concentrations of RecBCD
enzyme (0.025 functional RecBCD enzyme molecules/linear dsDNA end). Fig. 4shows that both the rate and the extent of
RecABCD-dependent joint molecule formation by the wild type enzyme are
increased due to the presence of
sites in the linear dsDNA
(labeled
dsDNA) (see also Eggleston and
Kowalczykowski (1993b)); the maximal rate and extent of joint molecule
formation are increased approximately 2.3- and 1.2-fold, respectively,
compared with the
° reaction (Table I). The extent of
joint molecule formation reaches a maximum in about 8 min for the
-containing reactions, whereas for the non
-containing
reactions, it reaches a maximum in about 15 min. The slow decrease in
joint molecule formation seen at the extended time points reflects the
RecA protein-dependent dissociation of joint molecules (Shibata et
al., 1982; Roman and Kowalczykowski, 1989a). Both mutant enzymes
unwind DNA (data not shown), and equivalent amounts of functional
enzyme (based on the apparent stoichiometry derived from DNA helicase
activity shown in Table II) were used in experiments. In agreement
with both in vivo observations and with their capacity to
unwind dsDNA, both the RecBC*D enzyme (Fig. 4) and the RecBCD
enzyme (Fig. 5) promote RecA protein- and SSB
protein-dependent joint molecule formation between linear and
supercoiled dsDNA. Joint molecule formation was slightly lower for
RecBC*D and slightly higher for RecBCD
, but in neither case
was pairing affected by the presence of
sites. Table Isummarizes the extents and rates of joint molecule
formation with the mutant enzymes for reactions containing
or
° dsDNA.
Figure 4:
Effect of the mutant RecBC*D enzyme on
joint molecule formation. Joint molecule formation was detected by
filter binding assays described under ``Materials and
Methods.'' Standard RecABCD-dependent reaction conditions
containing 10 µM nucleotides (2.3 nM dsDNA ends)
of either NdeI-pBR322 ° (
° dsDNA, circles) or NdeI-pBR322
FH (
dsDNA, <
i>triangles) and 5 µM
H-labeled
supercoiled pBR322
° DNA were used (see Fig. 1 for diagrams
of DNA substrates). Reactions were initiated by the addition of 0.31
nM RecBCD enzyme (filledsymbols) or 0.17
nM RecBC*D enzyme (opensymbols), which is
equivalent to 0.025 functional enzymes/dsDNA end for both sets. The
percentage of joint molecules formed is based on the total input
supercoiled DNA contained in a reaction; the values reported are the
average of three experiments, and errorbars indicate
the standard deviation.
Figure 5:
Effect of the mutant RecBCD enzyme on joint molecule formation. Standard
RecABCD-dependent filter binding reactions were performed and analyzed
in a manner identical to that described in the legend to Fig. 4. Linear
dsDNA substrates were NdeI-pBR322
° (
° dsDNA, circles) and NdeI-pBR322
FH (
dsDNA, triangles). Opensymbols represent reactions done in the presence of 1.8 nM RecBCD
enzyme. Data with the wild type RecBCD enzyme from Fig. 4 is
shown with closedsymbols for reference. Errorbars indicate the standard deviation of three
experiments.
The failure of
the mutant enzymes to display stimulation by in the joint
molecule formation assays suggested that they were defective in
recognition. To assess this possibility more directly, the production
of
-specific ssDNA fragments was assayed (Dixon and
Kowalczykowski, 1991). Fig. 6shows that the RecBC*D enzyme is
unable to produce a
-dependent ssDNA fragment, implying that
either
recognition or attenuation of nuclease activity is
defective. The RecBCD
enzyme is able to produce a limited
amount of
-dependent ssDNA (about one-third of the amount
produced by wild type RecBCD enzyme). The production of full-length
ssDNA by the RecBC*D enzyme is approximately equal to that produced by
the wild type enzyme; however, the RecBCD
enzyme produces
approximately 3.5 times more full-length ssDNA than the wild type
enzyme. The residual ability of RecBCD
enzyme to recognize
and to degrade dsDNA might arise from the presence of
undetectable amounts of full-length RecD subunit that may result from
limited suppression of the presumed nonsense mutation in the recD1011 allele, or it may be an intrinsic property of the
truncated RecD polypeptide. Regardless, the findings presented here are
consistent with results using crude cell extracts of recBC*D and recBCD
mutant strains (Amundsen et
al., 1986; Chaudhury and Smith, 1984; Ponticelli et al.,
1985; Schultz et al., 1983; Taylor, 1988).
Figure 6:
Ability of RecBC*D and RecBCD enzymes to recognize
. Specific
-recognition
assays contained the standard DNA unwinding reaction mixture; the
linear dsDNA StyI-pBR322
H (bottomoffigure) was radioactively end-labeled at the
5`-end. Reactions were performed and analyzed in a manner similar to
that described in the legend to Fig. 3. Reactions were initiated with
0.31, 0.17, and 1.8 nM wild type RecBCD, RecBC*D, and RecBCD
enzymes, respectively (0.025 functional enzyme/dsDNA end
based on the observed stoichiometry shown in Table II). The unwound
full-length ssDNA (4316 nucleotides) and
-dependent ssDNA (2129
nucleotides;
-ssDNA) are
indicated.
Detection by gel
electrophoresis of the joint molecules produced in RecABCD-dependent
joint molecule formation using the mutant RecBC*D and RecBCD enzymes is shown in Fig. 7. Reactions were similar to
those in Fig. 4and Fig. 5except that the linear dsDNA
contains a single
sequence (EcoRI-pBR322
F). Wild type enzyme shows the characteristic
formation of both
-independent (due to invasion by full-length
ssDNA) and
-dependent (due to invasion by the ssDNA fragment
downstream of
) joint molecules. In contrast, no discrete joint
molecules are produced in reactions with the RecBC*D enzyme at early
times; after 10 min, only
-independent (both full-length and
heterogeneous length) joint molecules are detected. In reactions
containing the RecBCD
enzyme, nearly all of the unwound DNA
is full-length and, consequently, the predominant joint molecule
produced is derived from invasion by the full-length ssDNA (Fig. 7,
-indep. JM).
Figure 7:
Ability of the mutant RecBC*D and RecBCD enzymes to form
-dependent and independent joint
molecules. Standard RecABCD-reaction conditions were used, except all
DNA and protein components were increased by a factor of 4. The donor
linear dsDNA EcoRI-pBR322
F
(
dsDNA) was radioactively end-labeled at the
5`-end. Reactions were initiated with the addition of 1.25 nM wild type RecBCD enzyme, 0.70 nM RecBC*D enzyme, or 7.08
nM RecBCD
enzyme where indicated (0.025 functional
enzyme/dsDNA end based on the observed stoichiometry shown in Table I).
All values reported are a percentage of the total input linear dsDNA
contained in a reaction that is unwound. Specific
-dependent (
-dep.JM) and
-independent (
-indep.
JM) joint molecules are indicated as well as the
-dependent
ssDNA fragment (
-ssDNA) and full-length
ssDNA.
The formation of homologously paired joint molecules by the
concerted action of RecA, RecBCD, and SSB proteins is clearly
stimulated by the presence of the recombination hotspot, .
promotes an increase in both the rate and extent of overall joint
molecule formation when present in the donor linear dsDNA (Dixon and
Kowalczykowski, 1991; Eggleston and Kowalczykowski, 1993b). Here we
refine this conclusion by demonstrating that 1) the relative amount of
-specific joint molecule formation exceeds that expected based
solely on the increased level of
-fragment production that
results from attenuation of nuclease activity, suggesting an additional
role for
; 2) the pattern of ssDNA product formation is highly
dependent on Mg
concentration and, for
DNA,
-specific ssDNA fragments derived
from both the
-containing and
-complementary strands are
detected; and 3) the in vitro behavior of two additional
mutant RecBCD enzymes in
-dependent reactions is consistent with
the expectations of the nuclease attenuation model (Dixon and
Kowalczykowski, 1991, 1993; Eggleston and Kowalczykowski, 1993a, 1993b;
Kowalczykowski, 1994).
A common observation in the -dependent
RecABCD reactions is the enhanced formation of
-dependent joint
molecules relative to
-independent joint molecules (e.g. see Fig. 1). Since binding of the ssDNA fragments by RecA
protein is required for joint molecule formation, the observed
stimulation of
-dependent joint molecule formation can therefore
result from an increase in the concentration of
-specific ssDNA
fragments relative to that of the nonspecific fragments as a
consequence of nuclease attenuation, from an increased kinetic ability
of RecA protein to bind to the
-containing ssDNA as it is being
produced by RecBCD enzyme, or from an increased stability of the RecA
protein-
-specific ssDNA complexes. The first possibility is the
simplest explanation for the increased invasion by
-specific
ssDNA fragments. Since
attenuates the nuclease activity of
RecBCD enzyme (Dixon and Kowalczykowski, 1991; Dixon and
Kowalczykowski, 1993), the concentration of
-specific fragments
is greater than that of any other specific-length ssDNA fragments
produced in the presence or in the absence of
. This hypothesis,
however, does not fully explain the bias displayed for invasion by
-specific fragments over the full-length linear ssDNA in these
reactions, since especially when normalized for the amount produced,
formation of
-dependent joint molecules is 4-5-fold more
efficient than formation of
-independent joint molecules. The
kinetic explanation is based on the fact that the 3`-end of ssDNA is
more invasive than the 5`-end (Dixon and Kowalczykowski, 1991, Konforti
and Davis, 1987, 1990); therefore, a simple explanation for the bias in
joint molecule formation is that the 3`-end at
is formed first,
whereas the 3`-end of the complementary (5`-terminal) strand is distal
to
and is liberated as ssDNA later. Immediate binding of RecA
protein to the 3`-end at
permits rapid formation of a
presynaptic complex at that end. The outcome of such a temporal
sequence of events may result in an apparent increase in the rate of
invasion by the
-specific ssDNA fragments, resulting in a greater
yield of stable (plectonemic) joint molecules. However, this hypothesis
does not explain why the total pool of heterogeneous ssDNA fragments,
which must possess a 3`-end, does not contribute to an equally
significant population of heterogeneously sized joint molecules. The
final explanation, which is that the
sequence itself may
interact preferentially with RecA protein and/or with its target DNA
sequence, seems the most unlikely. However, although both the increased
survival of
-specific fragments (relative to other discrete ssDNA
fragments) and their appearance as the first and most prevalent unique
3`-terminal ssDNA species under DNA pairing conditions may be
sufficient to explain their predominance as substrates for joint
molecule formation, recent results support the possibility that
-like DNA sequences behave anomalously in RecA protein-promoted
pairing reactions
(
)and will be descri
bed
elsewhere.
The second major outcome of this work reported here was
uncovered in the course of examining the effect of various reaction
conditions on the RecABCD-promoted -dependent pairing reaction;
two distinct conclusions emerged from the effects of Mg
concentration on the reaction. The first is that the minimum
Mg
concentration required for
-dependent joint
molecule formation is dictated by the Mg
dependence
of recA protein-promoted pairing since, at concentrations of
Mg
that are too low to support RecABCD-dependent
joint molecule formation (<4 mM Mg
),
there nevertheless is sufficient
-specific ssDNA fragment
production by RecBCD enzyme. The second conclusion is that the amount
of
-specific ssDNA fragment production increases with decreasing
Mg
concentration, to a point ( Fig. 3and Fig. 5). As elaborated elsewhere (Kowalczykowski, 1994;
Kowalczykowski et al., 1994), this behavior is readily
explained by the reduction of nuclease activity that accompanies a
decrease in the free Mg
concentration (Eggleston and
Kowalczykowski, 1993a) and by the following simple but comprehensive
view of the relationship between the nuclease and helicase activities
of RecBCD enzyme (Fig. 8).
Figure 8:
Pattern
of ssDNA fragment production by RecBCD enzyme and at various
free Mg
concentrations. The pattern of ssDNA fragment
production is strongly dependent on the free Mg
concentration. At the lowest free Mg
concentrations, nuclease activity is nearly comple
tely suppressed
but helicase activity is not, resulting in the production of
predominantly full-length ssDNA regardless of the presence of a
sequence. The pause at
increases the probability of cleavage at
, resulting in formation of both upstream and downstream
-specific fragments at higher Mg
concentrations. A further increase in Mg
concentration increases nuclease activity, resulting in cleavage
of the bottom DNA strand containing the
complement, and in
degradation of the top upstream fragment; the downstream
-specific fragment is preserved due to attenuation of nuclease
activity elicited by interaction with
. Continued increase in the
free Mg
concentration results in loss of all
-specific fragments due to the increased probability of random
endonucleolytic cleavage during DNA
unwinding.
The nuclease activity of RecBCD
enzyme is envisioned to act on ssDNA endonucleolytically during the
course of DNA unwinding (translocation), and the nuclease and helicase
activities are considered to be independent of one another. The
helicase activity is relatively insensitive to Mg concentration (Roman and Kowalczykowski, 1989a), but the
frequency of endonucleolytic cleavage is quite sensitive (Eggleston and
Kowalczykowski, 1993b). The probability of recovering an ssDNA species
of a given size is a function of both translocation rate and cleavage
frequency, with the average size produced increasing as the
translocation rate increases and the cleavage frequency decreases. If
the enzyme pauses at a sequence, as it does at
(Dixon and
Kowalczykowski, 1993), then the probability of cleaving
(nonspecifically) at that sequence is increased in proportion to the
length of the pause. Furthermore, despite the attenuation of nuclease
activity that is elicited by
, some nuclease activity remains
``downstream'' of
. This occasional nicking after
by the attenuated enzyme results in a lower net yield of the
-specific ssDNA fragments. Reduction of the free Mg
concentration reduces the overall nucleolytic activity of RecBCD
enzyme, resulting in fewer cleavage events both upstream and downstream
of
. However, further reduction of nuclease activity by reducing
the concentration of Mg
to below 125 µM diminishes the overall yield of
-specific fragment; this is
due to a decrease in the probability of cleavage by RecBCD enzyme when
it is paused at
. Thus, fewer DNA molecules are cleaved in the
vicinity of
and, despite the fact that fewer molecules would be
degraded downstream of
, the net yield of
-specific
fragments is reduced; instead, because of the overall lower level of
nuclease activity, more full-length ssDNA is produced.
This general
idea that the detection of -specific ssDNA fragments in vitro is a trade-off between the frequency of cleaving (nonspecifically)
in the vicinity of
while paused and the frequency of cleaving
(nonspecifically) while translocating through the DNA explains another
feature of Fig. 1. At the lower Mg
concentrations, the
-specific fragment derived from the DNA
strand that is 3` at the entry site (the ``upstream''
fragment) is detected (Ponticelli et al., 1985). This follows
because when both the frequency of nonspecific degradation is low and
the distance to
is short, the probability of cleavage in the
``upstream'' region is low. This behavior, coupled with a
reasonable probability of cleavage at
, permits detection of the
upstream
-specific ssDNA fragment under these conditions.
Finally, the data in Fig. 1illustrate the formation of a
novel -specific fragment at the lower Mg
concentration. This fragment has a mobility consistent with
cleavage of the lower, non-
-containing (i.e.
complement) DNA strand in the vicinity of the
sequence. Recent
studies verify the identity of this species as
-specific, and
both its formation and properties will be described elsewhere.
(
)
The third major outcome of the findings presented
here is the demonstration that the mutant RecBC*D and RecBCD enzymes are unresponsive to the presence of
within
linear dsDNA, in agreement with their phenotypic behavior (Chaudhury
and Smith, 1984; Schultz et al., 1983). Although the mutant
enzymes are sufficiently processive to unwind completely the dsDNA
substrates used ( Fig. 6and Fig. 7), they are less
efficient than the wild type enzyme at joint molecule formation in the
presence of
( Fig. 4and Fig. 5; Table I), and
few or no
-dependent joint molecules are formed (Fig. 7).
However, the reason for the absence of
-specific species is
different for each mutant enzyme.
The inability of the RecBC*D
enzyme to stimulate joint molecule formation in the presence of
is most simply explained by a failure of the enzyme to recognize
, to attenuate its nuclease activity, or both (Fig. 7).
Since the mutation maps to the recC gene (Schultz et
al., 1983), these findings suggest that at least one of the
domains responsible for
recognition or attenuation of nuclease
activity resides in the RecC subunit. To explain the ability of this
class of mutants to promote modest levels of recombination in the
absence of
recognition, it was proposed that they are capable of
recognizing and stimulating recombination at undefined sequences other
than 5`-GCTGGTGG-3` (Schultz et al., 1983). Evidence for this
possible explanation, however, was not seen here, perhaps because the
linear donor DNA used (pBR322) lacked this postulated sequence. Thus,
the phenotypic behavior of the RecBC*D enzyme resembles, to a less
severe degree, that of the RecB
CD enzyme, which also
fails to recognize
and/or to attenuate its nuclease activity
(Eggleston and Kowalczykowski, 1993a, 1993b).
In contrast, although
the RecBCD enzyme failed to produce
-specific species,
its ability to form joint molecules was nearly identical with that of
the wild type enzyme. But, as seen in Fig. 7, this ability is due
to the substantially greater amount of full-length ssDNA produced and,
consequently, of joint-molecules containing full-length ssDNA. Thus,
the RecBCD
enzyme lacks most of the nuclease activity
associated with the wild type enzyme and, hence, behaves like a
constitutively
-activated, nuclease-attenuated enzyme. Due to the
nature of enhanced recombination observed in recD cells
(Chaudhury and Smith, 1984; Lovett et al., 1988; Thaler et
al., 1989), the inability of RecBCD
enzyme to promote
-specific joint molecule formation was anticipated. Genetic
studies led to the view that recD mutations resulted in a
recombinogenic form of the enzyme, ostensibly the RecBC enzyme, that
was equivalent to the
-activated form of the RecBCD enzyme
(Thaler et al., 1989). The reconstituted RecBC enzyme, devoid
of the RecD subunit, is a DNA helicase that neither degrades DNA nor
interacts with
sequences (Boehmer and Emmerson, 1991; Masterson et al., 1992; Korangy and Julin, 1993; Dixon et al.,
1994). Subsequent biochemical studies established the equivalence of
the RecBC and the
-activated RecBCD enzymes (Dixon et
al., 1994). Since a majority of the recBCD
mutations are amber mutations producing a severely
truncated or undetectable RecD polypeptide, it is reasonable that the
RecBCD
and the RecBC enzymes are nearly functionally
equivalent.
The findings presented here suggest that may have
an additional role in the stimulation of homologous pairing that goes
beyond the crucial regulation of the nuclease activity of RecBCD
enzyme. Although regulation of nuclease activity is required, as
demonstrated with the RecBC*D and RecB
CD enzymes, the
constitutive attenuation of nuclease activity inherent in RecBCD
enzyme does not allow the same level of pairing in vitro that is observed with wild type RecBCD enzyme and
. Though
the RecBCD
enzyme produces even greater amounts of
full-length ssDNA, the extent of joint molecule formation (when
normalized for the amount of ssDNA produced) is less than that observed
for
-dependent joint molecule formation by the wild type enzyme
(see Fig. 7). Thus, the possibility remains that either the
manner by which
-specific ssDNA is presented by RecBCD to RecA
protein or the interaction of RecA protein with
-specific ssDNA
fragments is somehow unique.
Table: Effect of on joint molecule formation by
wild-type and mutant RecBCD enzymes
Reactions were performed as
described under ``Materials and Methods'' using standard
RecABCD reaction conditions containing recipient H-labeled
supercoiled pBR322
° DNA.
Table: 0p4in
Due to the low specific activity
of the RecBCD
enzyme preparation, saturating rates of dsDNA
unwinding could not be attained with an experimentally accessible
RecBCD
concentration. Thus, the observed stoichiometry and
apparent k
are minimal estimates based on
highest rate of DNA unwinding (135 nM base pairs/s) that was
experimentally attainable; the highest rate obtained was about of the
plateau value obtained for both wild type and RecBC*D enzymes
(400-450 nM base pairs/s).