(Received for publication, May 15, 1995)
From the
RecBCD enzyme is essential for the major pathway of homologous
recombination of linear DNA in Escherichia coli. It is a
potent nuclease and helicase and, during its unwinding of
double-stranded DNA, makes single-strand scissions in the vicinity of
Chi recombination hot spots. We report here that both the strand that
is cut and the position of the cuts relative to Chi depended on the ATP
to Mg ratio. With ATP in excess, Chi-dependent nicks
occurred, as we have previously reported, four to six nucleotides to
the 3`-side of the Chi octamer (5`-GCTGGTGG-3`) and were detected only
on the strand bearing that sequence. Three differences were seen with
Mg
in excess. 1) Chi-dependent 3`-ends were produced
on the GCTGGTGG-containing strand closer to and within the Chi octamer.
2) Chi-dependent cuts occurred on the complementary DNA strand. 3)
RecBCD enzyme destroyed the 3`-terminated strand of DNA from its entry
point up to the vicinity of the Chi site, as others have previously
reported. We show here that, with Mg
in excess, the
enzyme continued to travel along DNA, after encountering a Chi site,
releasing both strands of the DNA distal to Chi as single strands. We
discuss potential biological consequences of these two modes of RecBCD
enzyme-Chi interaction.
RecBCD enzyme (EC 3.1.11.5), encoded by the recB, recC, and recD genes of Escherichia coli, is
the best characterized enzyme specific to the major (RecBCD) pathway of
homologous recombination in E. coli conjugation and
transduction (reviewed in (1) ). Purified RecBCD has many
activities. It has potent ATP-dependent ss ()and ds DNA
exonuclease activities and a weak ATP-stimulated endonuclease activity
on ss, but not ds, circular DNA. Under conditions that reduce these
nuclease activities (e.g. 5 mM ATP and 1 mM Mg
) the enzyme totally unwinds ds DNA, up to 40
kilobase pairs or longer in the presence of SSB (2, 3, 4) . Electron microscopy revealed that
the enzyme travels unidirectionally and highly processively from a ds
DNA end through the DNA, with either transient or permanent unwinding
of the DNA behind the enzyme(5, 6) . Under such
conditions the enzyme has a potent site-specific cleavage activity at
Chi sequences (5`-GCTGGTGG-3`)(7) , a recombinational hot spot
specific to the RecBCD pathway (reviewed in (8) ). This
cleavage occurs only as the enzyme is unwinding the DNA from right to
left, as Chi is written here(9) . The enzyme makes
Chi-dependent ss nicks a few bases to the 3`-side of Chi on the
GCTGGTGG-containing (``Upper'') strand (
)but makes
no detectable Chi-dependent nicks on the complementary
strand(7, 9) (Fig. 1, left). The
activities of the purified enzyme, under these conditions, correlate
well with the properties of Chi deduced from genetic studies and
support a previously proposed model of Chi-stimulated recombination by
the RecBCD pathway (8, 10) (see
``Discussion'').
Figure 1:
Action of
RecBCD enzyme on ds DNA containing a Chi site. RecBCD enzyme is shown
acting on Chi-containing ds DNA under Low Mg conditions ([ATP] >
[Mg
]) (7) or High Mg
conditions ([Mg
] >
[ATP])(13) . SSB, necessarily present under High
Mg
conditions, is omitted for clarity. The previously
unreported Chidependent nicking of the Lower strand at Chi is described
in this report.
RecBCD enzyme has been coupled with RecA
and SSB proteins to produce Chi-dependent joint molecules from linear
Chi-containing ds DNA and supercoiled Chi-free ds
DNA(11, 12, 13) . This reaction requires
[Mg] to be in excess over [ATP] (e.g. 8 mM Mg
and 1 mM ATP) to allow RecA protein to promote homologous strand exchange
between the linear DNA unwound by RecBCD enzyme and the supercoiled ds
DNA. Under this condition RecBCD enzyme has a different reaction at
Chi: it degrades the Upper strand from its 3`-end up to Chi, which
attenuates the exonuclease active under this condition (Fig. 1, right). Unwinding continues, to produce ss DNA with a 3`-end
near Chi, which is used by RecA and SSB proteins to make a joint
molecule.
Under both reaction conditions ([ATP] >
[Mg]) or [Mg
]
> [ATP]) RecBCD enzyme thus produces ss DNA with a 3`-end
near Chi, the postulated substrate for RecA protein. With
[ATP] > [Mg
] RecBCD enzyme
unwinds DNA up to Chi, cuts the Upper strand at Chi, and continues
unwinding to produce ss DNA with Chi near its 3`-end (Fig. 1, left). With [Mg
] >
[ATP] the enzyme degrades the Upper strand up to Chi, ceases
degradation, and continues unwinding to produce a similar ss DNA
fragment (Fig. 1, right). To explore which reaction
more nearly reflects that in E. coli cells, we have further
characterized the reaction products under the two conditions. Markedly
different products were observed, and we discuss their relationship to
the types of genetic recombinants produced by the RecBCD pathway.
During the course of
this work we examined the differences between these two reaction
conditions, using Chi cleavage assays similar to those described below.
We found that, in the High Mg reactions, MOPS buffers
with pH values between 7.0 and 7.5 would substitute for the Tris
acetate and that omission of the ATP-regenerating system did not affect
the results. Likewise we observed that omission of SSB from the Low
Mg
reactions did not alter the products
observed(23) . (
)
Using a 3`-end-labeled
substrate, we examined the survival of ds DNA, and its Chi-dependent
cleavage products, under conditions related to High Mg conditions.
Neither DNA unwound by RecBCD enzyme nor
Chi-dependent ss fragments were observed if Mg
exceeded ATP either at high ATP concentrations (5 mM ATP, 8 mM Mg
) or at low ATP
concentrations (1 mM ATP, 8 mM Mg
),
even in the presence of SSB, as observed by Dixon and
Kowalczykowski(13) . When ATP was in excess, ss DNA reaction
products survived both at high ATP concentration (5 mM ATP, 3
mM Mg
) and at low ATP concentration (1
mM ATP, 0.5 mM Mg
). The ratio of
ATP to Mg
(presumably free Mg
ion)
is thus the salient feature distinguishing the two reaction conditions,
in accord with previous observations(22, 24) . In the
experiments reported below, either 5` or internal
P labels
were used, to allow detection of Chi-specific reaction products under
either reaction condition.
Figure 2:
Reaction of linear ds DNA with RecBCD
enzyme under High Mg conditions. A,
Chi-dependent cleavage of both strands of duplex DNA. DNA substrates
were plasmid pBR322, and its
E or
F derivatives, linearized at the unique NdeI site and
P labeled at both 5`-ends. RecBCD
enzyme was incubated with DNA, in reaction mixtures lacking both ATP
and heparin, to allow binding of enzyme to DNA ends. After 5 min or
more at 37 °C, reactions were started by the addition of a mixture
of ATP and heparin and were stopped after 90 s. The reaction mixture
(10 µl) contained 1 nM DNA and 1 mg/ml heparin in High
Mg
reaction buffer (see ``Experimental
Procedures'') and the indicated concentrations of RecBCD enzyme.
In lanes 1, 8, and 20, heparin was added to
the reaction mixture prior to addition of RecBCD enzyme. Size markers,
chosen to produce fragments approximately the same size as those
produced by RecBCD enzyme cutting at Chi sites, were restriction
digests of labeled substrate DNA. They were denatured before
electrophoresis, as were the unreacted DNA samples in lanes 2, 9, and 19. EagI (lane 7) cuts pBR322 at base
pair 940, close to the
E site (nt
984-992). In this figure, but not in other experiments (not
shown), overdigestion by PpuMI (lane 14) produced
cuts at sites other than the canonical site at base pair 1481, near the
F site (nt 1493-1500). Samples
were analyzed on a 1.2% native agarose gel. RecBCD per DNA denotes the ratio of RecBCD enzyme molecules to DNA molecules. B, release of reaction products as single strands. Reactions
(as above but without heparin) were for 60 s with 0.5 nM RecBCD enzyme and 1 nM
E DNA. RecBCD enzyme was omitted from lanes 25 and 26. The indicated samples were denatured by boiling
immediately before analysis on a 1.2% native agarose gel. Lanes marked M contain marker DNA, an EagI digest of pBR322, as in A.
The Chi sequence is uniquely determined by the sequence
5`-GCTGGTGG-3` (18) , and no significant effects of flanking
sequences have previously been found for the nicks on the Upper
strand(7, 9, 23) . Fig. 2shows that
the two Chi sequences tested, which are about 500 base pairs apart and
share no obvious flanking sequence similarities(18) ,
stimulated equivalent levels of cutting on the Lower strand of the DNA
under High Mg conditions.
The fragments just
described were Chi-dependent, since they were produced at much lower
levels from DNA devoid of Chi sites (lanes 10-13). In
the absence of Chi the DNA was degraded, even in the presence of SSB,
to generate both intermediates (mostly less than 800 nt long) and the
final products of degradation by the enzyme (short oligonucleotides,
seen at the bottom of the gel). Fragments apparently equivalent in size
to those resulting from Lower strand nicking at
F were seen in the
° lanes
but were severalfold enhanced by
F.
The products shown in Fig. 2A resulted from a single round of reaction of RecBCD enzyme with ds DNA. Heparin prevents binding of RecBCD enzyme to the ends of ds DNA but does not disrupt preformed complexes(25) . In the experiments in Fig. 2, RecBCD enzyme was bound to DNA ends in the absence of ATP, and the reactions were started by addition of a mixture of ATP and heparin. In the control lanes (1, 8, and 20), addition of heparin to the DNA prior to the addition of RecBCD enzyme prevented any detectable reaction on ds DNA. It is unlikely that the reaction products resulted from the subsequent action of RecBCD enzyme on ss products released in the first round of reaction, as SSB strongly inhibits the activities of the enzyme on ss DNA(2) .
The yields of the Chi-dependent fragments depended upon the RecBCD enzyme concentration. The yield of fragments resulting from nicking at Chi on the Upper strand was maximal when there was approximately one RecBCD enzyme per DNA molecule. Presumably, at higher enzyme concentrations the reaction products were destroyed during the collision between enzyme molecules traversing the DNA from opposite directions, as previously observed(13) .
Products resulting from the cutting of the Lower strand at Chi persisted, even at the highest enzyme concentrations used. We infer that the ss product was released by the enzyme, prior to its collision with another enzyme molecule, and was resistant to degradation by RecBCD enzyme, due to its coating of SSB. The maximal yield of the Lower strand cut fragment appears to be at least equivalent to that of the Upper strand cut.
In a previous report on Chi cleavage under these conditions, only faint bands are visible (Fig. 3, A and B, of (13) ) in positions consistent with cutting on the Lower strand as reported here. More recently, fragments corresponding to Lower strand cuts at Chi were reported(24) ; these fragments were Chi-dependent (cited in (24) ). Our results appear more consistent with the latter than with the former report.
Figure 3:
Resistance of hairpin ends to RecBCD
enzyme degradation. A, construction of the substrates. The
self-complementary oligonucleotide shown was 5`-end labeled with P, and some of it was self-ligated(14) . Monomer
and dimer length molecules were purified by gel electrophoresis. Some
of the purified dimer DNA was treated with ClaI or HaeIII to produce linear ds DNA with internal labels. The intermolecular dimer shown results from isomerization of the
hairpin monomer material and is not separated from the ligated dimer on
gel purification. B, reaction of hairpin DNAs with RecBCD
enzyme. Reactions (10 µl) contained, in High Mg
reaction mix lacking SSB, the indicated concentrations of RecBCD
enzyme and monomer (0.11 nM) or dimer (0.09 nM)
hairpin DNAs or restricted dimer hairpin DNAs (0.14 nM). After
10 min of incubation at 37 °C, the reactions were analyzed by
trichloroacetic acid precipitation(3) . Precipitate and
supernatant fractions were counted by Cerenkov radiation. The graph
plots the percentage of the counts precipitable before RecBCD enzyme
reaction subsequently released by RecBCD
enzyme.
Given the ss nature of the products, the comigration of the Chi-dependent species with the ss size markers confirms that the Chi-dependent cuts are in the immediate vicinity of the Chi sequences. Their exact locations are described below.
We tested this possibility by using synthetic
hairpin-shaped oligonucleotides ligated onto one end of the DNA
substrates. Hairpins prevent the entry of RecBCD enzyme under Low
Mg reaction conditions(14) . We first tested
whether such hairpin-shaped molecules were resistant to RecBCD enzyme
under High Mg
conditions in the absence of SSB.
Hairpin oligonucleotides, labeled at their 5`-termini, were
self-ligated, and the double-length products purified (Fig. 3A). Removal of the tips, by restriction enzyme
digestion, provided positive controls for RecBCD enzyme sensitivity.
The ``double-length'' DNA was reacted with increasing
concentrations of RecBCD enzyme and reaction products assayed by
trichloroacetic acid precipitation (Fig. 3B). The small
fraction of
P released by low concentrations of RecBCD
enzyme presumably came from double-length open-ended ds DNA molecules
formed by annealing of two hairpin molecules (as shown in Fig. 3A, top). However, the remainder of the
DNA was resistant to 100 times the concentration of RecBCD enzyme
needed to solubilize the control DNA species (unligated hairpin
oligonucleotides or double-length molecules whose tips had been removed
by restriction enzyme digestion). Thus, hairpin oligonucleotide caps
are indeed resistant to cleavage by RecBCD enzyme under High
Mg
conditions, even in the absence of SSB.
Substrates for Chi cutting were made by ligating hairpin
oligonucleotides onto fragments of pBR322 with or without Chi sites (Fig. 4) to produce DNA molecules in which RecBCD enzyme could
approach Chi only in the active orientation. DNA molecules were
prepared uniquely labeled in the Upper or the Lower
``strand'' of the DNA, in the position labeled A or T in Fig. 4, to allow separate examination of the two strands of the
DNA. In the E substrates the
P label was between the ds DNA end and Chi, allowing
detection of fragments extending from Chi toward the ends of the DNA
strands, while in the
F substrates the
P label was distal to Chi, allowing detection of fragments
extending from Chi toward the hairpin.
Figure 4:
Cleavage at Chi during unidirectional
travel by RecBCD enzyme. DNA substrates were 2300-base pair fragments
of pBR322, bearing the indicated Chi sites (A, °; B,
E; C,
F), extending clockwise from the EcoRI site (nt 4360) to the NdeI site (nt 2297), with
a synthetic oligonucleotide cap (Fig. 3A) ligated onto
the EcoRI site. Substrates bore
P labels on one
or the other strand of the DNA, immediately 5` to one or both of the A
or T nucleotides in the StyI site at nt 1371, as indicated in
the diagram. DNA (50 pM) was reacted for 1 min with RecBCD
enzyme under High or Low Mg
conditions as indicated
and analyzed on a 1% alkaline agarose gel. Size markers (lanes 7 and 14) were a 3`-end-labeled BstEII digest of
phage
DNA. RecBCD:DNA denotes the ratio of RecBCD enzyme
molecules to DNA molecules. The diagram below the figure shows the
locations of the Chi sites and
P labels, and their
distances in nucleotides from the hairpin. The small diagrams next to
the autoradiograms denote the parts of the molecules present in the
adjacent fragments. Half-length molecules, cut at the hairpin, are not
indicated in Panels B and C.
Reaction of these substrates
under Low Mg conditions produced products similar to
those previously observed using substrates labeled on both
strands(15) . Reaction of either
DNA
substrate produced half-length (2300 nt) molecules, resulting from
RecBCD enzyme molecules passing Chi without cutting it, then cutting
the hairpin from within; fragments of this length were the only
specific reaction products seen with
° substrates (Fig. 4A, lanes 4-6 and 11-13). Such fragments were seen with substrates labeled
either in the Upper or the Lower strand, confirming that there is
little or no preferential degradation of either strand under Low
Mg
conditions. Under High Mg
conditions, however, the half-length fragments are seen with
Lower (5`-terminated) strand labeled substrates (lanes 2 and 3) but not with Upper (3`-terminated) strand labeled
substrates (lanes 9 and 10), confirming the
strand-specific degradation previously reported under these
conditions(13) .
Nicking of the Upper strand at
E under Low Mg
conditions produced a 1300-nt fragment with the T-labeled
substrate (Fig. 4B, lanes 11-13) and a
3300-nt fragment with the Alabeled DNA (Panel B, lanes
4-6). The absence of a specific 1300-nt fragment with
A-labeled DNA under Low Mg
conditions (Panel
B, lanes 4-6) is further evidence of the lack of
Lower strand cutting under these conditions.
Under High
Mg conditions, a 1300-nt fragment was produced with
A-labeled DNA (Fig. 4B, lanes 2 and 3), demonstrating that RecBCD enzyme molecule(s) that
encounter Chi from only one direction can indeed produce Lower strand
cuts at Chi. The product was Chi-dependent (compare lanes 2 and 3 of Panel B with those of Panel A)
and was produced at low enzyme concentrations (lane 2 with 2
enzyme molecules per DNA end).
Experiments with 3`-end-labeled DNA
molecules bearing Chi sites have previously been interpreted as
evidence that RecBCD enzyme degrades the 3`-terminated strand until it
encounters a Chi sequence(13) . Such data were consistent with
the alternative view that RecBCD enzyme removed only a few
3`-terminal-labeled nucleotides, rendering invisible (in the reported
experiments) a postulated DNA fragment extending from the Chi site to
near the 3`-terminus. Results in Fig. 4B, with a
E substrate labeled internally in the
3`-terminated strand, under High Mg
conditions,
failed to reveal either any fragment of the postulated size (1300 nt)
or any Chi-dependent smear indicative of limited degradation. The
experiment thus shows that degradation of the 3`-terminated strand
extends at least 900 nt from the terminus, and presumably up to Chi.
If the RecBCD enzyme molecule that nicked the Lower strand at
E under High Mg
conditions also failed to degrade the 3`-terminated strand, then
a 3300-nt T-labeled fragment would be observed (analogous to the
3300-nt A labeled fragment seen in lanes 4-6 of Panel B under Low Mg
conditions). The
failure to observe such a fragment (Panel B, lanes 9 and 10) implies that RecBCD enzyme molecules that cut the
Lower strand do indeed also degrade at least part of the Upper strand
of the DNA.
A capped DNA substrate, with the label distal to the Chi
site, permits investigation of the nuclease activities of RecBCD enzyme
after it has encountered Chi (F in Fig. 4C). The oligonucleotide cap is essential to
prevent RecBCD enzyme molecules approaching from the opposite direction
and destroying the 3`-end of the Lower strand. Cleavage of the Upper
strand at
F would produce, in the
absence of other cuts, a 3800-nt fragment. Such fragments were the most
prominent product observed, under Low Mg
conditions,
with the
P label in either strand (Panel C, lanes 4-6 and 11-13). Fragments of this
size were much less prominent under High Mg
conditions (Panel C, lanes 2, 3, 9, and 10) and may be no more frequent than a
contaminant band present in the substrate (lanes 1 and 8). The most prominent reaction product with
F DNA, under High Mg
conditions, was 1500 nt long, resulting from RecBCD enzyme
cutting both at
F and at the tip of the
DNA. The occurrence of this 1500-nt fragment, and the absence of a
2300-nt fragment with Upper strand labeled DNA, confirms that the
nuclease activity of RecBCD enzyme on 3`-terminated strands is
attenuated at Chi(13) . The yield of this fragment was,
however, much lower than that of equivalent 3800-nt fragment produced
under Low Mg
conditions, showing that the attenuation
under High Mg
conditions is only partial (Panel
C, lanes 9 and 10 versus lanes 11-13).
Observation of the 1500-nt fragment with Lower strand labeled DNA shows
that RecBCD enzyme neither gains a nuclease activity nor loses its
hairpin cutting activity upon nicking the Lower strand at Chi.
In
summary, the experiments in Fig. 4demonstrate that nicking of
the Lower strand at Chi, under High Mg conditions, is
not a result of collisions between RecBCD enzyme molecules coming from
opposite directions, or from enzyme molecules encountering Chi from the
``opposite'' direction. These results, when taken with the
results from Fig. 2, imply that a single RecBCD enzyme,
encountering a Chi in the ``active'' orientation, is
sufficient to catalyze nicking of the Lower strand of the DNA at Chi.
The scission at Chi on the Lower strand is indeed a nick (or two or
more closely spaced nicks), as shown by the recovery of fragments to
both sides of it, while that on the Upper strand results from
degradation of the 3`-terminated strand up to Chi.
Figure 5:
Cleavage sites at Chi on the Upper strand
of DNA. Substrates were fragments of pBR322 and its
F derivative, extending clockwise from
the unique AvaI site (at nt 1426) to the unique PvuII
site at nt 2067, bearing a 5`-
P label on the AvaI
end. DNA (1 nM) was reacted with 0.25 nM RecBCD
enzyme for 1 min at 37 °C, purified as described under
``Experimental Procedures,'' and analyzed on an 8%
polyacrylamide-urea gel. Reactions in lanes 1-12 were
under High Mg
conditions, but with the indicated ATP
and Mg
concentrations and with MOPS-KOH buffer (pH
7.6) substituted for the Tris acetate buffer. Those in lanes
16-24 used Low Mg
conditions with the
indicated ATP and Mg
concentrations. Maxam-Gilbert
degradation products of the
° DNA were run in lanes marked G+A or C+T. Lanes
designated M contained a mixture of several restriction
digests of the
° substrate, including HpaII and HinfI. The Maxam-Gilbert ladder is identified over lanes
21 and 22, and the corresponding
F sequence is aligned next to the gel.
The gray and black arrows are a qualitative
representation of the relative levels of Chi-enhanced cutting under Low
Mg
and High Mg
conditions,
respectively.
The High Mg and
Low Mg
reactions were carried out with different
buffers and at different pH values, and with different ATP
concentrations, but the salient difference was the ATP to
Mg
ratio. As the ATP concentration in a High
Mg
reaction was increased (lanes 1 through 12 versus lane 23), the Chi-dependent cut sites moved toward
the positions seen in the Low Mg
reactions.
Similarly, when the ATP concentration in a Low Mg
reaction was decreased, the reaction products changed to resemble
those of a High Mg
reaction (lanes 16-24
versus lane 2).
A qualitative summary of the cut sites under
High and Low Mg conditions is shown on the right of Fig. 5and is later compared to Lower strand cuts.
Figure 6:
Cleavage sites at Chi on the Lower strand
of DNA. Substrates were fragments of pBR322, and its
F derivative, extending clockwise from
the unique EagI site at nt 940 to the DdeI site at nt
1582, bearing a 5`-
P label on the DdeI end. A, DNA (0.25 nM) was reacted with 0.1 nM RecBCD enzyme for 1 min at 37 °C, using High Mg
conditions, but with 10 mM ATP, prior to electrophoresis
on an 8% polyacrylamide sequencing gel. B, DNA (2 nM)
was reacted with RecBCD enzyme under High Mg
conditions, using heparin to prevent reinitiation as in Fig. 2. In lanes 8, 9, 27, and 28, heparin was omitted, while in lanes 5 and 6 it was added prior to RecBCD enzyme. A mixture of restriction
enzyme fragments of the substrate was run in each of the unmarked
lanes. Lanes 16 and 17 contain Maxam-Gilbert
degradation products of the Chi° substrate, with its sequence
superimposed (white and black letters are used merely
to aid legibility). The corresponding Chi
sequence is
given to the right of the figure. C, PhosphorImager analysis
of Chi-enhanced cuts (lanes 24 and 25). The entire
width of each lane was analyzed, using area analysis and manual
baseline adjustment (ImageQuant Version 3.3, Molecular Dynamics Inc.).
Numbers on the graph represent the enhancement of Chi cutting at each
position, as measured by the relative areas under the Chi
and Chi° peaks at each position. The widths of the arrows in
part B are proportional to the relative magnitude of the Chi
enhancement at each position, as determined by subtracting the area
under each Chi
peak from that under the corresponding
Chi° peak.
The results of PhosphorImager analysis of the °
and
F reaction products at the highest
RecBCD enzyme concentration are shown in Fig. 6C.
Maximal Chi-enhanced cleavage (
3-fold) was after the C (in the
center of the Chi sequence) at nt 1497, with enhancement disappearing
within 9 nt to either side. These results are shown schematically in Fig. 6and are compared, in Fig. 7, to the positions of
Upper strand cuts. Similar quantitation showed a 15-fold enhancement by
Chi of the major band on the Upper strand under Low Mg
conditions and a 4.5-fold enhancement of the major band produced
on the Upper strand under High Mg
reaction
conditions.
Figure 7:
A compilation of the Chi-enhanced nicks on
the two DNA strands near F. The Chi
sequence is in bold type. The relative sizes of the lines
depicting nicks on the Upper strand are a visual impression of the
relative amounts of nicking, from Fig. 5. The widths of the
lines below the sequence are proportional to the relative magnitude of
the Chi enhancement of Lower strand nicking at each position, as
determined in Fig. 6.
RecBCD enzyme rapidly
destroys DNA molecules with ds DNA ends generated within E. coli by the intracellular action of a type I restriction
enzyme(36) , by rolling circle plasmid replication(37, 38) or by the intracellular induction of phage 's
terminase(39) . Such degradation is reduced by Chi on the
linear DNA molecules(37, 38, 39) . The
requirement for RecA protein (37, 38, 39) and
SSB (39) suggests that RecBCD-mediated homologous
recombination, rather than simply the inactivation of the exonuclease
activity of RecBCD enzyme by Chi or the titration of the enzyme by the
large number of ds DNA ends in the cell, is responsible for the
apparent loss of exonuclease activity.
Two groups have investigated
the trans effect of a plasmid bearing a Chi site on the
survival of a compatible Chi-free plasmid in the same cell. Zaman and
Boles (38) did not find extensive protection of a Chi°
plasmid by a compatible Chi plasmid in the same cell.
Kuzminov et al.(39) , however, found that
intracellular linearization of a plasmid bearing Chi largely prevented
the degradation of an unrelated linearized Chi° plasmid in the same
cell. If, in contrast to the conclusion in the preceding paragraph,
this protection reflects inactivation of RecBCD enzyme, such
inactivation must be long lived. Since, however, the Chi-mediated
inactivation of purified RecBCD enzyme is rapidly reversed by High
Mg
conditions(35) , the conditions within the
cell may more nearly resemble the Low Mg
conditions
described here.
Figure 8:
Models for recombination stimulated by
RecBCD enzyme and Chi. The models are discussed in the text. The Chi
symbol () denotes the strand bearing the 5`-GCTGGTGG-3` sequence
and is omitted after the first panel. DNA synthesis in model B, third panel from the top, renders it identical to Panel A, and so the two models are combined at that point. In
all models the top panel shows the production of an invasive
single strand by the action of RecBCD enzyme on a Chi site, and the second panel shows the synapsis of this strand, under the
action of RecA protein and SSB, with a homologous DNA molecule that
need not carry a Chi. Subsequent panels show the resolution of these
intermediates to a complete recombinant or pair of
recombinants.
First, in Fig. 8A we diagram the essence of the previously proposed model, under conditions in which the Upper, but not the Lower, strand is nicked. RecBCD enzyme enters the right end of the black parental ds DNA, and travels along the DNA, unwinding and rewinding it (5) . This rewinding occurs more frequently with long (>10 kilobase pairs) DNA used for electron microscopy(5) , and likely with the DNA substrates typical in E. coli recombination, than with the short (<5 kilobase pairs) DNA used here and in other studies with Chi and purified RecBCD enzyme(7, 9, 12, 13, 14, 15, 23, 24, 35) . When the enzyme encounters Chi, it nicks the Upper strand of the DNA, and continues to unwind the DNA, resulting in extrusion of a 3`-ended single strand. SSB and RecA protein then promote invasion of the homologous region of the gray parental ds DNA, to form a D-loop. Cleavage of the D-loop, followed by annealing and ligations, produces a Holliday junction (central panel) which in turn is cleaved by one or more enzymes in E. coli to produce two reciprocal recombinants (40) .
Second, if intracellular conditions allow the exonuclease activity of RecBCD enzyme to degrade the Upper strand up to the 3`-side of Chi, the same basic model still holds (Fig. 8B). After the Holliday junction has formed, the 3`-terminated strand of gray DNA can prime DNA synthesis, resulting in the recreation of the degraded 3`-terminated strand. Hence reciprocal recombinants could be formed, regardless of the intracellular conditions, if the Chi-mediated strand cleavages are restricted to the Upper strand of the DNA.
Third, if the Lower strand, but not the
Upper strand, is nicked at Chi, then recombination could occur as shown
in Fig. 8A, except that the invading single strand
extending to the left of Chi will have a 5`-end, rather than a 3`-end.
Experiments with purified RecA protein and substrates similar to those
in Fig. 8A suggest that the 3`-terminus is preferred in
such reactions(41) . Cleavage of the Lower strand produces a
3`-ended fragment extending to the right of Chi (Fig. 8C), which would lead to Chi stimulation of
recombination to the right of Chi; this has not been observed (e.g. see (42, 43, 44) ). These observations
question whether Lower strand cutting and, hence, High Mg conditions do indeed occur in E. coli.
Fourth, Chi-mediated cuts on both strands (Fig. 8C) preclude the recovery of reciprocal recombinants, for markers bracketing Chi, from just the two DNA duplexes in the diagram. If the Upper strand is degraded up to Chi (or merely nicked at Chi) and the Lower strand is nicked at Chi, then a Holliday junction can still be formed (Fig. 8C). However, as the joint molecule is missing part of one of the parental DNAs (black DNA to the right of Chi), reciprocal recombinants cannot be formed but could be recovered if the incomplete arm of the Holliday junction recombines with a third ds DNA(45) . The partially unwound DNA structure in the upper panel of Fig. 8C, produced by continued unwinding by RecBCD enzyme after cutting both strands at Chi, is equivalent to the ``split end'' structure, hypothesized to be a recombination intermediate(46) .
In summary, the models in Fig. 8, A and B, are essentially equivalent to that
previously proposed(10) , which can account for conjugational
and transductional recombination in E. coli(47) and
the stimulation at and to the left of Chi (e.g. see (42, 43, 44) ). The model in Fig. 8C does not allow reciprocal recombinants to
emerge from a single interaction between only two parental DNA
molecules but reciprocal recombinants could arise when three or more
DNA molecules are involved, as in phage crosses(45) .
This model could also account for the integration of ds DNA fragments
into the E. coli chromosome during conjugation or
transduction. The model in Fig. 8C, however, predicts
stimulation to the right of Chi, which has not been detected (e.g. see (42, 43, 44) ).
Following the fate of DNA molecules during recombination in E. coli, for example by Southern blot hybridization of DNA extracted from cells, may reveal which mode of RecBCD enzyme action prevails in E. coli and, hence, support one or another model of recombination. Knowledge of the products of RecBCD enzyme reaction on Chi-containing DNA, as reported here, will aid the design and interpretation of such experiments.