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.

EXPERIMENTAL PROCEDURES

Proteins

DNA Substrates

Plasmids

Numbering and Strand Designation Scheme

5`-End-labeled Full-length pBR322

Fragments with 5`-End Label on One Strand

Strand-specific Mid-labeled Substrates

Size Standards

Reaction Conditions

Sample Preparation and Electrophoresis

Native Gels

Denaturing (Alkaline) Gels

``Sequencing'' Gels

Identification of Cleavage Sites

RESULTS

Mg to ATP Ratio Determines Nuclease Activity

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.

Cuts on Both Strands at Chi under High Mg Conditions

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.

Reaction Products Are Released as ss DNA

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.

Strand Nicking during Unidirectional Travel by RecBCD Enzyme

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.

Position of Upper Strand Cuts at 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.

Position of Lower Strand Cuts at Chi

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.

DISCUSSION

Summary of Observations

ATP and Mg Levels in E. coli

Protection of Linear DNA by Chi in E. coli

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.

Genetic Consequences of Upper versus Lower Strand Cuts at Chi

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.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants GM31693 and GM32194. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence and reprint requests should be addressed: Fred Hutchinson Cancer Research Center, 1124 Columbia St., Seattle, WA 98104. Tel.: 206-667-4438; Fax: 206-667-6497.

()

The abbreviations used are: ss, single-stranded; ds, double-stranded; MOPS, 3-[N-morpholino]propanesulfonic acid; nt, nucleotides; SSB; single stranded DNA binding protein from E. coli.

()

The strand nicked at Chi under these conditions (that containing 5`-GCTGGTGG-3`) is designated the ``Upper'' strand. The other strand is dubbed the ``Lower'' strand.

()

A. F. Taylor, unpublished results.

ACKNOWLEDGEMENTS

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