The Role of Negative Superhelicity and Length of Homology in the Formation of Paranemic Joints Promoted by RecA Protein*

Brian C. WongDagger §, Sung-Kay ChiuDagger , and Samson A. Chowparallel **

From the Dagger  Department of Biochemistry, University of Hong Kong, Hong Kong and the parallel  Department of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, California 90095

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Escherichia coli RecA protein pairs homologous DNA molecules to form paranemic joints when there is an absence of a free end in the region of homologous contact. Paranemic joints are a key intermediate in homologous recombination and are important in understanding the mechanism for a search of homology. The efficiency of paranemic joint formation depended on the length of homology and the topological forms of the duplex DNA. The presence of negative superhelicity increased the pairing efficiency and reduced the minimal length of homology required for paranemic joint formation. Negative superhelicity stimulated joint formation by favoring the initial unwinding of duplex DNA that occurred during the homology search and was not essential in the maintenance of the paired structure. Regardless of length of homology, formation of paranemic joints using circular duplex DNA required the presence of more than six negative supercoils. Above six negative turns, an increasing degree of negative superhelicity resulted in a linear increase in the pairing efficiency. These results support a model of two distinct kinds of DNA unwinding occurring in paranemic joint formation: an initial unwinding caused by heterologous contacts during synapsis and a later one during pairing of the homologous molecules.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Escherichia coli RecA protein is a prototype of a class of recombination proteins that are widespread among prokaryotes and eukaryotes (for reviews, see Refs. 1-4). RecA protein is essential in homologous genetic recombination. In vitro, RecA protein polymerizes on single-stranded DNA to form a helical nucleoprotein filament that mediates homologous pairing with duplex DNA and subsequent strand exchange between the two parental molecules. Much of the information on RecA-promoted homologous pairing is derived from a model reaction involving circular single-stranded DNA and linear duplex DNA. In such a system wherein a free DNA end is available, the incoming single strand and its complement in the duplex DNA are free to rotate around each other. The heteroduplex DNA produced has the classical intertwined or plectonemic structure of duplex DNA (5). Since a free end only represents a small fraction of the entire molecule, the apposition of two DNA molecules, especially in vivo, is likely to occur within the interior region at a distance from the ends. RecA protein also promotes the homologous alignment of two DNA substrates in the absence of a free end. The resulting joint is termed paranemic: the DNA strands from the two parental molecules are paired in the homologous region but not topologically linked, and the joint is unstable in the absence of RecA protein (5, 6). Experimentally, paranemic joints can be studied free from plectonemic joints by pairing circular single-stranded DNA with either superhelical DNA or linear "buried homology" duplex DNA in which the homologous region is flanked at both sides by stretches of heterologous sequences (5, 7). Neither combination has net intertwining of the aligned DNA strands nor complete displacement of the strand of like polarity within the original duplex.

A poorly understood event in homologous recombination is homology recognition. Since a paranemic joint is a key intermediate in homologous pairing and the joint formation takes place at or immediately after homology recognition (5, 6), studies on factors critical for paranemic joint formation may provide clues on the mechanism for the search of homology. One such factor is negative superhelicity. During joint formation, the double-stranded DNA is unwound (8, 9), which is defined here as a reduction in twist without a change in linking number. DNA unwinding mediated by RecA protein involves a disruption of base stacking but presumably not the base pairing. The extensive unwinding of duplex DNA is probably a result of binding and alignment to the incoming nucleoprotein filament, the axial spacing of which is extended about 1.5 times that in native B-form DNA (10, 11). Because of the unwinding, the topology of the duplex DNA affects the efficiency of paranemic joint formation. Negatively superhelical DNA is a better substrate in forming paranemic joints than positively superhelical DNA and relaxed circular duplex DNA (12, 13). Negative superhelicity also plays an essential role in allowing the formation of paranemic joints between imperfectly homologous molecules (5). The mechanism by which negative superhelicity stimulates paranemic joint formation is unclear.

Besides substrate topology, the length of homology also influences homologous recombination (14-17). Since homologous recombination takes place with little sequence preference, the process requires a minimal length of homology to differentiate between homologous and heterologous contacts (18). Depending on the pathway involved, genetic studies showed that the minimal length for homologous recombination is about 20-100 bp1 (15, 16). Above the minimal length, the frequency of homologous recombination increases with the length of homology. When homologous pairing is restricted to DNA ends in vitro, formation of stable joint molecules by RecA protein requires a homology length of about 30 bp (19, 20). Although the size of paranemic joints ranges from a few hundred to several thousand bp (5, 9, 21-23), the minimal homology length required and the effect of homology length on paranemic joint formation have not been fully examined.

In this report, we showed that the efficiency of paranemic joint formation was dependent on the length of homology and the topological forms of the duplex DNA substrate. The minimal length of homology required varied with the topological forms of the duplex. We found that negative superhelicity stimulated joint formation by favoring the initial underwinding of duplex DNA but was not involved in the stabilization of the paranemic joint itself.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials

RecA protein was purified as described (24). The concentration of RecA protein was determined by absorbance using a value for E277 nm1% (corrected for light scattering) of 6.33 (25). E. coli topoisomerase I was a gift from Dr. Leory Liu at Robert Wood Johnson Medical School, and wheat germ topoisomerase I was purchased from Promega. E. coli single-stranded DNA-binding protein was purchased from Amersham Pharmacia Biotech. Creatine phosphokinase (Type I), S1 nuclease, and proteinase K were from Sigma. Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs. Nuclease-free bovine serum albumin was purchased from Life Technologies, Inc. [methyl-3H]thymidine (6.7 Ci/mmol) was obtained from NEN Life Science Products. ATP was from Amersham Pharmacia Biotech, and spermidine and creatine phosphate were from Sigma. All other chemicals were purchased from Sigma or BDH Laboratory Supplies.

DNA Substrates

Cloning of Phage DNA into Plasmid DNA-- To prepare chimeric DNA molecules, various lengths (59-662 bp) of phage DNA (phi X174 or G4) were inserted into the multiple cloning site of plasmid IBI20 (International Biotechnology Inc.) or Bluescript II KS+ (Stratagene) using standard cloning procedures (26). The construction of the chimeric plasmids is described in the legend to Fig. 1. For convenience, the chimeric plasmids were named pX/Y-Z, where X refers to the source of the plasmid vector, Y refers to the source of the phage insert, and Z refers to the length of the insert DNA.

Preparation of Single-stranded and Double-stranded DNA-- Circular single-stranded (viral or plus strand) DNA and supercoiled [3H]DNA from phages phi X174, G4, M13, and the chimeric phage M13Gori1 were prepared as described (24). Circular single-stranded DNA of the phagemid Bluescript II KS+ was prepared as suggested by the manufacturer. The purities of the circular single-stranded and supercoiled DNA preparations were checked by agarose gel electrophoresis, and the preparations were found to contain less than 5% linear and nicked molecules, respectively. The concentrations of single-stranded DNA and double-stranded DNA were measured by absorbance at 260 nm with an extinction coefficient of 8.3 and 6.7, respectively. Unless otherwise stated, the concentration of DNA was expressed in mol of nucleotide residues.

Preparation of Relaxed Circular Duplex DNA-- Relaxed circular duplex DNA was prepared by incubating 10 µg of the negatively supercoiled DNA with 10 units of wheat germ topoisomerase I (Promega) at 37 °C for 30 min under conditions described by the manufacturer. Such treatment resulted in the relaxation of greater than 95% of the negatively supercoiled DNA as determined by agarose gel electrophoresis.

Preparation of Supercoiled DNA with a Defined Negative Superhelicity-- To produce a set of superhelical DNA with different numbers of superhelical turns, negatively superhelical DNA of pKS/G4-138 (3,074 bp) or pKS/G4-662 (3,571 bp) isolated from E. coli strain HB101 was treated with wheat germ topoisomerase I in the presence of various amounts of ethidium bromide. The amount of ethidium bromide used was adjusted such that for each superhelical turn to be retained after the enzyme treatment, 14 molecules of ethidium bromide was added for each plasmid duplex DNA molecule (27, 28). The concentration of the ethidium bromide solution was determined by absorbance at 480 nm using an extinction coefficient of 5.6 mM-1 cm-1 (29).

Except for the presence of ethidium bromide, the reaction conditions were the same as that described earlier for the production of relaxed circular duplex DNA. After treatment with topoisomerase I, the reaction mixture was treated with 0.1% SDS, and 300 µg/ml proteinase K at 37 °C for 15 min. The ethidium bromide and proteins were then removed by three rounds of extraction with phenol/chloroform/isoamyl alcohol (25:24:1) and followed by ethanol precipitation. The DNA was then redissolved in 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA. The production of superhelical DNA containing different superhelical densities was confirmed by two-dimensional agarose gel electrophoresis (30). The DNA bands were visualized by staining with 0.5 µg/ml ethidium bromide. The mobility of the DNA products increased with increasing amount of ethidium bromide present in the reaction mixture, corresponding to an increasing degree of negative superhelicity (27). As a negative control, supercoiled DNA was incubated in the complete reaction mixture without wheat germ topoisomerase I, and the mobility of the DNA remained unchanged. The superhelical density of untreated plasmid DNA in our preparation was about -0.07.

Standard Reaction Conditions

Unless otherwise stated, the reaction mixture contained a final concentration of 33 mM Tris-HCl (pH 7.5), 1.3 mM ATP, 6 mM creatine phosphate, 10 units/ml creatine phosphokinase, 88 µg/ml bovine serum albumin (nuclease-free), 1.8 mM dithiothreitol, 1 mM MgCl2, and circular single-stranded DNA. The mixture was incubated for 2 min at 37 °C. RecA protein was then added, and the incubation was continued for another 10 min at 37 °C. The amount of RecA protein was added in a ratio of 1 mol of RecA protein for every 2 mol of total nucleotide residues available for RecA binding. The pairing reaction was initiated by adding 4 µM duplex [3H]DNA and adjusting the final concentration of MgCl2 to 13 mM. Unless otherwise specified, the number of single-stranded DNAs in the pairing reaction was in 4-fold excess over the number of duplex DNA molecules. The concentration of single-stranded DNA (ss) used was calculated as follows: [ss] = 4 ([duplex]/2)(length of ss/length of duplex).

Nitrocellulose Filter Assay for Joint Molecules

The formation of paranemic joints was measured by a P-loop assay (24), which is modified from the D-loop assay (31). Both assays measure the retention by nitrocellulose filters of duplex DNA that has attached to single-stranded or partially single-stranded DNA. However, in the P-loop assay, the removal of RecA protein by high salt concentrations is minimized in order to preserve the metastable joints. At appropriate times, 10-µl aliquots of the reaction mixture were directly diluted in 4.2 ml of ice-cold 1.5 M NaCl, 0.15 M sodium citrate, 10 mM EDTA (pH 7.5). The solution was immediately filtered through a nitrocellulose filter (Sartorius type SM11306, 0.45-µm pore size) that had been soaked in 1.5 M NaCl, 0.15 M sodium citrate (pH 7.5). The filters were then rinsed three times with 2 ml of ice-cold 1.5 M NaCl, 0.15 M sodium citrate (pH 7.5), dried under a heat lamp, and put into vials with 5 ml of toluene containing 0.5% 2,5-diphenyloxazole and 0.01% 1,4-bis[2-(5-phenyloxazolyl)]benzene. The radioactivity was determined in a scintillation counter.

To measure the formation of joint molecules that were stable in the absence of RecA protein (32), at appropriate times we added to the sample aliquots a final concentration of 1% SDS and 20 mM EDTA. The samples were then incubated at 4 °C for 6 min prior to the nitrocellulose filter assay as described above. Alternatively, we added to the sample aliquots a final concentration of 0.1% SDS and 300 µg/ml proteinase K. The mixture was incubated at 37 °C for 5 min before passing through nitrocellulose filters.

Unwinding Assay

Unwinding of duplex DNA was examined by pairing circular single-stranded DNA with relaxed closed circular duplex DNA. The three-stranded pairing reaction was carried out as described under "Standard Reaction Conditions." Twenty minutes after the addition of 8 µM relaxed circular duplex DNA, a 100-µl aliquot was removed, and 3 units of wheat germ topoisomerase I was added to the mixture. After a further incubation at 37 °C for 2 min, the reaction was terminated by adjusting the mixture to 300 µg/ml proteinase K, 20 mM EDTA, and 0.1% SDS. The mixture was then incubated at 37 °C for 15 min, and the products were analyzed by two-dimensional electrophoresis on a 1.0% agarose gel (30). The samples were separated in the first dimension at 7 V/cm for 5 h in the absence of any intercalating agent. The gel was then soaked in 2 liters of 0.09 M Tris borate, 2 mM EDTA (pH 8.0) containing 4 µM of chloroquine for 16 h with gentle shaking. In the second dimension, the gel was placed at 90° to the previous dimension, and electrophoresis was carried out at 7 V/cm for 5 h with the soaking buffer used as the running buffer. After gel electrophoresis, the gel was soaked in distilled water for 16 h to remove chloroquine prior to staining in 0.5 µg/ml ethidium bromide for 15-30 min. The gel was then destained in 10 mM MgSO4 solution prior to UV photography.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

DNA Substrates for the Formation of Paranemic Joints-- RecA protein promotes the pairing between circular single-stranded and circular double-stranded DNA to form paranemic joints in which the DNA strands are paired without net intertwining (5). In this study, we used two combinations of DNA substrates for the formation of paranemic joints: pairing of a circular single-stranded DNA with either a chimeric closed circular duplex DNA or a linear "buried homology" duplex DNA in which the homologous region is flanked on both sides by stretches of heterologous sequences (see Fig. 1). Except for the chimeric phage M13Gori1, the chimeric duplex DNA was constructed by inserting different lengths of phage G4 or phi X174 DNA into plasmid DNA that are completely heterologous to the phage DNA sequences (Fig. 1).


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Fig. 1.   DNA substrates. A, construction of chimeric plasmids containing different lengths of phage sequences. The plasmid vector was either pIBI20 (International Biotechnology Inc.) or pBluescript II KS+ (Stratagene). The phage DNA was inserted into the polylinker region of the plasmid. E1 and E2 denote the restriction enzymes used to cleave the plasmid DNA. E3 and E4 denote the restriction enzymes used for isolating the phage DNA fragments used in the subsequent cloning step. ClaI and ScaI were the enzymes used to linearize the chimeric plasmids derived from pIBI20 and pBluescript II KS+, respectively, to produce linear "buried homology" DNA substrates. For simplicity, the chimeric plasmids were termed pX/Y-Z, where X refers to the plasmid vector, Y represents the source of phage DNA, and Z refers to the length of the phage DNA insert. Paranemic joints were formed by pairing the chimeric, double-stranded plasmid DNA with the single-stranded DNA of the phage from which the insert was derived. B, a map of the chimeric phage M13Gori1 showing the restriction sites used to produce linear "buried homology" substrates. Paranemic joints were formed by pairing the BamHI-cleaved M13Gori1 duplex DNA (substrate a) with circular G4 single strands (2,216 bp of homology) or the XhoI-cleaved M13Gori1 duplex DNA (substrate b) with circular M13 single strands (6,407 bp of homology). Thick lines represent G4 DNA sequences; thin lines represent M13 DNA sequences. In both A and B, the numbers in parentheses denote the lengths in bp of the region by which they appear, and the numbers without parentheses denote the lengths in bp of the entire molecule.

The chimeric, double-stranded DNA was paired with the single-stranded DNA of the parental phage to form paranemic joints. In one combination of DNA substrates, circular single strands and circular duplex, there was a complete absence of DNA ends. In the other combination, the duplex DNA lacked a DNA end that was homologous to the circular single-stranded DNA. Therefore, in both combinations, net intertwining of DNA strands was prohibited, and the pairing between the two DNA molecules led to the formation of paranemic joints (5, 6).

Efficiency of Paranemic Joint Formation Depends on Homology Length-- When superhelical or linear "buried homology" duplex DNA containing different lengths of phage DNA sequences were paired with the circular single-stranded phage DNA, the formation of joint molecules increased correspondingly with the length of homology shared between the two DNA substrates (Fig. 2). When the duplex DNA was superhelical, the maximal efficiency of paranemic joint formation was reached at a homology length of 2,200 bp (Fig. 2, A and C), whereas linear "buried homology" duplex DNA required a homology length of 6,400 bp to reach the plateau (Fig. 2, B and C). For any particular length of homology between 140 and 2,200 bp, superhelical duplex DNA exhibited a higher efficiency of joint formation than that of linear "buried homology" duplex DNA (Fig. 2C). At a homology length of 6,400 bp, the efficiencies of joint formation of the two kinds of duplexes were indistinguishable. The minimal length of homology required for paranemic joint formation also differed between superhelical and linear "buried homology" duplex DNA. The minimal lengths required were between 59 and 138 bp for superhelical DNA and between 497 and 662 bp for linear duplex DNA (Fig. 2).


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Fig. 2.   Effects of homology length and superhelicity on paranemic joint formation. A, superhelical DNA. B, linear duplex DNA with "buried homology." Duplex DNA (4 µM) sharing different lengths of homology with the incoming circular single-stranded DNA was used for the formation of paranemic joints. Presynaptic filaments were formed by incubating circular single-stranded DNA of phages phi X174, G4, or M13 (in 4-fold molar excess over the number of duplex molecules) with RecA protein for 10 min at 37 °C as described under "Experimental Procedures." The pairing reaction was started by adding duplex DNA and adjusting the final concentration of MgCl2 to 13 mM. The lengths of homology in bp shared between single strands and duplex molecules in various DNA combinations (see Fig. 1) were 59 (black-square), 138 (bullet ), 321 (triangle ), 497 (black-triangle), 662 (square ), 2,216 (diamond ), and 6,407 (black-diamond ). The heterologous control (open circle ) was the pairing reaction between circular single-stranded DNA of phage G4 and the parental pBluescript II KS+ plasmid DNA. The extent of joint formation was monitored by the P-loop assay. C, yield of paranemic joints as a function of homology length. The yields of paranemic joint formation obtained in A and B using superhelical (black-down-triangle ) and linear "buried homology" duplex DNA (down-triangle), respectively, at 40 min after the start of the reaction were plotted against the homology length shared between the single- and double-stranded DNA.

Negative Superhelicity Stimulates Paranemic Joint Formation-- The finding that negatively superhelical DNA generally produced a higher yield of joint molecules than their linear counterparts indicated that negative superhelicity enhanced the formation of paranemic joints. Previous studies have shown that negative superhelicity, perhaps by stabilizing the nascent structure made by RecA protein, plays a determining role in permitting the formation of paranemic joints between imperfectly homologous molecules (5) and between gapped and fully double-stranded DNA (33-35). To further investigate the effect of DNA topology on the formation of paranemic joints, we paired circular single-stranded G4 or pBluescript II KS+ DNA with various topological forms of pKS/G4-662 duplex DNA (Fig. 1A, e). In this experiment, the length of homology depends on the source of the single-stranded DNA: 662 bp for G4 and 2,950 bp for pBluescript II KS+. The results showed that, among negatively superhelical, linear, and relaxed circular duplex DNA, superhelical DNA had the highest efficiency, while relaxed closed circular duplex DNA had the poorest efficiency in the formation of paranemic joints (Fig. 3, A and B). The difference in pairing efficiency between linear and relaxed closed circular duplex DNA was more prominent with DNA substrates that shared a longer length of homology (Fig. 3B). Furthermore, with relaxed closed circular duplex DNA, the yield of joint formation was the same regardless of whether the duplex DNA shared 662 or 2,910 bp of homology with the incoming single-stranded DNA (Fig. 3, A and B).


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Fig. 3.   Efficiency of paranemic joint formation depends on the topological forms of duplex DNA. A, 662 bp of homology. Circular single-stranded DNA of phage G4 (6.4 µM) was incubated with 4.2 µM RecA protein and 1 mM MgCl2 at 37 °C for 10 min as described under "Experimental Procedures." The reaction mixture was then divided into three aliquots. To each aliquot, 4 µM of pKS/G4-662 DNA of different topological forms was added, and the final concentration of MgCl2 was raised to 13 mM to start the pairing reaction. The topological forms of the duplex DNA tested were negatively superhelical (bullet ), linear (black-square), and relaxed closed circular (black-triangle). The formation of paranemic joints was monitored by the P-loop assay. B, 2,910 bp of homology. The reaction was identical to that shown in panel A except that the circular single-stranded DNA was from phagemid pBluescript II KS+ (4 µM). The symbols are the same as in A. C, activation of relaxed closed circular duplex DNA by cleavage with a restriction enzyme. Circular single-stranded DNA of phage G4 (6.4 µM) was incubated with 1 mM MgCl2 and 4.2 µM RecA protein for 10 min at 37 °C. Four µM of relaxed closed circular pKS/G4-662 DNA (black-triangle) was added, and the final concentration of MgCl2 was then raised to 13 mM. Fifteen minutes after the addition of duplex DNA (indicated by an arrow), the restriction endonuclease KpnI (triangle ) or enzyme buffer (black-triangle) was added, and the incubation was continued for another 25 min. The formation of joint molecules was monitored by the P-loop assay.

The pairing efficiency of the relaxed circular duplex DNA could be drastically increased when the DNA was cleaved by the restriction enzyme KpnI, which introduces a single cut and generates a 3'-end complementary to the single-stranded DNA (see substrate e in Fig. 1A). When the enzyme was added to an ongoing pairing reaction involving circular single-stranded G4 DNA and relaxed closed circular duplex of pKS/G4-662, there was a prompt increase in joint formation (Fig. 3C). This experiment showed that the poor pairing efficiency of the relaxed closed circular duplex DNA was due to a topological constraint and not to an inactivation of the duplex during its preparation or to the presence of inhibitors.

Similar to our observation presented here, Rould et al. (13) reported that relaxed closed circular duplex DNA formed paranemic joints less efficiently than linear duplex. However, they found that negatively superhelical duplex DNA paired less efficiently than linear "buried homology" duplex DNA. We believe that the discrepancy may be due to a difference in the superhelical density of the duplex DNA used in the two studies. Below a certain density of negative superhelicity, we showed that superhelical duplex DNA paired less efficiently than linear duplex DNA (see Fig. 7 and "Discussion").

Negative Superhelicity Is Not Essential for the Maintenance of Paranemic Joints-- The result shown in Fig. 2 clearly indicated that, with substrates that contained short homology lengths, negative superhelicity was required for paranemic joint formation. It was not clear, however, whether negative superhelicity was required continuously for the stability of the paranemic joints once it was formed. To address this question, we formed paranemic joints by pairing negatively superhelical DNA of pKS/G4-138 with circular single-stranded G4 DNA (Fig. 4). We chose this combination of DNA substrates because with a homology length of 138 bp, there was a large difference in paranemic joint formation between the superhelical and linear DNA (Fig. 2). At various times after the start of the pairing reaction, the circular duplex DNA was converted to linear "buried homology" DNA by the addition of the restriction enzyme ScaI, which cleaves the duplex DNA once in the heterologous region (Fig. 1A). The circular duplex DNA was completely linearized within 2 min of ScaI incubation as determined by the filter assay of Kuhnlein et al. (36) (Fig. 4B) and by gel electrophoresis (data not shown).


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Fig. 4.   Superhelicity is not required for the maintenance of paranemic joints. A, survival of paranemic joints after linearization of the superhelical DNA. Circular single-stranded DNA of phage G4 (6 µM) was incubated with 4.3 µM of RecA protein at 37 °C for 10 min as described under "Experimental Procedures." Superhelical DNA of pKS/G4-138 (6 µM) was added, and the concentration of MgCl2 was adjusted to 13 mM to start the pairing reaction. The extent of formation of paranemic joints was measured by the P-loop assay (open circle ). At 0 (diamond ), 20 (square , black-square), or 50 min (triangle , black-triangle) (indicated by arrows) after the addition of superhelical DNA, restriction enzyme ScaI, which cleaves once in the heterologous region of the chimeric plasmid DNA, was added at an amount of 0.4 unit/ng of DNA to an aliquot of the reaction mixture. At various times after the addition of the enzyme, the formation of paranemic joints was measured by the P-loop assay (diamond , square , triangle ), and the formation of protein-free joints was measured by incubating an aliquot of the reaction mixture at 0 °C in 1% SDS for 6 min before the P-loop assay (black-square, black-triangle). B, time course of linearization. At 50 min after the start of the pairing reaction between the circular single-stranded DNA of phage G4 and pKS/G4-138 duplex DNA, an aliquot of the reaction mixture was removed for enzyme digestion as described above. The extents of cleavage after different times of digestion were monitored by the filter assay of Kuhnlein et al. (36). The open bar represents a negative control in which enzyme digestion buffer was added. The hatched bar represents a reaction in which the restriction enzyme ScaI was added.

When the circular duplex DNA was linearized during the pairing reaction, a substantial fraction of the joint molecules remained, and the level was unchanged for at least 25 min after the addition of the restriction enzyme (Fig. 4A). The final yield of paranemic joints was determined by the time of addition of the restriction enzyme. For instance, when the enzyme was added at the start of the pairing reaction, about 10% of the duplex was in paranemic joints throughout the course of the reaction, whereas about 60% of the duplex DNA was detected as joint molecules when the restriction enzyme was added 50 min after the start of the pairing reaction. Therefore, once the supercoiled DNA was linearized, it became incapable of joint formation. However, when the supercoiled DNA was linearized after joint formation, a majority of the joints remained even in the absence of negative superhelicity. To confirm that the joints that survived restriction enzyme treatment remained paranemic in nature, we incubated the mixture at 4 °C in 1% SDS for 6 min to remove RecA protein before the filter assay (32). No joint molecules were detected after deproteinization (Fig. 4A), indicating that these joints were indeed paranemic. Thus, we conclude that negative superhelicity promotes the formation of paranemic joints but is not necessary for the maintenance of the paranemic joints.

Linear "Buried Homology" Duplex DNA with Homology Length Shorter than 662 bp Does Not Form Paranemic Joints-- Since no paranemic joint was detected between circular single-stranded DNA and linear duplex with a homology length shorter than 662 bp (Fig. 2), it may reflect the minimal requirement of 662 bp of homology between the described substrates for paranemic joint formation. Alternatively, it was possible that linear duplex with homology length shorter than 662 bp could form paranemic joints but that they were too unstable to survive the filter assay.

Previous works using DNA substrates that share 2,216 bp of homology showed that E. coli topoisomerase I converts a metastable paranemic joint to a stable hemicatenate structure that survives heat treatment and increases the amount of joint molecules detected by the nitrocellulose filter assay (5, 37). Since E. coli topoisomerase I itself does not promote the formation of joint molecules, the observation suggests that, in the absence of the enzyme, some of the joint molecules dissociate during the filter binding assay. We reasoned that if the inefficiency of the joint molecule formation with linear duplex containing less than 662 bp of homology was due to their dissociation during the filter assay, then the addition of E. coli topoisomerase I might stabilize these nascent joint molecules by converting them to a hemicatenate structure that is stable in the filter assay.

When we added E. coli topoisomerase I to the reaction mixture immediately after the addition of linear duplex DNA, we found that E. coli topoisomerase I did not increase the yield of joint molecules from pairing reactions between circular single strands and linear "buried homology" duplex that shared 138 or 321 bp of homology (Fig. 5, A and B). On the contrary, as a positive control, the addition of E. coli topoisomerase I to a pairing reaction using ScaI-linearized pKS/G4-662 DNA, which shared 662 bp of homology with phage G4 single-stranded DNA, resulted in a slight increase in the formation of paranemic joints as well as the production of stable hemicatenate molecules (Fig. 5C). The result suggested that linear duplex DNA with a short homology did not form paranemic joints.


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Fig. 5.   Absence of paranemic joint formation with linear duplex DNA containing short lengths of "buried homology." Circular single-stranded DNA of phage G4 was incubated with RecA protein and 1 mM MgCl2 for 10 min at 37 °C. ScaI-linearized duplex DNA (4 µM) of the chimeric plasmid pKS/G4-138 (A), pKS/G4-321 (B), or pKS/G4-662 (C) was added, and the MgCl2 concentration was adjusted to 13 mM to start the pairing reaction. At the time of the addition of the duplex DNA, 11 nM E. coli topoisomerase I (open circle , bullet ) or an equal volume of enzyme buffer (triangle , black-triangle) was included. The extent of joint molecule formation was monitored by the P-loop assay (open circle , triangle ). The extent of hemicatenate formation (bullet , black-triangle) was determined by incubating the reaction mixture in 0.1% SDS and 300 µg/ml proteinase K at 37 °C for 5 min prior to the filter assay. The amount of DNA retained by nitrocellulose filters after treatment with SDS and proteinase K was the same as that obtained by treating the reaction mixture at 80 °C for 5 min in the presence of 0.15 M NaCl, 15 mM sodium citrate (data not shown; Refs. 5 and 37).

It remained possible that E. coli topoisomerase I failed to recognize paranemic joints formed from DNA molecules that shared 138 or 321 bp of homology. In a separate experiment, we detected the formation of hemicatenate when we added E. coli topoisomerase I to the paranemic joints formed from superhelical pKS/G4-138 DNA and circular single-stranded DNA of phage G4 (Fig. 6). This observation indicated that paranemic joints formed from as short as 138 bp of homology could be recognized by E. coli topoisomerase I. Moreover, we showed earlier that paranemic joints formed from superhelical duplex DNA with 138 bp of homology remained detectable by the same filter assay after the duplex was linearized (Fig. 4), indicating that the filter assay is capable of detecting paranemic joints with 138 bp of homology in the absence of negative superhelicity. Taken together, these results showed that, with linear duplex DNA containing less than 662 bp of homology, the absence of paranemic joints was primarily due to an insufficient length of homology and not to joint dissociation under the assay condition.


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Fig. 6.   Paranemic joints with a 138-bp homology length can be converted into hemicatenates by E. coli topoisomerase I. Paranemic joints were formed by pairing 10.8 µM circular single-stranded DNA of phage G4 with 4 µM superhelical DNA of pKS/G4-138 in the presence of 6.4 µM RecA protein. The extent of joint molecule formation was monitored by the P-loop assay (open circle ), and the formation of deproteinized joint molecules was measured by incubating the mixture in 0.1% SDS and 300 µg/ml proteinase K at 37 °C for 5 min prior to the P-loop assay (triangle ). Thirty minutes after the start of the reaction (indicated by an arrow), the reaction mixture was split into two equal aliquots. To one aliquot, 11 nM of E. coli topoisomerase I (bullet , black-triangle) was added, while an equal volume of enzyme buffer was added to the second aliquot. The reaction mixtures were then incubated at 37 °C, and the formation of paranemic joints (open circle , bullet ) and protein-free joints (triangle , black-triangle) was monitored for another 20 min as described earlier.

To examine whether the ability of linear duplex DNA containing short lengths of "buried homology" to form paranemic joints could be enhanced by other factors, we repeated the pairing reaction between circular single strands of phage G4 and linear "buried homology" duplex of pKS/G4-138 and pKS/G4-321 under a variety of reaction conditions. These included varying the molar ratio between single-stranded and double-stranded DNA from 1:1 to 6:1; adding E. coli single-stranded DNA-binding protein and changing the order of addition of single-stranded DNA-binding protein and RecA protein; replacing ATP with ATPgamma S; increasing the concentration of MgCl2 from 13 to 25 mM; or replacing MgCl2 with magnesium acetate. None of these conditions improved the efficiency of formation of paranemic joints (data not shown).

Unwinding of Duplex DNA during the Formation of Paranemic Joints-- Formation of paranemic joints is accompanied by an unwinding or a local reduction in twist of duplex DNA (9). Unwinding of duplex DNA during the formation of joint molecules can be detected by treating the pairing products between a circular single-stranded DNA and a relaxed closed circular duplex DNA with wheat germ topoisomerase I. We confirmed this observation by pairing a relaxed closed duplex DNA of pKS/G4-662 with a homologous circular single-stranded DNA (data not shown). Furthermore, two-dimensional gel electrophoresis showed that the degree of unwinding of the DNA species produced was proportional to the length of homology (9).2 The observation suggests that, depending on the length of homology, there may be a minimal negative superhelicity that is required for homologous pairing.

To further substantiate that homologous pairing involves unwinding of duplex DNA and to define the minimal superhelicity required for a particular length of homology, we prepared double-stranded DNA of pKS/G4-138 and pKS/G4-662 containing different numbers of negative superhelical turns and paired these substrates with circular single-stranded DNA of either phage G4 or pBluescript II KS+ to form paranemic joints (Fig. 7). The lengths of homology shared among these substrates were 138 (G4 single strands and pKS/G4-138), 662 (G4 single strands and pKS/G4-662), 2,910 (pBluescript KS II+ single strands and pKS/G4-662), and 2,936 bp (pBluescript KS II+ single strands and pKS/G4-138). Consistent with the observation that homologous pairing involves unwinding, we found that the efficiency of paranemic joint formation was proportional to the number of negative superhelical turns. In all reactions using substrates that shared various lengths of homology, relaxed closed circular DNA (zero superhelical turn) had the lowest efficiency in joint formation, and the yield of joint formation remained at the background level although there were six negatively superhelical turns present in the duplex DNA. Regardless of the length of homology, above six superhelical turns, the efficiency of joint formation increased steadily with increasing negative superhelicity. At the highest level of superhelicity tested (36 turns), nearly all of the duplex DNA was included in the paranemic joint.


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Fig. 7.   Dependence of paranemic joint formation on the level of negative superhelicity. Circular single-stranded DNA of phage G4 (7.2 µM; square , black-square) or circular single-stranded DNA from phagemid pBluescript II KS+ (4.2 µM; open circle , bullet ) was incubated with 5 µM RecA protein and 1 mM MgCl2 at 37 °C for 10 min. The pairing reaction was started by adding 6 µM pKS/G4-138 (square , open circle ) or pKS/G4-662 DNA (black-square, bullet ) containing different negatively superhelical turns and adjusting the final concentration of MgCl2 to 13 mM. The formation of paranemic joints was monitored by the P-loop assay at 40 min after the start of the reaction.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Two factors critical in the formation of paranemic joints were characterized: length of homology and negative superhelicity. We found that the efficiency of paranemic joint formation depends on the length of homology and the topological forms of the duplex DNA. With a linear duplex DNA, efficient formation of paranemic joints requires a homology length of about 700 bp. In the presence of negative superhelicity, the minimal length of homology required is reduced to 140 bp.

Our findings are consistent with the current understandings of the RecA-catalyzed homologous pairing reaction. Since the single-stranded DNA in a presynaptic nucleoprotein filament is extended upon binding to RecA protein (38), the duplex DNA has to be unwound in order to match the increased axial spacing of the single-stranded DNA during homologous pairing (8, 9). Unwinding of duplex DNA is energetically and topologically unfavorable. The efficiency of joint molecule formation therefore depends on factors that can stabilize the nascent joint, such as base pairing or protein-DNA interactions, or can accommodate the unfavorable effect of duplex unwinding, such as negative superhelicity. The presence of negative superhelicity allows duplex unwinding using the torsional energy available. Thus, when compared with the linear "buried homology" duplex, negatively superhelical duplex DNA requires a shorter homology length for paranemic joint formation. When compared with the linear "buried homology" duplex with the same length of homology (between 140 and 2,000 bp), negatively superhelical duplex has a higher efficiency of joint formation. In the absence of the torsional energy to aid unwinding, linear duplex DNA requires minimally about 700 bp of "buried homology" to provide sufficient stabilization (via base pairing and RecA protein-DNA interactions) to balance the unfavorable unwinding involved in homologous pairing.

Interestingly, although negative superhelicity stimulates the formation of paranemic joints, it is not essential in the maintenance of the joint. A majority of the paranemic joint remained when negative superhelicity was removed after the formation of the joint. This observation supports the hypothesis that negative superhelicity promotes joint formation primarily by lowering the energy barrier required for the unwinding of duplex DNA during joint formation and not for stabilizing the joint after its formation.

Relative to linear duplex DNA, the stimulation seen with negative supercoiled DNA and the inhibition observed with relaxed circular duplex DNA are also a result of DNA unwinding during paranemic joint formation. With circular duplex DNA, unwinding of duplex DNA during homologous pairing generates compensatory positively superhelical turns outside the region of pairing. These energetically unfavorable positive turns make the relaxed circular duplex DNA a poor substrate for the homologous pairing reaction catalyzed by RecA protein (13). Consistent with this explanation is the finding that the low pairing efficiency of the relaxed circular duplex can be enhanced by the addition of eukaryotic topoisomerase I, which removes the positive turns (13). On the other hand, the ability of linear duplex DNA to dissipate the compensatory turns by rotation about the DNA ends make it a better substrate than relaxed circular duplex DNA for joint formation.

Our results suggest that unwinding of duplex DNA upon interaction with presynaptic complexes is a major determinant in the requirement of a certain homology length for joint formation. In order to better understand the relationship between unwinding and homology length, the pairing reaction was carried out using circular duplex DNA with different negatively superhelical densities and lengths of homology. We found that, regardless of the length of homology, a minimum of six negatively superhelical turns are required for paranemic joint formation. Above six negatively superhelical turns, the joint formation increased with increasing superhelicity. Considering that duplex DNA bound with RecA protein is partially unwound with a helical repeat of 18 bp/turn and the average rotation/bp changes from 34.3° for B-form DNA to 19.4° (39), six superhelical turns correspond to a paired region of 140 bp ((6 × 360°)/(34.3° - 19.4°)/bp). This size also approximates the minimal length of homology required to form paranemic joints using superhelical DNA with 21 negative turns (Fig. 2). In the presence of ATP, the minimal size of plectonemic joints, wherein the DNA strands between two pairing DNA substrates are truly intertwined, is estimated to be 60 bp or less (14, 19, 20).2 If the length of homology recognition is less than 60 bp, what are the determinants that define the minimal size of paranemic joints? We hypothesized that one major factor is homology-independent unwinding of duplex DNA (13), produced by nonspecific contacts between presynaptic complexes and duplex DNA (40-42).

Our model proposes that, irrespective of the available length of homology, about 140 bp of the duplex DNA is unwound upon contact with the presynaptic filament during the search for homologous DNA sequences. Using a different assay, a similar degree of unwinding produced by heterologous contacts has also been reported (13). The homology-independent unwinding thus generates six compensatory positive turns, which are inhibitory to paranemic joint formation. We reasoned that the poor pairing efficiency of circular duplex DNA with six or fewer negatively superhelical turns is due to an insufficient number of negative supercoils needed to compensate for the unfavorable effect of positive supercoils generated by the homology-independent unwinding. The inhibition can be minimally overcome by the presence of six negatively superhelical turns, which lowers the torsional stress imposed by the positive turns and provides 140 bp of base pairing and RecA-DNA interactions, after which the presence of an increasing negative superhelicity proportionally stimulates the efficiency of joint formation as the torsional energy is progressively increased.

One possible mechanism by which the presence of an increased negative superhelicity enhances the pairing reaction is to facilitate strand separation. In the RecA-mediated pairing and strand exchange reaction, the single-stranded DNA within the nucleoprotein filament binds in the minor groove of the extended Watson-Crick duplex (43-45). One proposed model for recognition of homology and formation of a nascent joint involves local opening of the base pairs of the duplex, followed by Watson-Crick pairing of the incoming single strand with its complementary strand. Since it requires a transient opening of the duplex DNA, such a mechanism is likely to be facilitated by negative superhelicity.

Thus, the present study shows that formation of paranemic joints in the RecA-promoted pairing reaction involves two distinct unwindings. One is heterologous unwinding, which is independent of homology, has a limited size, and occurs during synapsis. The other is homologous unwinding, which depends on homology length, can be extensive in size, and occurs during joint formation. Consistent with the hypothesis that pairing involves an initial homology-independent unwinding of duplex DNA is the observation that the pairing efficiency of superhelical DNA below a certain negative superhelical density was actually lower than that of the linear "buried homology" duplex DNA, with the two substrates containing the same length of homology (compare Fig. 2B with Fig. 7). Since linear DNA is topologically unconstrained, the pairing efficiency is not affected by heterologous unwinding. In contrast, superhelical DNA with low degree of negative supercoils will have insufficient torsional energy needed to counter the compensatory positive turns generated from heterologous unwinding and joint formation. The proficiency of superhelical DNA to form paranemic joints is therefore dependent on the superhelical density. The dependence on superhelical density may explain an earlier result showing that the pairing efficiency of superhelical DNA is lower than that of linear DNA (13).

An obligatory step in general recombination is recognition of homology, which can occur anywhere along the entire length of homologous molecules. A key intermediate in the search of homology is a paranemic joint, which serves to increase local DNA concentration and align homologous DNA sequences via significant but unstable interactions (46-48). The formation of paranemic joints thus increases the possibility of locating a free DNA end in the homologous region, which leads to strand exchange and formation of a stable plectonemic joint. A paranemic joint can also be converted into a plectonemic joint by the action of topoisomerase I or nucleases (37, 49). The present study shows that the proficiency of duplex DNA in forming paranemic joints is affected by both DNA topology and homology length, with the two factors interacting in a dynamic way. Therefore, homologous recombination, like many other cellular processes, may be regulated by altering the topological status of the DNA substrates.

    ACKNOWLEDGEMENTS

We thank Charles Radding, Stephen Chung, Paul Berg, and Mai Har Sham for helpful discussions and Michael Cox, Steve Kowalczykowski, and Charles Radding for careful reading of the manuscript.

    FOOTNOTES

* This work was supported in part by grants from the Croucher Foundation, the Committee on Research and Conference Grants, and the Medical Faculty Research Grant Fund (University of Hong Kong) and Department of Energy Grant DE-FC03-87-ER60615.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Wong Ching Yee Medical Scholarship and a Sir Edward Youde Memorial Fellowship.

Recipient of a Mary Sun Scholarship and a Sir Edward Youde Memorial Fellowship. Present address: Dept. of Biochemistry, Beckman Center, Stanford University, Stanford, CA 94305.

** To whom correspondence should be addressed: Dept. of Molecular and Medical Pharmacology, UCLA School of Medicine, Los Angeles, CA 90095. Tel.: 310-825-9600; Fax: 310-825-6267; E-mail: schow{at}pharm.medsch.ucla.edu.

1 The abbreviations used are: bp, base pair(s); ATPgamma S, adenosine 5'-O-(thiotriphosphate).

2 B. C. Wong, S.-K. Chiu, and S. A. Chow, unpublished results.

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Abstract
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Results
Discussion
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