RecA Protein-mediated DNA Strand Exchange Research Articles

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Optimal conditions for RecA protein-mediated DNA strand exchange include 6-8 mm Mg(2+) in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg(2+)) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although single-stranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg(2+) results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg(2+) and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg(2+) levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.

A distinctive single-strand DNA-binding protein from the Archaeon Sulfolobus solfataricus.

Single-stranded DNA binding proteins (SSBs) have been identified in all three domains of life. Here, we report the identification of a novel crenarchaeal SSB protein that is distinctly different from its euryarchaeal counterparts. Rather than comprising four DNA-binding domains and a zinc-finger motif within a single polypeptide of 645 amino acids, as for Methanococcus jannaschii, the Sulfolobus solfataricus SSB protein (SsoSSB) has a single DNA-binding domain in a polypeptide of just 148 amino acids with a eubacterial-like acidic C-terminus. SsoSSB protein was purified to homogeneity and found to form tetramers in solution, suggesting a quaternary structure analogous to that of E. coli SSB protein,despite possessing DNA-binding domains more similar to those of eukaryotic Replication Protein A (RPA). We demonstrate distributive binding of SsoSSB to ssDNA at high temperature with an apparent site size of approximately five nucleotides (nt)per monomer. Additionally, the protein is functional both in vitro and in vivo, stimulating RecA protein-mediated DNA strand-exchange and rescuing the ssb-1 lethal mutation of E. coli respectively. We discuss possible evolutionary relationships amongst the various members of the SSB/RPA family.

RecA as a motor protein. Testing models for the role of ATP hydrolysis in DNA strand exchange.

ATP hydrolysis (by RecA protein) fundamentally alters the properties of RecA protein-mediated DNA strand exchange reactions. ATP hydrolysis renders DNA strand exchange unidirectional, greatly increases the lengths of hybrid DNA created, permits the bypass of heterologous DNA insertions in one or both DNA substrates, and is absolutely required for exchange reactions involving four DNA strands. There are at least two viable models to explain how ATP hydrolysis is coupled to DNA strand exchange so as to bring about these effects. The first couples ATP hydrolysis to a redistribution of RecA monomers within a RecA filament. The second couples ATP hydrolysis to a facilitated rotation of the DNA substrates. The RecA monomer redistribution model makes the prediction that heterology bypass should not occur if the single-stranded DNA substrate is linear. The facilitated DNA rotation model predicts that RecA protein should promote the separation of paired DNA strands within a RecA filament if one of them is contiguous with a length of DNA being rotated about the filament exterior. Here, a facile bypass of heterologous insertions with linear DNA substrates is demonstrated, providing evidence against a role for RecA monomer redistribution in heterology bypass. In addition, we demonstrate that following a four-strand DNA exchange reaction, a distal segment of DNA hundreds of base pairs in length can be unwound in a nonreciprocal phase of the reaction, consistent with the direct coupling of an ATP hydrolytic motor to the proposed DNA rotation.

RecA Filament Dynamics during DNA Strand Exchange Reactions

The role of ATP hydrolysis in RecA protein-mediated DNA strand exchange reactions remains controversial. Competing models suggest that ATP hydrolysis is coupled either to a simple redistribution of RecA monomers within a filament to repair filament discontinuities, or more directly to rotation of the DNA substrates to drive branch movement unidirectionally. Here, we test key predictions of the RecA redistribution idea. When ATP is hydrolyzed, DNA strand exchange is accompanied by a RecA exchange reaction, between free and bound RecA protomers in the interior of RecA filaments, that meets a central prediction of the model. The RecA protomer exchange is not required for, and does not occur during, the "search for homology" in which the single-stranded DNA within a RecA-ssDNA nucleoprotein filament is homologously aligned with the duplex DNA. Instead, the RecA exchange is triggered by the completion of strand exchange (a strand switch to generate a hybrid DNA product) in any given segment of the filament. In effect, formation of hybrid DNA leads to a change in filament conformation to one with properties approximating those of RecA filaments bound to double-stranded DNA. Addition of the RecA K72R mutant protein to a reaction with the wild type protein leads to the formation of mixed filaments and a poisoning of the DNA strand exchange reaction. Under some conditions, a facile RecA protomer exchange is observed, and significant ATP is hydrolyzed, even though DNA strand exchange is entirely blocked by the mutant protein. A redistribution of RecA protomers coupled to ATP hydrolysis is not sufficient in itself to explain how ATP hydrolysis facilitates DNA strand exchange. A RecA protomer exchange may nevertheless play an important role in the DNA strand exchange process.

RecA Protein Dynamics in the Interior of RecA Nucleoprotein Filaments

We characterize aspects of the conformation and dynamic state of RecA filaments when bound to dsDNA that are specifically linked to the presence of the second of the two bound DNA strands. Filaments bound to dsDNA exhibit a facile exchange between free and bound RecA monomers or oligomers in the filament interior that is not seen on ssDNA. The RecA mutant K72R, which binds but does not hydrolyze ATP, forms mixed filaments with wild type RecA protein under some conditions. In the presence of dATP, mixed filaments are formed on dsDNA or ssDNA in which the RecA K72R content approximately reflects the proportion of the K72R mutant in the total RecA protein present when the filament is formed. In the presence of ATP, mixed filaments are formed on dsDNA, but the mutant protein strongly inhibits the binding of wtRecA protein to single-stranded DNA. When RecA K72R is added to pre-formed filaments containing only wild-type RecA protein on single-stranded DNA, little of the mutant protein exchanges into the filament. Exchange occurs readily, however, when the filament is bound to double-stranded DNA. The presence of a second DNA strand in RecA-dsDNA filaments produces as altered and more dynamic filament state relative to filaments formed on single-stranded DNA. The results point to a substantial alteration in filament state when synapsis occurs during RecA protein-mediated DNA strand exchange.

Evidence for the Coupling of ATP Hydrolysis to the Final (Extension) Phase of RecA Protein-mediated DNA Strand Exchange

RecA protein promotes a limited DNA strand exchange reaction, without ATP hydrolysis, that typically results in formation of short (1-2 kilobase pairs) regions of hybrid DNA. This nascent hybrid DNA is extended in a reaction that can be coupled to ATP hydrolysis. When ATP is hydrolyzed, the extension phase is progressive and its rate is 380 +/- 20 bp min-1 at 37 degrees C. A single RecA nucleoprotein filament can participate in multiple DNA strand exchange reactions concurrently (involving duplex DNA fragments that are homologous to different segments of the DNA within a nucleoprotein filament), with no effect on the observed rate of ATP hydrolysis. The ATP hydrolytic and hybrid DNA extension activities exhibit a dependence on temperature between 25 and 45 degrees C that is, within experimental error, identical. This provides new evidence that the two processes are coupled. Arrhenius activation energies derived from the work are 13.3 +/- 1.1 kcal mole-1 for DNA strand exchange, and 14.4 +/- 1.4 kcal mole-1 for ATP hydrolysis during strand exchange. The rate of branch movement in the extension phase (base pair min-1) is related to the kcat for ATP hydrolysis during strand exchange (min-1) by a factor equivalent to 18 bp throughout the temperature range examined. The 18-base pair factor conforms to a quantitative prediction derived from a model in which ATP hydrolysis is coupled to a facilitated rotation of the DNA substrates. RecA filaments possess an intrinsic capacity for DNA strand exchange, mediated by binding energy rather than ATP hydrolysis, that is augmented by an ATP-dependent molecular motor.

Blocked RecA Protein-mediated DNA Strand Exchange Reactions Are Reversed by the RuvA and RuvB Proteins

RecA protein is unable to complete a DNA strand exchange reaction between a circular single-stranded DNA and a linear duplex DNA substrate with heterologous sequences of 375 base pairs at the distal end. Instead, it generates a branched intermediate in which strand exchange has proceeded up to the homology/heterology junction. Addition of the RuvA and RuvB proteins to these stalled intermediates leads to the rapid conversion of intermediates back to the original substrates. The reversal reaction is initiated at the branch, and the hybrid DNA is unwound in the direction opposite to that of the RecA reaction that created it. Under optimal conditions the rate of the reaction exhibits only a modest dependence on the length of hybrid DNA that must be unwound. Products of the reversal reaction are detected within minutes after addition of RuvAB, and appear with an apparent first order progress curve, exhibiting a t1/2 in the range of 6-12 min under optimal conditions. Few molecules that have undergone only partial reversal are detected. This suggests that the assembly or activation of RuvAB on the branched substrate is rate-limiting, while any migration of RuvAB on the DNA to effect unwinding of the hybrid DNA (and reformation of substrate DNA) is very fast. The results are discussed in context of the role of RuvA and RuvB proteins in recombinational DNA repair. We suggest that one function of the RuvAB proteins is to act as an antirecombinase, to eliminate intragenomic crossovers between homologous segments of the bacterial chromosome that might otherwise lead to deleterious inversions or deletions.

RuvA and RuvB proteins facilitate the bypass of heterologous DNA insertions during RecA protein-mediated DNA strand exchange.

RecA protein-mediated DNA strand exchange between circular single-stranded DNA and linear duplex DNA readily bypasses short (up to 100 base pairs) heterologous inserts in one of the DNA substrates. Larger heterologous inserts are bypassed with decreasing efficiency, and inserts larger than 200 base pairs substantially block RecA-mediated DNA strand exchange. The RuvA and RuvB proteins dramatically facilitate the bypass of larger heterologous inserts. When the RuvA and RuvB proteins are added to an ongoing RecA protein-mediated strand exchange reaction, interior heterologous inserts of 1 kilobase pair are bypassed at significant frequencies. The RuvA, RuvB, and RecA proteins are all required for this activity. Bypass occurs only when homologous sequences are present on both sides of the insert. When the heterologous insert is positioned at either end of the linear duplex substrate, the RuvA and RuvB proteins do not significantly increase product formation in RecA protein-mediated DNA strand exchange reactions. The results suggest an important role for RuvA and RuvB in the bypass of DNA structural barriers during recombinational DNA repair.

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. III. Unidirectional branch migration and extensive hybrid DNA formation.

We have identified two functions for RecA-mediated ATP hydrolysis during DNA strand exchange. First, ATP hydrolysis renders RecA protein-mediated DNA strand exchange unidirectional (5' to 3' with respect to the single-stranded DNA). In the presence of a nonhydrolyzable analog adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), DNA strand exchange is bidirectional. Second, ATP hydrolysis is required for extensive formation of hybrid DNA. In the presence of ATP hydrolysis, the length of the exchanged region is limited only by the available homology, whereas in the absence of ATP hydrolysis, only 2 kilobase pairs or less of hybrid DNA are formed before branch migration is blocked in the majority of paired intermediates. Both of these functions of RecA protein-mediated ATP hydrolysis are crucial in ensuring the effectiveness of recombinational DNA repair, especially when the lesion to be repaired is distant from the initial crossover point.

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. I. Bypassing a short heterologous insert in one DNA substrate.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski, J.P., Bear, D.G., and Kowalczykowski, S.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 21-25), calling into question the role of ATP hydrolysis in the strand exchange reaction. Here, we demonstrate that the ATP gamma S-mediated reaction can go to completion when the duplex DNA substrate is only 1.3 kilobase pairs in length. The ATP gamma S-mediated reaction, however, is completely blocked by a 52-base pair heterologous insertion in either DNA substrate. This same barrier is readily bypassed when ATP replaces ATP gamma S. This indicates that at least one function of recA-mediated ATP hydrolysis is to bypass structural barriers in one or both DNA substrates during strand exchange. This suggests that ATP hydrolysis is directly coupled to the branch migration phase of strand exchange, not to promote strand exchange between homologous DNA substrates during recombination, but instead to facilitate the bypass of structural barriers likely to be encountered during recombinational DNA repair.

On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. II. Four-strand exchanges.

RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski et al., 1990), calling into question the role of ATP hydrolysis in this reaction. We demonstrate here that the ATP gamma S-mediated process is restricted to homologous strand exchange reactions involving three strands. In four-strand exchanges between a gapped duplex circle and a second linear duplex, joint molecules are formed in the gap but are not extended into the four-strand region when ATP gamma S is present. This result provides evidence that one function of ATP hydrolysis in the recA system is to facilitate reciprocal DNA strand exchange involving four strands. Implications with respect to the role of four-stranded pairing intermediates and the mechanistic relationship between three- and four-strand exchange reactions are discussed.

Putative three-stranded DNA pairing intermediate in recA protein-mediated DNA strand exchange: no role for guanine N-7.

As an early step in DNA strand exchange reactions, the recA protein aligns homologous sequences within two DNA molecules to form a putative triple-stranded intermediate. In virtually all models for three-stranded DNA proposed to date, hydrogen bonds involving the N-7 position of guanine have played a prominent structural role. To determine whether the N-7 position of guanine is required for triple helix and heteroduplex formation in the recA protein-mediated DNA pairing reaction, guanine was completely replaced by the base analog 7-deazaguanine in both strands of the duplex DNA substrate using polymerase chain reaction. This modified double-strand DNA was reacted with unmodified single-strand DNA in vitro. The 7-deazaguanine-substituted DNA functioned as well as the unsubstituted DNA in recA protein-mediated DNA three-strand exchange reactions. Strand exchange reactions involving four strands also proceeded normally when three of the four strands contained 7-deazaguanine rather than guanine. In fact, the rate of strand exchange improved somewhat when the modified DNA substrates were used. This indicates either that the N-7 position of guanine is not essential for the formation of the putative triple-stranded DNA pairing intermediate, or that a three-stranded (or four-stranded) structure is not an obligate intermediate in recA protein-mediated DNA strand exchange.

On RecA protein-mediated homologous alignment of two DNA molecules. Three strands versus four strands.

The recA protein from Escherichia coli can homologously align two duplex DNA molecules; however, this interaction is much less efficient than the alignment of a single strand and a duplex. Three strand paranemic joints are readily detected. In contrast, duplex-duplex pairing is detected only when the incoming (second) duplex is negatively supercoiled, and even here the pairing is inefficient. The recA protein-promoted four strand exchange reaction is initiated in a three strand region, with efficiency increasing with the length of potential three strand pairing available for initiation. This indicates that a paranemic joint involving three DNA strands may be an important intermediate in all recA protein-mediated DNA strand exchange reactions and that the presence of three strands rather than four is a fundamental structural parameter of paranemic joints.

Assembly and disassembly of RecA protein filaments occur at opposite filament ends. Relationship to DNA strand exchange.

RecA protein primarily associates with and dissociates from opposite ends of nucleoprotein filaments formed on linear duplex DNA. RecA nucleoprotein filaments that are hydrolyzing ATP therefore engage in a dynamic process under some conditions that has some of the properties of treadmilling. We have also investigated whether the net polymerization of recA protein at one end of the filament and/or a net depolymerization at the other end drives unidirectional strand exchange. There is no demonstrable correlation between recA protein association/dissociation and the strand exchange reaction. RecA protein-mediated DNA strand exchange is affected minimally by changes in reaction conditions (dilution, pH shift, or addition of small amounts of adenosine-5'-O-(3-thiotriphosphate) that have large and demonstrable effects on recA protein association, dissociation, or both. Rather than driving strand exchange, these assembly and disassembly processes may simply represent the mechanism by which recA nucleoprotein filaments are recycled in the cell.