Formation of covalently closed heteroduplex DNA by the combined action of gyrase and RecA protein.

The concerted action of DNA gyrase and RecA protein of Escherichia coli on intact and gapped homologous or partially homologous plasmid DNA molecules leads to the formation of covalently closed DNA containing one strand of each parental molecule. Large regions of non-homology can be incorporated into the closed circular duplex. Both proteins are essential for the reaction to take place, and type I topoisomerase cannot substitute for DNA gyrase.     

Homologous pairing and the formation of nascent synaptic intermediates between regions of duplex DNA by RecA protein

The RecA protein from E. coli gains access to duplex DNA, by nucleation from a short single-stranded gap, to form a spiral nucleoprotein filament that is capable of interaction with homologous duplex DNA. The observations described here demonstrate that any part of the nucleoprotein filament, whether it contains single- or double-stranded DNA, is capable of pairing with homologous duplex DNA. Homologous contacts between regions of duplex DNA lead to an increase in the initial rate and final extent of joint molecule formation. The experiments indicate that pairing is facilitated by the formation of nascent synaptic intermediates between duplex DNA sequences. Using chimeric form I DNA, which is incapable of forming an inter-wound or plectonemic joint with the gapped DNA due to the presence of flanking heterologous sequences, we show that these duplex-duplex pairing reactions involve extensive underwinding of the double helix.     

DNA sequence dependence of ATP hydrolysis by RecA protein

The DNA sequence dependence of the ATPase activity of RecA protein has been investigated for a variety of single strand octamer and hexadecamer homopolymers and alternating copolymers. Under assay conditions where the single strand DNA concentration exceeds the RecA protein concentration, significant differences in the rates of ATP hydrolysis for the various single strand DNA oligomer cofactors are observed. Under the conditions examined, the order of efficiency of the DNA cofactors in inducing RecA mediated ATPase activity is found to be: dA16 greater than dT16 greater than d(TC)16 greater than dT8 greater than dC16 greater than dA8 = dG8 greater than dG16 greater than dC8 greater than d(AG)16. These results demonstrate not only a dependence of RecA ATPase activity on the sequence composition of short single strand DNA they further reveal ATPase activity can be affected by the nearest neighbor nucleotide sequence of short DNA cofactors.     

Underwinding of DNA associated with duplex-duplex pairing by RecA protein

Homologous pairing between gapped circular and partially homologous form I DNA, catalyzed by Escherichia coli RecA protein, leads to the formation of nascent synaptic joints between regions of duplex DNA. These duplex-duplex interactions result in underwinding of the form I DNA, as detected by a topoisomerase assay. Underwound DNA species have been studied with regard to their formation, stability, and topological requirements. The synaptic joints are short-lived and of low frequency compared with those formed between single-stranded and duplex DNA. Measurement of the degree of underwinding indicates joints 300-400 base pairs in length, in which the two DNA molecules are presumed to be interwound within the RecA-nucleoprotein filament. Underwound DNA was not detected in reactions between gapped DNA and partially homologous nicked circular or relaxed covalently closed DNA. We have also investigated the requirements for the initiation of strand exchange. Previous results have shown that strand exchange requires a homologous 3'-terminus complementary to the gapped region. We now show that the minimum length of overlap required for efficient initiation of strand exchange is one to two turns of DNA within the RecA-DNA nucleoprotein filament.     

Twisting and untwisting a single DNA molecule covered by RecA protein

We study dsDNA-RecA interactions by exerting forces in the pN range on single DNA molecules while the interstrand topological state is controlled owing to a magnetic tweezers setup. We show that unwinding a duplex DNA molecule induces RecA polymerization even at moderate force. Once initial polymerization has nucleated, the extent of RecA coverage still depends on the degree of supercoiling: exerting a positive or negative torsional constraint on the fiber forces partial depolymerization, with a strikingly greater stability when ATPgammaS is used as a cofactor instead of ATP. This nucleofilament's sensitivity to topology might be a way for the bacterial cell to limit consumption of precious RecA monomers when DNA damage is addressed through homologous recombination repair.     

Binding of SSB and RecA protein to DNA-containing stem loop structures: SSB ensures the polarity of RecA polymerization on single-stranded DNA

Single-stranded DNA-binding proteins play an important role in homologous pairing and strand exchange promoted by the class of RecA-like proteins. It is presumed that SSB facilitates binding of RecA on to ssDNA by melting secondary structure, but direct physical evidence for the disruption of secondary structure by either SSB or RecA is still lacking. Using a series of oligonucleotides with increasing amounts of secondary structure, we show that stem loop structures impede contiguous binding of RecA and affect the rate of ATP hydrolysis. The electrophoretic mobility shift of a ternary complex of SSB-DNA-RecA and a binary complex of SSB-DNA are similar; however, the mechanism remains obscure. Binding of RecA on to stem loop is rapid in the presence of SSB or ATPgammaS and renders the complex resistant to cleavage by HaeIII, to higher amounts of competitor DNA or low temperature. The elongation of RecA filament in a 5' to 3' direction is halted at the proximal end of the stem. Consequently, RecA nucleates at the loop and cooperative binding propagates the RecA filament down the stem causing its disruption. The pattern of modification of thymine residues in the loop region indicates that RecA monomer is the minimum binding unit. Together, these results suggest that SSB plays a novel role in ensuring the directionality of RecA polymerization across stem loop in ssDNA. These observations have fundamental implications on the role of SSB in multiple aspects of cellular DNA metabolism.     

Fibers of RecA protein and complexes of RecA protein and single-stranded phi X174 DNA as visualized by negative-stain electron microscopy

Monomers of purified RecA protein polymerize into helical fibers whose pitch is 7.2 nm to 7.5 nm and whose diameter is 11 nm. Either short (approximately 0.2 micron), single fibers, or bundles of aligned, longer fibers, can be formed preferentially, by varying the Mg2+ concentration. When RecA protein is bound to circular, single-stranded phi X174 DNA it forms helical fibers of different classes of contour lengths, ranging from 0.98 micron, depending upon the conditions of assembly. Two different helical pitches are found, one of 9.3 nm when the incubation buffer contains, besides the obligatory Mg2+, either ATP gamma S or ATP accompanied by single-strand binding protein, and one of 5.5 nm when the latter additives are omitted. Preformed fibers of the compact type can be converted to open ones of 9.3 nm pitch upon addition of ATP gamma S, even after the removal of unbound RecA. All signs of helicity are obliterated upon glutaraldehyde cross-linking except in those fibers whose assembly has been mediated by ATP gamma S. RecA protein and single-strand binding protein are competitively bound to single-stranded DNA. Composite complexes, however, are not encountered unless ATP gamma S is present. Otherwise, segments of DNA that are coated by one or the other protein are seen as separate regions. When the assembly of complexes of single-stranded DNA and RecA is mediated by single-strand binding protein and ATP, the axial separation between successive bases is 0 X 42 nm, somewhat greater than the axial distance between bases in one strand of duplex DNA in the B form. It is proposed that the bases of the single-stranded DNA in the complex are located near its inner surface, and that base-pairing with double-stranded DNA takes place following invasion of the central cavity of the complex.     

Formation of heteroduplex DNA during mammalian intrachromosomal gene conversion.

We have studied intrachromosomal gene conversion in mouse Ltk- cells with a substrate designed to provide genetic evidence for heteroduplex DNA. Our recombination substrate consists of two defective chicken thymidine kinase genes arranged so as to favor the selection of gene conversion products. The gene intended to serve as the recipient in gene conversion differs from the donor sequence by virtue of a palindromic insertion that creates silent restriction site polymorphisms between the two genes. While selection for gene conversion at a XhoI linker insertion within the recipient gene results in coconversion of the nearby palindromic site in more than half of the convertants, 4% of convertant colonies show both parental and nonparental genotypes at the polymorphic site. We consider these mixed colonies to be the result of genotypic sectoring and interpret this sectoring to be a consequence of unrepaired heteroduplex DNA at the polymorphic palindromic site. DNA replication through the heteroduplex recombination intermediate generates genetically distinct daughter cells that comprise a single colony. We believe that the data provide the first compelling genetic evidence for the presence of heteroduplex DNA during chromosomal gene conversion in mammalian cells.     

Concerted strand exchange and formation of Holliday structures by E. coli RecA protein

RecA protein makes stable joint molecules from fully duplex DNA and molecules that are partially single-stranded; the latter may be either duplex molecules with an internal gap in one strand or molecules with single-stranded ends. Stable joint molecules form only when the end of at least one strand is in a homologous region. When RecA protein pairs linear duplex molecules and tailed molecules that share the same sequence end to end, the joints, which are located away from the single-stranded tails in most instances, have the electron microscopic appearance associated with the Holliday structure resulting from the reciprocal exchange of strands. The reaction leading to reciprocal strand exchange involves the concerted displacement of a strand from the end of the duplex molecule. These observations support the view that RecA protein makes stable joint molecules only by transferring strands and not by the side-by-side pairing of duplex regions.     

Kinetics of DNA renaturation catalyzed by the RecA protein of Escherichia coli

The recA enzyme of Escherichia coli catalyzes renaturation of DNA coupled to hydrolysis of ATP. The rate of enzymatic renaturation is linearly dependent on recA protein concentration and shows saturation kinetics with respect to DNA concentration. The kinetic analysis of the reaction indicates that the Km for DNA is 65 microM while the kcat is approximately 48 pmol of duplex formed (pmol of recA)-1 (20 min)-1. RecA protein catalyzed renaturation has been characterized with respect to salt sensitivity, Mg2+ ion and pH optima, requirements for nucleoside triphosphates, and inhibition by nonhydrolyzable nucleoside triphosphates and analogues. These results are consistent with a Michaelis-Menten mechanism for DNA renaturation catalyzed by recA protein. A model is described in which oligomers of recA protein bind rapidly to single-stranded DNA, and in the presence of ATP, these nucleoprotein intermediates aggregate to bring complementary sequences into close proximity for homologous pairing. As with other DNA pairing reactions catalyzed by recA protein, ongoing DNA hydrolysis is required for renaturation. However, unlike the strand assimilation or transfer reaction, renaturation is inhibited by E. coli helix-destabilizing protein.     

Location of functional regions of the Escherichia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants.

In previous work (E. S. Tessman and P. K. Peterson, J. Bacteriol. 163:677-687 and 688-695, 1985), we isolated many novel protease-constitutive (Prtc) recA mutants, i.e., mutants in which the RecA protein was always in the protease state without the usual need for DNA damage to activate it. Most Prtc mutants were recombinase positive and were designated Prtc Rec+; only a few Prtc mutants were recombinase negative, and those were designated Prtc Rec-. We report changes in DNA sequence of the recA gene for several of these mutants. The mutational changes clustered at three regions on the linear RecA polypeptide. Region 1 includes amino acid residues 25 through 39, region 2 includes amino acid residues 157 through 184, and region 3 includes amino acid residues 298 through 301. The in vivo response of these Prtc mutants to different effectors suggests that the RecA effector-binding sites have been altered. In particular we propose that the mutations may define single-stranded DNA- and nucleoside triphosphate-binding domains of RecA, that polypeptide regions 1 and 3 comprise part of the single-stranded DNA-binding domain, and that polypeptide regions 2 and 3 comprise part of the nucleoside triphosphate-binding domain. The overlapping of single-stranded DNA- and nucleoside triphosphate-binding domains in region 3 can explain previously known complex allosteric effects. Each of four Prtc Rec- mutants sequenced was found to contain a single amino acid change, showing that the change of just one amino acid can affect both the protease and recombinase activities and indicating that the functional domains for these two activities of RecA overlap. A recA promoter-down mutation was isolated by its ability to suppress the RecA protease activity of one of our strong Prtc mutants.     

Formation of heteroduplex DNA promoted by the combined activities of Escherichia coli recA and recBCD proteins

We have established an in vitro reaction in which heteroduplex DNA formation is dependent on the concerted actions of recA and recBCD proteins, the major components of the recBCD pathway of genetic recombination in vivo. We find that heteroduplex DNA formation requires three distinct enzymatic functions: first, the helicase activity of recBCD enzyme initiates heteroduplex DNA formation by unwinding the linear double-stranded DNA molecule to transiently form single-stranded DNA (ssDNA); second, recA protein traps this ssDNA before it reanneals; third, recA protein catalyzes the pairing of this ssDNA molecule with another homologous ssDNA molecule, followed by the renaturation of these molecules to form heteroduplex DNA. The first two functions should be important to all in vitro reactions involving recA and recBCD proteins, whereas the third will be specific to the DNA substrates used. The rate-limiting step of heteroduplex DNA formation is the trapping by recA protein of the ssDNA produced by recBCD enzyme. A model for this reaction is described.     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

DNA polymerase V and RecA protein, a minimal mutasome

A hallmark of the Escherichia coli SOS response is the large increase in mutations caused by translesion synthesis (TLS). TLS requires DNA polymerase V (UmuD'2C) and RecA. Here, we show that pol V and RecA interact by two distinct mechanisms. First, pol V binds to RecA in the absence of DNA and ATP and second, through its UmuD' subunit, requiring DNA and ATP without ATP hydrolysis. TLS occurs in the absence of a RecA nucleoprotein filament but is inhibited in its presence. Therefore, a RecA nucleoprotein filament is unlikely to be required for SOS mutagenesis. Pol V activity is severely diminished in the absence of RecA or in the presence of RecA1730, a mutant defective for pol V mutagenesis in vivo. Pol V activity is strongly enhanced with RecA mutants constitutive for mutagenesis in vivo, suggesting that RecA is an obligate accessory factor that activates pol V for SOS mutagenesis.     

General mechanism for RecA protein binding to duplex DNA

RecA protein binding to duplex DNA occurs by a multi-step process. The tau analysis, originally developed to examine the binding of RNA polymerase to promoter DNA, is adapted here to study two kinetically distinguishable reaction segments of RecA-double stranded (ds) DNA complex formation in greater detail. One, which is probably a rapid preequilibrium in which RecA protein binds weakly to native dsDNA, is found to have the following properties: (1) a sensitivity to pH, involving a net release of approximately one proton; (2) a sensitivity to salts; (3) little or no dependence on temperature; (4) little or no dependence on DNA length. The second reaction segment, the rate-limiting nucleation of nucleoprotein filament formation accompanied by partial DNA unwinding, is found to have the following properties: (1) a sensitivity to pH, involving a net uptake of approximately three protons; (2) a sensitivity to salts; (3) a relatively large dependence on temperature, with an Arrhenius activation energy of 39 kcal mol(-1); (4) a sensitivity to DNA topology; (5) a dependence on DNA length. These results contribute to a general mechanism for RecA protein binding to duplex DNA, which can provide a rationale for the apparent preferential binding to altered DNA structures such as pyrimidine dimers and Z-DNA.     

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4',6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPgammaS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.     

Networks of DNA and RecA protein are intermediates in homologous pairing

Partial coating of single-stranded DNA by recA protein causes its aggregation, but conditions that promote complete coating inhibit independent aggregation of single strands and, instead, cause the mutually dependent conjunction of single- and double-stranded DNA in complexes that sediment at more than 10 000 S. This coaggregation is independent of homology but otherwise shares key properties of homologous pairing of single strands with duplex DNA: both processes require ATP, MgCl2, and stoichiometric amounts of recA protein; both are very sensitive to inhibition by salt and ADP. Coaggregates are closed domains that are intermediates in homologous pairing: they form faster than joint molecules, they include virtually all of the DNA in the reaction mixture, and they yield joint molecules nearly an order of magnitude faster than they exchange DNA molecules with the surrounding solution. The independent aggregation of single-stranded DNA differs in all respects except the requirement for Mg2+, and its properties correlate instead with those associated with the renaturation of complementary single strands by recA protein.     

A possible interaction of single-strand binding protein and RecA protein during post-ultraviolet DNA synthesis

The mechanism of DNA replication in ultraviolet (UV)-irradiated Escherichia coli is proposed. Immediately after UV exposure, the replisome aided by single-strand DNA-binding protein (SSB) can proceed past UV-induced pyrimidine dimers without insertion of nucleotides. Polymerisation eventually resumes somewhere downstream of the dimer sites. Due to the limited supply of SSB, only a few dimers can be bypassed in this way. Nevertheless, this early DNA synthesis is of great biological importance because it generates single-stranded DNA regions. Single-stranded DNA can bind and activate RecA protein, thus leading to induction of the SOS response. During the SOS response, the cellular level of RecA protein increases dramatically. Due to the simultaneous increase in the concentration of ATP, RecA protein achieves the high-affinity state for single-stranded DNA. Therefore it is able to displace DNA-bound SSB. The cycling of SSB on and off DNA enables the replisome to bypass a large number of dimers at late post-UV times. During this late replication, the stoichiometric amounts of RecA protein needed for recombination are involved in the process of postreplication repair.     

Force and ATP hydrolysis dependent regulation of RecA nucleoprotein filament by single-stranded DNA binding protein

In Escherichia coli, the filament of RecA formed on single-stranded DNA (ssDNA) is essential for recombinational DNA repair. Although ssDNA-binding protein (SSB) plays a complicated role in RecA reactions in vivo, much of our understanding of the mechanism is based on RecA binding directly to ssDNA. Here we investigate the role of SSB in the regulation of RecA polymerization on ssDNA, based on the differential force responses of a single 576-nucleotide-long ssDNA associated with RecA and SSB. We find that SSB outcompetes higher concentrations of RecA, resulting in inhibition of RecA nucleation. In addition, we find that pre-formed RecA filaments de-polymerize at low force in an ATP hydrolysis- and SSB-dependent manner. At higher forces, re-polymerization takes place, which displaces SSB from ssDNA. These findings provide a physical picture of the competition between RecA and SSB under tension on the scale of the entire nucleoprotein SSB array, which have broad biological implications particularly with regard to competitive molecular binding.