Chanchal DasGupta

Homologous pairing in genetic recombination: recA protein makes joint molecules of gapped circular DNA and closed circular DNA

The recA protein, which is essential for genetic recombination in E. coli, promotes the homologous pairing of double-stranded DNA and linear single-stranded DNA, thereby forming a three-stranded joint molecule called a D loop. Single-stranded DNA stimulates recA protein to unwind double-stranded DNA. By a presumably related mechanism, recA protein promoted the homologous pairing of two circular double-stranded molecules when one of them has a gap in one strand. The two molecules were joined at homologous sites by noncovalent bonds. The covalently closed molecule remained intact and was not topologically linked to the intact circular strand of the gapped substrate. Electron microscopy showed that molecules were usually linked at two or more nearby points. The junctions in most molecules were shorter than 300 nucleotides. Sometimes the region between two extreme points was separated into two arms, producing an ellipsoidal loop (called an eye loop). The junctions in these biparental joint molecules were frequently remote from the site of the gap. We infer that a free end of the interrupted strand crosseover to form a structure like a D loop which moved away from the gap by branch migration.

The topology of homologous pairing promoted by RecA protein

In addition to catalyzing the pairing of linear single-stranded DNA with homologous duplex DNA, recA protein promotes the pairing of circular single strands with linear duplex DNA or nicked circular duplex DNA, and of gapped circular duplex DNA with superhelical DNA. RecA protein will thus produce joint molecules of DNA at a high frequency from a pair of homologous molecules if one of them is single-stranded or partially single-stranded, and if either one has a free end. The structure made from a linear single strand and duplex DNA is a D loop. The joint molecule made from circular single-stranded DNA and linear duplex DNA is a branched structure in which the circular strand has displaced a strand from one end of the duplex molecule. In these structures, the heteroduplex regions reach sizes approaching that of full-length fd DNA. When we used restriction fragments of duplex fd DNA that were approximately half-length, we found circular molecules that were half duplex and half single-stranded. Similarly, single-stranded circles displaced a strand from nicked circular duplex DNA, yielding structures related to those made with linear duplex DNA, as well as other structures. Our observations indicate that purified recA protein catalyzes a concerted strand transfer with several features of particular biological interest, including the initiation of a strand crossover (in some cases perhaps the crossing back of a strand as well) and the production of long heteroduplex joints by a kind of branch migration. While a free end permits interwinding of DNA strands and the formation of joints containing stable right-handed helices, the free end is not essential for the promotion of homologous pairing by recA protein. When we mixed phage G4 double-stranded DNA and recA protein with single-stranded circular M13 DNA containing an insert of 274 bases of G4 DNA, we observed by electron microscopy the formation of a few percent of complexes in which single-stranded circular DNA and duplex DNA were joined side by side in the region of shared sequence homology. The frequency of such complexes was twenty to thirty times greater than that observed in a control mixture of G4 duplex DNA and single-stranded circular fd DNA, molecules which do not share a region of extensive homology. We conclude that recA protein can promote homologous association of a single strand and duplex DNA without the plectonemic colling that characterizes the normal Watson-Crick structure of DNA.

Synapsis and the formation of paranemic joints by E. coli RecA protein

E. coli RecA protein promotes the homologous pairing of a single strand with duplex DNA even when certain features of the substrates, such as circularity, prohibit the true intertwining of the newly paired strands. The formation of such nonintertwined or paranemic joints does not require superhelicity, and indeed can occur with relaxed closed circular DNA. E. coli topoisomerase I can intertwine the incoming single strand in the paranemic joint with its complement, thereby topologically linking single-stranded DNA to all of the duplex molecules in the reaction mixture. The efficiency of formation of paranemic joints, the time course, and estimates of their length, all suggest that they represent true synaptic intermediates in the pairing reaction promoted by RecA protein.

Homologous pairing and topological linkage of DNA molecules by combined action of E. coli recA protein and topoisomerase I

E. coli RecA protein and topoisomerase I, acting on superhelical DNA and circular single strands in the presence of ATP and Mg2+, topologically link single-stranded molecules to one another, and single-stranded molecules to duplex DNA. When superhelical DNA is relaxed by prior incubation with topoisomerase, it is a poor substrate for catenation. Extensive homology stimulates the catenation of circular single-stranded DNA and superhelical DNA, whereas little reaction occurs between these forms of the closely related DNAs of phages phi X174 and G4, indicating that, in conjunction with topoisomerase I, RecA protein can discriminate perfect or nearly perfect homology from a high degree of relatedness. Circular single-stranded G4 DNA reacts with superhelical DNA of chimeric phage, M13G ori 1, to form catenanes, at least half of which survive heating at 80 degrees C following restriction cleavage in the M13 region, but few of which survive following restriction cleavage in the G4 region. Electron microscopic examination of catenated molecules cleaved in the M13 region reveals that in most cases the single-stranded G4 DNA is joined to the linear duplex M13(G4) DNA in the homologous G4 region. The junction frequently has the appearance of a D loop, with an extent equivalent to 100 or more bp. We conclude that a significant fraction of catenanes were hemicatenanes, in which the single-stranded circle was topologically linked, probably by multiple turns, to its complementary strand in the duplex DNA. These observations support the previous conclusion that RecA protein can pair a single strand with its complementary strand in duplex DNA in a side-by-side fashion without a free end in any of the three strands.

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.

Formation of nascent heteroduplex structures by RecA protein and DNA

E. coli RecA protein promotes homologous pairing in two distinguishable phases: synapsis and strand exchange. With circular single strands (plus strand only) and linear duplex DNA, polarized or unidirectional strand exchange appeared to cause heteroduplex joints to form and grow from a unique end of the duplex DNA. However, a variety of other pairs of substrates appeared to form joint molecules without regard to the polarity of the strands involved. This paradox has been resolved by observations that show that synapsis is fast, nonpolar and sensitive to inhibition by ADP, whereas strand exchange is slow, directional and relatively insensitive to inhibition by ADP. Thus a heteroduplex joint initiated at one end of the duplex DNA grows by continued strand exchange, whereas a joint initiated at the other end dissociates and is unable to start again because accumulating ADP inhibits synapsis. RecA protein appears to form a nascent protein-DNA structure, the RecA synaptic structure, in which at least 100-300 bp in the duplex molecule are held in an unwound configuration and in which the incoming strand is aligned with its complement.

REC A PROTEIN OF E. COLI PROMOTES HOMOLOGOUS PAIRING OF DNA MOLECULES BY A NOVEL MECHANISM

ABSTRACT Using the energy of ATP, recA protein cata-lyzes the homologous pairing of DNA molecules. One such pairing reaction is the formation of a D-loop from duplex DNA and homologous single-stranded frag-ments. This reaction is distinctly different from the renaturation of complementary single strands that is promoted either by heat or by helix destabilizing pro-teins, both of which favor nucleation of homologous sequences by unfolding single strands. In the presence of ATP, recA protein forms a ternary complex with single and double-stranded DNA. Unlike annealing reactions, which are second order, the kinetics of synthesis of D-loops by recA protein resemble classi-cal Michaelis-Menten kinetics, which confirms that a ternary complex consisting of recA protein and two DNA molecules is a precursor whose conversion to a D-loop limits the rate of the reaction.