General Genetic Recombination Research Articles

Helical repeat of DNA in the region of homologous pairing.

The process of DNA strand exchange during general genetic recombination is initiated within protein-stabilized synaptic filaments containing homologous regions of interacting DNA molecules. The RecA protein in bacteria and its analogs in eukaryotic organisms start this process by forming helical filamentous complexes on single-stranded or partially single-stranded DNA molecules. These complexes then progressively bind homologous double-stranded DNA molecules so that homologous regions of single- and double-stranded DNA molecules become aligned in register while presumably winding around common axis. The topological assay presented herein allows us to conclude that in synaptic complexes containing homologous single- and double-stranded DNA molecules, all three DNA strands have a helicity of approximately 19 nt per turn.

Characterization of the DNA-binding domain of β protein, a component of phage λ Red-pathway, by UV catalyzed cross-linking

β protein, a key component of Red-pathway of phage λ is necessary for its growth and general genetic recombination in recombination-deficient mutants of Escherichia coli To facilitate studies on structure-function relationships, we overexpressed β protein and purified it to homogeneity. A chemical cross-linking reagent, glutaraldehyde, was used to stabilize the physical association of β protein in solution. A 67-kDa band, corresponding to homodimer, was identified after separation by SDS-polyacrylamide gel electrophoresis. Stoichiometric measurements indicated a site-size of 1 monomer of β protein/5 nucleotide residues. Electrophoretic gel mobility shift assays suggested that β protein formed stable nucleoprotein complexes with 36-mer, but not with 27-or 17-mer DNA. Interestingly, the interaction of β protein with DNA and the stability of nucleoprotein complexes was dependent on the presence of MgC1 2, and the binding was abolished by 250 mM NaCl. The K d of β protein binding to 36-mer DNA was on the order of 1.8 × 10 −6 M. Photochemical cross-linking of native β protein or its fragments, generated by chymotrypsin, to 36-mer DNA was performed to identify its DNA-binding domain. Characterization of the cross-linked peptide disclosed that amino acids required for DNA-binding specificity resided within a 20-kDa peptide at the N-terminal end. These findings provide a basis for further understanding of the structure and function of β protein.

Locations of functional domains in the RecA protein. Overlap of domains and regulation of activities.

We review the locations of various functional domains of the RecA protein of Escherichia coli, including how they have been assigned, and discuss the potential regulatory roles of spatial overlap between different domains. RecA is a multifunctional and ubiquitous protein involved both in general genetic recombination and in DNA repair: it regulates the synthesis and activity of DNA repair enzymes (SOS induction) and catalyses homologous recombination and mutagenesis. For these activities RecA interacts with a nucleotide cofactor, single-stranded and double-stranded DNAs, the LexA repressor, UmuD protein, the UmuD'2C complex as well as with RecA itself in forming the catalytically active nucleofilament. Attempts to locate the respective interaction sites have been advanced in order to understand the various functions of RecA. An intriguing question is how these numerous functional sites are contained within this rather small protein (38 kDa). To assess more clearly the roles of the respective sites and to what extent the sites may be interacting with each other, we review and compare the results obtained from various biological, biochemical and physico-chemical approaches. From a three-dimensional model it is concluded that all sites are concentrated to one part of the protein. As a consequence there are significant overlaps between the sites and it is speculated that corresponding interactions may play important roles in regulating RecA activities.

Bypass of DNA Heterologies During RuvAB-mediated Three- and Four-strand Branch Migration

During general genetic recombination and recombinational DNA repair, DNA damages and heterologies are often encountered which must be efficiently processed by the cellular recombination machinery. In RecA-mediated three-strand exchange reactions between single-stranded circular and linear duplex DNA, or four-strand exchange reactions between gapped circular and linear duplex DNA, heterologies can only be bypassedin vitrowhen they are short in length and are followed by homologous DNA downstream. Larger DNA inserts block RecA-mediated strand exchange, indicating that effective bypass requires other components of the recombination machinery. The RuvA and RuvB proteins ofEscherichia coliform an important part of this machinery. In this work, we have analysed the ability of RuvA and RuvB to bypass large tracts of DNA heterology in both three- and four-strand exchange reactions, using recombination intermediates made by theE. coliRecA protein. Under optimal reaction conditions for RuvAB, up to 1000 bp of DNA heterology can by bypassed in three-strand reactions and 300 bp of DNA heterology can be bypassed in four-strand reactions. Whereas high concentrations of RuvB (in the absence of RuvA) can promote homologous branch migration, we find that RuvB alone is unable to catalyse heterologous bypass, indicating an essential role for both proteins in homologous recombination and recombinational DNA repair processes. Under certain conditions, the bypass of heterology is stimulated by the single-strand binding protein SSB.

Phage T4 homologous strand exchange: A DNA helicase, not the strand transferase, drives polar branch migration

Homologous strand exchange is a central step in general genetic recombination. A multiprotein complex composed of five purified bacteriophage T4 proteins (the products of the uvsX, uvsY, 32, 41, and 59 genes) that mediates strand exchange under physiologically relevant conditions has been reconstituted. One of these proteins, the product of the uvsY gene, is required for homologous pairing but strongly inhibits branch migration catalyzed by UvsX protein, the phage RecA analog. Branch migration is completely dependent on the gene 41 protein, a DNA helicase that also functions in phage replication. The helicase is delivered to the strand exchange complex by the gene 59 accessory protein in a strand-specific fashion through direct interactions between the gene 59 and gene 32 proteins. These data suggest that strand transferases such as UvsX protein are essential for homologous pairing in vivo, but that a DNA helicase drives polar branch migration.

On the Origin of Genetic Diversity

The organic diversity found in nature today is ultimately a consequence of a continuous cycle of building up genetic diversity, which is subsequently channeled by natural selection, chance events, and by geographic or other modes of speciation. Whereas modern evolutionary theory provides detailed explanations of the mechanisms channeling genetic diversity, little is known on the evolutionary forces responsible for the presence and the properties of processes that create genetic diversity. Conventionally, genetic diversity is viewed as the outcome of mutation mechanisms that are not themselves optimized by natural selection. In contrast, a great number of hypotheses are available on the adaptive significance of meiosis, sex and general genetic recombination in eukaryotes, which are mechanisms for the “management” of genetic diversity, but not for its production. In this contribution, we argue that bacteria have evolved elaborate mechanisms for the production of genetic diversity. These mechanisms could themselves be subject to adaptation by natural selection. A selection mechanism termed second-order selection is discussed which could account for the abundance of genes that cause random DNA sequence variation in bacteria.

Strand Exchange Through a DNA-Protein Complex Requires a DNA Helicase

The phage T4 uvsX and gene 32 proteins are capable of mediating homologous strand exchange, a central reaction in general genetic recombination, in vitro using naked DNA substrates. However, strand exchange is blocked by a sequence specific DNA-protein complex. Since protein-complexed substrates must be employed in vivo, this suggests that another factor(s) is required for strand exchange with protein-complexed DNAs. We show here that a DNA helicase, the T4 dda protein, allows the phage recombination machinery to drive branch migration through a RNA polymerase-promoter complex. This is the first observation of in vitro strand exchange using protein-bound substrates. These results suggest that a DNA helicase is a necessary component of the "protein machine" that mediates recombination in vivo.

Electron Microscopy and image analysis of nucleo-protein complexes

The RecA protein of E. coli has been shown to mediate genetic recombination, regulate its own synthesis, control the expression of other genes, act as a specific protease, form a helical polymer and have an ATPase activity, among other observed properties. The unusual filament formed by the RecA protein on DNA has not previously been shown to exist outside of bacteria. Within this filament, the 36 Å pitch of B-form DNA is extended to about 95 Å, the pitch of the RecA helix. We have now establishedthat similar nucleo-protein complexes are formed by bacteriophage and yeast proteins, and availableevidence suggests that this structure is universal across all of biology, including humans. Thus, understanding the function of the RecA protein will reveal basic mechanisms, in existence inall organisms, that are at the foundation of general genetic recombination and repair.Recombination at this moment is assuming an importance far greater than just pure biology. The association between chromosomal rearrangements and neoplasms has become stronger and stronger, and these rearrangements are most likely products of the recombinatory apparatus of the normal cell. Further, damage to DNA appears to be a major cause of cancer.

The role of protein-protein interactions in the assembly of the presynaptic filament for T4 homologous recombination.

The presynaptic filament is an obligatory intermediate in general genetic recombination. It is composed of a strand transferase protein polymerized along single-stranded DNA. In bacteriophage T4-infected cells, the presynaptic filament is composed of at least three proteins. In addition to the strand transferase (the uvsX protein), the uvsY (an accessory factor), and gene 32 (a helix-destabilizing factor), proteins also bind to the single-stranded DNA. In this report, we probe the assembly of the T4 presynaptic filament and the organization of the proteins in the complex. We find that interactions between the uvsY protein and the C terminus of the gene 32 protein are required to load UvsY onto gene 32 protein-covered DNA. Efficient binding of the uvsY protein to DNA is shown to be necessary for subsequent loading of the uvsX protein.

Inhibition of protein-mediated homologous pairing by a DNA helicase

Protein-mediated exchange of homologous DNA strands is a central reaction in general genetic recombination and the mechanism by which proteins mediate this process in vivo is a topic of keen interest. The dda protein of the bacteriophage T4 is a DNA helicase that has been shown to accelerate branch migration catalyzed by the phage uvsX and gene 32 proteins in vitro (Kodadek, T., and Alberts, B.M. (1987) Nature 326, 312-314). This study did not address the potential role of the helicase in protein-mediated homologous pairing, the first phase of the overall strand-exchange reaction. It is shown here that the dda protein inhibits uvsX protein-mediated pairing between homologous single and double-stranded DNAs. Experiments using deproteinized heteroduplex joints demonstrate that the dda helicase is capable of unwinding these structures to some extent and suggests that this activity may be responsible for the observed inhibition of pairing. It is found that the helicase also reduces the level of uvsX protein-mediated, single-stranded DNA-dependent ATP hydrolysis in the strand-exchange reactions, suggesting that the helicase may also act to destabilize the uvsX protein-DNA filaments that are important intermediates in the pairing reaction. Three other helicases are found to have no effect on the uvsX protein-mediated pairing reaction. A model rationalizing the ability of the dda protein to both inhibit homologous pairing and stimulate branch migration is presented and possible in vivo roles for this interesting activity are discussed.

Stoichiometry, base orientation, and nuclease accessibility of RecA.DNA complexes seen by polarized light in flow-oriented solution. Implications for the mechanism of genetic recombination.

By using flow linear dichroism, in combination with nuclease digestion and two spectroscopically distinguishable DNAs, we demonstrate the existence of two internal and one external DNA-binding sites in the RecA fiber. A number of different complexes between RecA and single- and double-stranded DNAs are characterized with respect to stoichiometry, location, and base orientation of each of the associated DNAs. Based on these results, we discuss important steps of the mechanism of general genetic recombination.

Homologous pairing in vitro initiated by DNA synthesis

A number of models have been proposed for the initiation of general genetic recombination. One of these, originally proposed by Meselson and Radding, imagines that the single-stranded 5′ tail that results from strand displacement DNA repair synthesis can initiate homologous recombination by invading a homologous duplex. The resultant D-loop intermediate is then processed into mature products. We demonstrate here that an in vitro system composed of the bacteriophage T4 uvsX protein (a RecA-like “strand transferase”) and part of the T4 DNA polymerase holoenzyme efficiently mediates pairing between nicked double-stranded circular and linear duplex DNAs, thereby demonstrating the feasibility of a key step in the Meselson-Radding model.

Gene conversion in Escherichia coli: the recF pathway for resolution of heteroduplex DNA.

The independent repair of mismatched nucleotides present in heteroduplex DNA has been used to explain gene conversion and map expansion after general genetic recombination. We have constructed and purified heteroduplex plasmid DNAs that contain heteroallelic 10-base-pair insertion-deletion mismatches. These DNA substrates are similar in structure to the heteroduplex DNA intermediates that have been proposed to be produced during the genetic recombination of plasmids. These DNA substrates were transformed into wild-type and mutant Escherichia coli strains, and the fate of the heteroduplex DNA was determined by both restriction mapping and genetic tests. Independent repair events that yielded a wild-type Tetr gene were observed at a frequency of approximately 1% in both wild-type and recB recC sbcB mutant E. coli strains. The independent repair of small insertion-deletion-type mismatches separated by 1,243 base pairs was found to be reduced by recF, recJ, and ssb single mutations in an otherwise wild-type genetic background and reduced by recF, recJ, and recO mutations in a recB recC sbcB genetic background (the ssb mutation was not tested in the latter background). Independent repair of small insertion-deletion-type mismatched nucleotides that were as close as 312 nucleotides apart was observed. There was no apparent bias in favor of the insertion or deletion of mutant sequences.

Effects of dominant-negative lac repressor mutations on operator specificity and protein stability

The specific binding of dominant-negative (I- d) lactose ( lac) repressers to wild-type (wt) as well as mutant ( O c) lac operators has been examined to explore the sequence-specific interaction of the lac repressor with its target. Mutant lacI genes encoding substitutions in the N-terminal 60 amino acids (aa) were cloned in a derivative of plasmid pBR322. Twelve of these lacI -d missense mutations were transferred from F' lac episomes using general genetic recombination and molecular cloning, and nine lacI missense mutations were recloned from M13- lacI phages [Mott et al., Nucl. Acids Res. 12 (1984) 4139–4152]. The mutant repressors were examined for polypeptide size and stability, for binding the inducer isopropyl-β- d-thiogalactoside (IPTG), as well as binding to wt operator. The mutant repressors' affinities for wt operator ranged from undetectable to about 1% that of wt repressor, and the mutant repressors varied in transdominance against represser expressed from a chromosomal lacI q gene. Six of the I- d repressors were partially degraded in vivo. All repressors bound IPTG with approximately the affinity of wt repressor. Repressers having significant affinity for wt operator or with substitutions in the presumed operator recognition helix (aa 17–25) were examined in vivo for their affinities for a series of single site O c operators. Whereas the Gly-18-, Ser-18- and Leu-18-substituted repressors showed altered specificity for position 7 of the operator [Ebright, Proc. Natl. Acad. Sci. USA 83 (1986) 303–307], the His-18 represser did not affect specificity. This result may be related to the greater side-chain length of histidine compared to the other amino acid substitutions.

The homologous recombination system of phage lambda. Pairing activities of beta protein.

The red genes of phage lambda specify two proteins, exonuclease and beta protein, which are essential for its general genetic recombination in recA- cells. These proteins seem to occur in vivo as an equimolar complex. In addition, beta protein forms a complex with another polypeptide, probably of phage origin, of Mr 70,000. The 70-kDa protein appears to be neither a precursor nor an aggregated form of either exonuclease or beta protein, since antibodies directed against the latter two proteins failed to react with 70-kDa protein on Ouchterlony double diffusion analysis. beta protein promotes Mg2+-dependent renaturation of complementary strands (Kmiec, E., and Holloman, W. K. (1981) J. Biol. Chem. 256, 12636-12639). To look for other pairing activities of beta protein, we developed methods of purification to free it of associated exonuclease. Exonuclease-free beta protein appeared unable to cause the pairing of a single strand with duplex DNA; however, like Escherichia coli single strand binding protein (SSB), beta protein stimulated formation of joint molecules by recA protein from linear duplex DNA and homologous circular single strands. Like recA protein, but unlike SSB, beta protein promoted the joining of the complementary single-stranded ends of phage lambda DNA. beta protein specifically protected single-stranded DNA from digestion by pancreatic DNase. The half-time for renaturation catalyzed by beta protein was independent of DNA concentration, unlike renaturation promoted by SSB and spontaneous renaturation, which are second order reactions. Thus, beta protein resembles recA protein in its ability to bring single-stranded DNA molecules together and resembles SSB in its ability to reduce secondary structure in single-stranded DNA.

Purification of a RecA protein analogue from Bacillus subtilis.

We have identified in Bacillus subtilis an analogue of the Escherichia coli RecA protein. Its activities suggest that it has a corresponding role in general genetic recombination and in regulation of SOS (DNA repair) functions. The B. subtilis protein (B. subtilis Rec) has a Mr of 42,000 and cross-reacts with antisera raised against E. coli RecA protein. Its level is significantly reduced in the recombination-deficient recE4 mutant. B. subtilis Rec is induced 10- to 20-fold in rec+ strains following treatment with mitomycin C, whereas it is not induced in the recombination-deficient mutants recE4, recE45, and recA1. We have purified B. subtilis Rec about 2000-fold to near homogeneity and we describe its activities. It catalyzes DNA-dependent hydrolysis of dATP at a rate comparable to that of E. coli RecA protein. However, B. subtilis Rec has a negligible ATPase activity, although ATP effectively inhibits dATP hydrolysis. In the presence of dATP, B. subtilis Rec catalyzes DNA strand transfer, assayed by the conversion of phi X174 linear duplex DNA and homologous circular single-stranded DNA to replicative form II (circular double-stranded DNA with a discontinuity in one strand). ATP does not support strand transfer by this protein. B. subtilis Rec catalyzes proteolytic cleavage of E. coli LexA repressor in a reaction that requires single-stranded DNA and nucleoside triphosphate. This result suggests that an SOS regulatory system like the E. coli system is present in B. subtilis. The B. subtilis enzyme does not promote any detectable cleavage of the E. coli bacteriophage lambda repressor.

General genetic recombination of bacterial plasmids.

Analysis of genetic recombination between 2 chromosomes, which differ from each other by at least 2 genetic markeŕs, is based mainly on the production of recombinants which are phenotypically distinguishable from their progenitors. The use of small DNA molecules such as bacterial plasmids as recombination substrates provides an additional handle for the investigation of recombination. It also allows the description of recombination events in molecular terms. Recombination of 2 plasmids, genetically distinguishable from each other, can be followed as recombination reactions in other systems by monitoring changes in the phenotypic expression of the plasmid genes (1,2,3). But in addition, plasmidic recombination may be investigated by analyzing changes of their molecular structures. Interplasmidic recombination leads to the formation of plasmid oligomers and intraplasmidic recombination between repeated sequences can lead to deletion of the region between the crossing-over sites, and, when the recombination substrate is an oligomer, to monomerization. When mutations affect restriction endonuclease sites, recombination products may be compared to recombination substrates by restriction endonculease analysis (2,4,5). The small size of bacterial plasmids also allows the isolation and characterization of molecular intermediates of the recombination pathway (6,7).

Role of RecA protein spiral filaments in genetic recombination.

Physical and enzymatic studies on RecA protein from Escherichia coli provide the basis for a molecular model of general genetic recombination, a novel feature of which is the role attributed to spiral filaments of RecA protein.

Resolution of Holliday structures by endonuclease VII as observed in interactions with cruciform DNA.

Exchange of genetic material during general genetic recombination is accomplished through a series of molecular events that are described in two fundamental models (Holliday 1964; Meselson and Radding 1975). An intrinsic part of each model is the formation of the Holliday structure. The structure consists of two aligned homologous double-stranded DNA molecules that, at some point, exchange strands of the same polarity. Because of branch migration, the structure can move away from its original site of formation before it is resolved into either patch or splice recombinants (Fig. 1). The existence of Holliday structures and their role in general genetic recombination has been demonstrated physically in a number of prokaryotic and eukaryotic systems (for review, see Dressler and Potter 1982). Remarkable progress has been made in understanding some of the enzymatic reactions leading to the formation of Holliday structures (West et al. 1983; for review, see Radding 1982). In contrast,...

Molecular mechanisms of general genetic recombination: the DNA-binding sites of RecA protein.

The RecA protein of Escherichia coli mediates homologous pairing and strand exchange reactions similar to those of general genetic recombination. In vitro, this protein acts on suitable DNA molecules from phages or plasmids, converting them to heteroduplex recombination intermediates. We describe studies on RecA-mediated, reciprocal strand exchanges between two duplexes. Homologous pairing can occur if one duplex is partially single stranded (gapped) and leads to a strand exchange capable of passing and contributing to the repair of a two-strand break in the fully duplex molecule. However, it fails to pass a break in the gapped duplex. In related studies, we show that homologous pairing leading to strand exchange can occur between DNA molecules at positions where both are duplex.