T4 UvsX Protein Research Articles

Role of Allosteric Switch Residue Histidine 195 in Maintaining Active-Site Asymmetry in Presynaptic Filaments of Bacteriophage T4 UvsX Recombinase

Recombinases of the highly conserved RecA/Rad51 family play central roles in homologous recombination and DNA double-stranded break repair. RecA/Rad51 enzymes form presynaptic filaments on single-stranded DNA (ssDNA) that are allosterically activated to catalyze ATPase and DNA strand-exchange reactions. Information is conveyed between DNA- and ATP-binding sites, in part, by a highly conserved glutamine residue (Gln194 in Escherichia coli RecA) that acts as an allosteric switch. The T4 UvsX protein is a divergent RecA ortholog and contains histidine (His195) in place of glutamine at the allosteric switch position. UvsX and RecA catalyze similar strand-exchange reactions, but differ in other properties. UvsX produces both ADP and AMP as products of its ssDNA-dependent ATPase activity--a property that is unique among characterized recombinases. Details of the kinetics of ssDNA-dependent ATP hydrolysis reactions indicate that UvsX-ssDNA presynaptic filaments are asymmetric and contain two classes of ATPase active sites: one that generates ADP, and another that generates AMP. Active-site asymmetry is reduced by mutations at the His195 position, since UvsX-H195Q and UvsX-H195A mutants both exhibit stronger ssDNA-dependent ATPase activity, with lower cooperativity and markedly higher ADP/AMP product ratios, than wild-type UvsX. Reduced active-site asymmetry correlates strongly with reduced ssDNA-binding affinity and DNA strand-exchange activity in both H195Q and H195A mutants. These and other results support a model in which allosteric switch residue His195 controls the formation of an asymmetric conformation of UvsX-ssDNA filaments that is active in DNA strand exchange. The implications of our findings for UvsX recombination functions, and for RecA functions in general, are discussed.

Complementation of bacteriophage induction and recombination defects in Escherichia coli RecA(-) mutants by expression of the cloned T4 bacteriophage uvsX gene.

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA(-) mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present.

Single-stranded DNA binding properties of the UvsX recombinase of bacteriophage T4: binding parameters and effects of nucleotides

Bacteriophage T4 provides an important model for the biochemistry and genetics of DNA metabolism. Phage-encoded proteins conduct all essential steps of T4 DNA replication, repair, and recombination. Central to these three processes is the T4 UvsX protein, a member of the filamentous, ATP-dependent class of general recombination enzymes typified by the Escherichia coli RecA protein. Like RecA, UvsX forms presynaptic filaments on single-stranded (ss) DNA, which are the obligatory nucleoprotein intermediates in recombination. Aspects of the T4 presynaptic filament are explored by quantitative characterization of the UvsX-ssDNA interaction using an etheno-derivitized single-stranded DNA molecule, ϵDNA, whose fluorescence is enhanced by UvsX binding. Studies with this model lattice show that UvsX exhibits a moderate level of cooperativity (ω = 100) when binding to ϵDNA with a binding-site size (n) equal to four nucleotide residues. Salt-stability studies of this complex reveal that the non-hydrolyzable ATP analog, ATPγS, induces a high-affinity binding mode that is distinguishable from complexes formed with ADP or in the absence of a nucleotide cofactor. With this new information, both functional relationships between the UvsX and RecA recombinases, and implications for UvsX interactions with the other proteins of the T4 presynaptic filament (UvsY and gp32) may be further explored.

Mechanisms of assembly of the enzyme-ssDNA complexes required for recombination-dependent DNA synthesis and repair in bacteriophage T4.

During late stages of bacteriophage T4 infection in E. coli, the initiation of phage DNA replication is dependent on the homologous recombination activity of the T4 uvsX protein. In vitro, uvsX protein initiates DNA synthesis on a duplex template by inserting the 3{prime} end of a homologous ssDNA molecule into the duplex. The resulting D-loop structure serves as a primer-template junction for the assembly of the T4 replication fork. Two key steps in this initiation process are (A) the assembly of uvsX-ssDNA complexes necessary for recombination activity and for the priming of lead-strand DNA synthesis, and (B) the assembly of the T4 primosome (gp41 helicase/gp61 primase complex) onto the single-stranded template for lagging-strand synthesis. Our laboratory is focusing on the mechanisms of these two different but related enzyme-ssDNA assembly processes. In this extended abstract, we describe recent efforts in our laboratory to elucidate the mechanism by which the gp41 helicase enzyme is assembled onto gp32-covered ssDNA, a process requiring the activity of a special helicase assembly factor, the T4 gp59 protein.

Overexpression, purification, sequence analysis, and characterization of the T4 bacteriophage dda DNA helicase.

The bacteriophage T4 dda protein is a 5'-3' DNA helicase that stimulates DNA replication and recombination reactions in vitro and seems to play a role in the initiation of T4 DNA replication in vivo. Oligonucleotide probes based on NH2-terminal amino acid sequence were used to precisely map the location of the dda gene on the T4 chromosome. Using polymerase chain reaction techniques, the dda gene was then cloned into an expression vector, and the overproduced protein was purified in two chromatography steps. Both the genomic and cloned dda genes were sequenced and found to be identical, encoding a protein of 439 amino acids. The dda protein contains amino acid sequences resembling those of other known helicases, and is most homologous to the Escherichia coli recD protein. Protein affinity chromatography was used to show a direct interaction between the dda protein and the T4 uvsX protein (a rec A-type DNA recombinase).

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.

Visualization of the homologous pairing of DNA catalyzed by the bacteriophage T4 UvsX protein.

The uvsX gene product is essential for DNA repair and general recombination in T4 bacteriophage. The ability of UvsX protein to catalyze the homologous pairing of single-stranded DNA (ssDNA) with double-stranded DNA (dsDNA) in vitro was examined by electron microscopic (EM), nitrocellulose filter binding, and gel electrophoretic methods. Optimal joining was observed at ratios of UvsX protein:ssDNA of 2 nucleotides/protein monomer. At this level, the ssDNA was fully covered by UvsX protein as seen by EM, while the dsDNA appeared protein-free. Using this stoichiometry, the pairing of circular ssDNA with homologous supertwisted dsDNA was found to produce a high frequency of complexes in which a supertwisted dsDNA molecule was joined to a UvsX protein-ssDNA filament over a distance of less than 100 base pairs. These joints were labile to deproteinization and must have been paranemic. Pairing of linear ssDNA containing buried homology to the dsDNA produced identical structures. Pairing of fully homologous linear ssDNA and supertwisted dsDNA yielded D-loop joints (plectonemic) as seen by EM following deproteinization. Both the paranemic and the plectonemic joints were at sites of homology, as demonstrated by restriction cleavage of the complexes. Visualization of the joined complexes prior to deproteinization showed that 50% of the joints had the architecture of the paranemic joints, whereas in the remainder, a topologically relaxed dsDNA circle merged with the UvsX protein-ssDNA filament for a distance of 450 base pairs. The structure of the filament was not visibly altered in this region. These observations are similar, but not identical, to findings in parallel studies utilizing the RecA protein of Escherichia coli.

Stimulation of protein-directed strand exchange by a DNA helicase.

The protein-mediated exchange of strands between a DNA double helix and a homologous DNA single strand involves both synapsis and branch migration, which are two important aspects of any general recombination reaction. Purified DNA-dependent ATPases from Escherichia coli (recA protein), Ustilago (rec 1 protein) and phage T4 (uvsX protein) have been shown to drive both synapsis and branch migration in vitro. The T4 gene 32 protein is a helix-destabilizing protein that greatly stimulates uvsX-protein-catalysed synapsis, and the E. coli SSB (single-strand binding) protein stimulates the analogous recA-protein-mediated reaction to a lesser degree. One suspects that several other proteins also play a role in the strand exchange process. For example, a DNA helicase could in principle accelerate branch migration rates by helping to melt the helix at the branch point. The T4 dda protein is a DNA helicase that is required to move the T4 replication fork past DNA template-bound proteins in vitro. Previously, we have shown that the dda protein binds to a column that contains immobilized T4 uvsX protein. We show here that this helicase specifically stimulates the branch migration reaction that the uvsX protein catalyses as a central part of the genetic recombination process in a T4 bacteriophage-infected cell.

Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvsX and uvsY.

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39 000 and 16 000, respectively. In the normal wild-type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single-stranded fd DNA fragment into superhelical double-stranded fd DNA (RFI). The reaction requires ATP and Mg2+ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.