Analytical model for determination of parameters of helical structures in solution by small angle scattering: comparison of RecA structures by SANS.

The filament structures of the self-polymers of RecA proteins from Escherichia coli and Pseudomonas aeruginosa, their complexes with ATPgammaS, phage M13 single-stranded DNA (ssDNA) and the tertiary complexes RecA::ATPgammaS::ssDNA were compared by small angle neutron scattering. A model was developed that allowed for an analytical solution for small angle scattering on a long helical filament, making it possible to obtain the helical pitch and the mean diameter of the protein filament from the scattering curves. The results suggest that the structure of the filaments formed by these two RecA proteins, and particularly their complexes with ATPgammaS, is conservative.

Recombinogenic activity of chimeric recA genes (Pseudomonas aeruginosa/Escherichia coli): a search for RecA protein regions responsible for this activity.

In the background of weak, if any, constitutive SOS function, RecA from Pseudomonas aeruginosa (RecAPa) shows a higher frequency of recombination exchange (FRE) per DNA unit length as compared to RecA from Escherichia coli (RecAEc). To understand the molecular basis for this observation and to determine which regions of the RecAPa polypeptide are responsible for this unusual activity, we analyzed recAX chimeras between the recAEc and recAPa genes. We chose 31 previously described recombination- and repair-proficient recAX hybrids and determined their FRE calculated from linkage frequency data and constitutive SOS function expression as measured by using the lacZ gene under control of an SOS-regulated promoter. Relative to recAEc, the FRE of recAPa was 6.5 times greater; the relative alterations of FRE for recAX genes varied from approximately 0.6 to 9.0. No quantitative correlation between the FRE increase and constitutive SOS function was observed. Single ([L29M] or [I102D]), double ([G136N, V142I]), and multiple substitutions in related pairs of chimeric RecAX proteins significantly altered their relative FRE values. The residue content of three separate regions within the N-terminal and central but not the C-terminal protein domains within the RecA molecule also influenced the FRE values. Critical amino acids in these regions were located close to previously identified sequences that comprise the two surfaces for subunit interactions in the RecA polymer. We suggest that the intensity of the interactions between the subunits is a key factor in determining the FRE promoted by RecA in vivo.

Overexpression of bacterial RecA protein stimulates homologous recombination in somatic mammalian cells.

The pairing of homologous molecules and strand exchange is a key event in homologous recombination promoted by RecA protein in Escherichia coli. Structural homologs of RecA are widely distributed in eukaryotes including mouse and man. As has been shown, human HsRad51 protein is not only structural but also functional homolog of RecA. The question arises whether the bacterial functional homolog of Rad51 can function in mammalian cells and increase the frequency of the homologous recombination. To investigate possible effects of bacterial RecA protein on the frequency of homologous recombination in mammalian cells, the E. coli RecA protein fused with a nuclear location signal from the large T antigen of simian virus 40 was overexpressed in the mouse F9 teratocarcinoma cells. We found that the frequency of gene targeting at the hprt locus was 10-fold increased in the mouse cells expressing the nucleus-targeted RecA protein. Southern blot analysis of individual clones that were generated by targeting recombination revealed predicted type of alterations in hprt gene. The data indicate that the bacterial nucleus-targeted RecA protein can stimulate homologous recombination in mammalian cells.

Fluorescence and excitation Escherichia coli RecA protein spectra analyzed separately for tyrosine and tryptophan residues.

The method for separation of emission (EM) and excitation (EX) spectra of a protein into EM and EX spectra of its tyrosine (Tyr) and tryptophan (Trp) residues was described. The method was applied to analysis of Escherichia coli RecA protein and its complexes with Mg(2+), ATPgammaS or ADP, and single-stranded DNA (ssDNA). RecA consists of a C-terminal domain containing two Trp and two Tyr residues, a major domain with five Tyr residues, and an N-terminal domain without these residues (R. M. Story, I. T. Weber, and T. A. Steitz (1992) Nature (London) 355, 374-376). Because the fluorescence of Tyr residues in the C-terminal domain was shown to be quenched by energy transfer to Trp residues, Trp and Tyr fluorescence of RecA was provided by the C-terminal and the major domains, respectively. Spectral analysis of Trp and Tyr constituents revealed that a relative spatial location of the C-terminal and the major domains in RecA monomers was different for their complexes with either ATPgammaS or ADP, whereas this location did not change upon additional interaction of these complexes with ssDNA. Homogeneous (that is, independent of EX wavelength) and nonhomogeneous (dependent on EX wavelength) types of Tyr and Trp fluorescence quenching were analyzed for RecA and its complexes with nucleotide cofactors and ssDNA. The former was expected to result from singlet-singlet energy transfer from these residues to adenine of ATPgammaS or ADP. By analogy, the latter was suggested to proceed through energy transfer from high vibrational levels of the excited state of Trp and Tyr residues to the adenine. In this case, for correct calculation of the overlap integral, Trp and Tyr donor emission spectra were substituted by the spectral function of convolution of emission and excitation spectra that resulted in a significant increase of the overlap integral and gave an explanation of the nonhomogeneous quenching of Trp residues in the C-terminal domain.

Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.

The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C).

Gene targeting for gene therapy: prospects.

Ideally, gene therapy involves the correction of genetic defects through the natural means of gene targeting. This therapy possesses a number of conceptual advantages. However, a major obstacle to successful gene therapy is the relative inefficiency of the targeting process in mammalian cells. Gene targeting may be accomplished by two different mechanisms: the homologous recombination and the mismatch correction of DNA heteroduplexes. Based on the model of homologous recombination for the well-studied prokaryotic and the less studied eukaryotic systems, three approaches have been employed to improve the efficiency and accuracy of homologous recombination events. These are: (1) artificial double-strand breaks in both the exogenous and the chromosomal DNA, (2) a contiguous long homology between the exogenous and chromosomal DNA, and (3) a transient overproduction of an active recombinase, the bacterial RecA or mammalian RecA-like proteins, in mammalian cell nuclei. Combining these approaches can result in more effective gene targeting protocols. The second mechanism has been improved based on recent observations of recombinogenic activity of oligonucleotides and, especially, specifically designed chimeric RNA/DNA oligonucleotides. The use of RecA-like proteins to stimulate searching for homology and forming stable DNA heteroduplexes between oligonucleotides and chromosomal DNA remains an attractive idea for additional improvement of gene targeting events.

Tn5/IS50 target recognition.

This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5/IS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.

Biochemical basis of hyper-recombinogenic activity of Pseudomonas aeruginosa RecA protein in Escherichia coli cells.

The replacement of Escherichia coli recA gene (recA[Ec]) with the Pseudomonas aeruginosa recA(Pa) gene in Escherichia coli cells results in constitutive hyper-recombination (high frequency of recombination exchanges per unit length of DNA) in the absence of constitutive SOS response. To understand the biochemical basis of this unusual in vivo phenotype, we compared in vitro the recombination properties of RecA(Pa) protein with those of RecA(Ec) protein. Consistent with hyper-recombination activity, RecA(Pa) protein appeared to be more proficient both in joint molecule formation, producing extensive DNA networks in strand exchange reaction, and in competition with single-stranded DNA binding (SSB) protein for single-stranded DNA (ssDNA) binding sites. The RecA(Pa) protein showed in vitro a normal ability for cleavage of the E. coli LexA repressor (a basic step in SOS regulon derepression) both in the absence and in the presence (i.e. even under suboptimal conditions for RecA(Ec) protein) of SSB protein. However, unlike other hyper-recombinogenic proteins, such as RecA441 and RecA730, RecA(Pa) protein displaced insufficient SSB protein from ssDNA at low magnesium concentration to induce the SOS response constitutively. In searching for particular characteristics of RecA(Pa) in comparison with RecA(Ec), RecA441 and RecA803 proteins, RecA(Pa) showed unusually high abilities: to be resistant to the displacement by SSB protein from poly(dT); to stabilize a ternary complex RecA::ATP::ssDNA to high salt concentrations; and to be much more rapid in both the nucleation of double-stranded DNA (dsDNA) and the steady-state rate of dsDNA-dependent ATP hydrolysis at pH7.5. We hypothesized that the high affinity of RecA(Pa) protein for ssDNA, and especially dsDNA, is the factor that directs the ternary complex to bind secondary DNA to initiate additional acts of recombination instead of to bind LexA repressor to induce constitutive SOS response.

A recombinational defect in the C-terminal domain of Escherichia coli RecA2278-5 protein is compensated by protein binding to ATP.

RecA2278-5 is a mutant RecA protein (RecAmut) bearing two amino acid substitutions, Gly-278 to Thr and Val-275 to Phe, in the alpha-helix H of the C-terminal subdomain of the protein. RecA2278-5 mutant cells are unusual in that they are thermosensitive for recombination but almost normal for DNA repair of UV damage and the SOS response. Biochemical analysis of purified RecAmut protein revealed that its temperature sensitivity is suppressed by prior binding of this protein to its ligand. In fact, the preheating of RecAmut protein for several minutes at a restrictive temperature (42 degrees C) in the absence of ATP resulted in inhibition at 42 degrees C of many activities related to homologous recombination including ss- and dsDNA binding, high-affinity binding for ATP, ss- or dsDNA-dependent ATPase, RecA-RecA interaction, and strand transfer capability. The binary complex RecAmut::ATP under the same conditions showed a decrease in only two activities, i.e. dsDNA binding and high-affinity binding for ATP. Besides ATP, sodium acetate (1.5 M) was shown to be another factor that can stabilize the RecAmut protein at 42 degrees C, judging by restoration of its DNA-free ATPase activity. The similarity of influence of high salt (with its non-specific binding) and ATP (binding specifically) on the apparent protein folding stability suggests that the structural stability of the RecA C-terminal domain is one of the conditions for correct interaction between RecA protein and ATP in the RecA::ATP::ssDNA presynaptic complex formation. The decrease in affinity for ATP was suggested to be the factor that determined a particular recombinational (but not repair) thermosensitivity of the RecA-mut protein. Finally, we show that the stability of C-terminal domain appeared to be necessary for the dsDNA-binding activity of the protein.

Genetic characteristics of new recA mutants of Escherichia coli K-12.

To search for functionally thermosensitive (FT) recA mutations, as well as mutations with differently affect RecA protein functions, seven new recA mutations in three different regions of the RecA protein structure proposed by Story et al. [R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318-325, 1992] were constructed. Additionally, the recA2283 allele responsible for the FT phenotype of the recA200 mutant was sequenced. Five single mutations (recA2277, recA2278, recA2283, recA2283E, and recA2284) and one double mutation (recA2278-5) generated, respectively, the amino acid substitutions L-277-->N, G-278-->P, L-283-->P, L-283-->E, I-284-->D, and G-278-->T plus V-275-->F in the alpha-helix H-beta-strand 9 region of the C-terminal domain of the RecA protein structure. According to recombination, repair, and SOS-inducible characteristics, these six mutations fall into four phenotypic classes: (i) an FT class, with either inhibition of all three analyzed functions at 42 degrees C (recA2283), preferable inhibition at 42 degrees C of recombination and the SOS response (recA2278), or inhibition at 42 degrees C of only recombination (recA2278-5); (ii) a moderately deficient class (recA2277); (iii) a nondeficient class (recA2283E); and (iv) a mutation with a null phenotype (recA2284). The recA2223 mutation generates an L-223-->M substitution in beta-strand 6 in a central domain of the RecA structure. This FT mutation shows preferable inhibition of the SOS response at 42 degrees C. The recA2183 mutation produces a K-183-->M substitution in alpha-helix F of the same domain. The Lys-183 position in the Escherichia coli RecA protein was found among positions which are important for interfilament interaction (R. M. Story, I. T. Weber, and T. A. Steitz, Nature (London) 355:318-325, 1992).

Partial purification and characterization of two types of homologous DNA pairing activity from rat testis nuclei.

We describe the partial purification and characterization of two different types of homologous DNA pairing activity from rat testis nuclear extracts. The activities are separated from each other by single-stranded DNA-cellulose affinity chromatography. One activity requires single-stranded DNA ends and promotes the homologous pairing of single-stranded DNA fragments with double-stranded circular DNA and has an apparent molecular mass of 100 kDa as determined by gel filtration chromatography. This pairing activity does not require the addition of exogenous ATP and is strongly Mg2+ -dependent. The second pairing activity promotes strand-transfer between single-stranded circular DNA and homologous double-stranded DNA fragments and has an apparent molecular mass of 30 kDa as determined by gel filtration chromatography. This pairing activity also does not require ATP but, in contrast to the former, is Mg2+ -independent.

Optimization of PCR-based diagnostics for human cytomegalovirus.

Various techniques of DNA template preparation for the PCR-based analysis of human CMV in biological fluids have been compared. Structural polymorphism of a CMV DNA segment (part of the major immediate early gene) in clinical isolates is described; the molecular markers (nucleotide substitutions, deletions, insertions) localized in the analyzed amplicon appear to be suitable for molecular-epidemiological studies. A scheme of spreading of the molecular markers in the population is suggested.

Functional characteristics of the recA gene from Serratia marcescens strain Sb.

The cloned recA gene from Serratia marcescens Sb was expressed and complemented defects in the UV repair, recombination, and SOS induction of an Escherichia coli host deleted for recA. Moreover, the Serratia gene, recA (Sm), supported the same frequency of recombination per unit length of DNA as did the homologous Escherichia coli gene, recA(Ec).

Postexcision transposition of the transposon Tn10 in Escherichia coli K12.

An experimental analysis of the fate of transposon Tn10 after excision from a proA::Tn10 site localized on the plasmid F' leads to the conclusions: 1. The precise excision is a progressive process. Its probability is estimated per time unit. 2. An excised Tn10 is always integrated into a different genetic locus. 2. An excised Tn10 is always integrated into a different genetic locus. 3. The kinetics of postexcision transposition are sometimes very slow. The excised transposon is inherited in one cell line in spite of cell multiplication. 4. The processes of excision and secondary insertion have no absolute requirement for the recA+ genotype but they are strongly enhanced in recA+ cells. 5. The kinetics of postexcision transposition are strongly dependent on the genetic site from which the transposon was excised. 6. The probability of postexcision transposition is fully determined by the probability of excision and depends on the genotype of the host and many other factors.

Interspecies recA protein substitution in Escherichia coli and Proteus mirabilis.

With the help of recombinant plasmids carrying the recA gene of Escherichia coli or of Proteus mirabilis the ability of the recA gene products to substitute functionally for each other was studied. The recA protein of each can function in recombination, repair, induction of mutations and prophages and in regulation of its own synthesis within the foreign host nearly equally well as in the natural host. It is, therefore, suggested that recA-dependent processes act similarly in E. coli and P. mirabilis.

Recombinational instability of F' plasmids in Escherichia coli K-12: localization of fre-sites.

The F' plasmids ORF-1 (purE+ tsxs proC+ lac+) and F'14 (argE+ metB+ ilv+) contain active regions of recombination, fre I and fre II correspondingly. The plasmid ORF-1 is stable in recF- cells (i.e., with the RecBC pathway of recombination) and decays in rec+ cells (RecBCF pathway) giving two types of product: F+ and plasmid pCK-1 (tsxs proC+ lac+) containing part of the initial DNA. They are extremely instable in the presence of the RecF pathway, (recBC- sbcB-), yielding F+ and plasmid pCK-2 (proC+ lac+). The instability of plasmids depends on a region of homology between the chromosome and the episome. The instability of ORF-1 shows the participation of IS3 elements (alpha 1 beta 3 and alpha 3 beta 1) in the recA, recF-dependent recombinational decay and allows localization of two active sites on the chromosome: fre I1 between purE and tsx markers and fre I2 between tsx and proC. The plasmid F'14, in accordance with published data, is able to yield F+ cells by recA-independent recombination. But eventually this plasmid may undergo a recA, recF-dependent decay. Genetic analysis of these events allows localization of an active point of recombination, freII1, between argE and metB. Another active point is localized inside the F factor. The recA-dependent decay of plasmid F-14 is also excluded on the RecBC pathway (recF- strains).

The process of general recombination in Escherichia coli K-12: structure of intermediate products.

A review of the data on the genetic determination of general recombination in Escherichia coli introduces three alternative pathways of recombination, RecBC, RecF, and RecBCF. One recBC-dependent pathway is functional in recF- cells. An initiating endonuclease is involved, acting on the chi-sites of DNA. The second is recF-dependent, acting in the double mutant recBC sbcB. The corresponding endonuclease uses the fre-sites as a substrate. A third pathway acting in wild-type cells is mixed. Both enzymatic systems participate in the overall process. We shall call it RecBCF. Using the thermosensitive recA44 mutant it became possible to study the kinetics of integration of donor DNA into the recipient chromosome via the RecF and RecBCF pathways of recombination. The RecF pathway is characterized by delayed recombination; not less than 14 h being needed to complete the process at 35 degrees C. By the RecBCF pathway (wild-type recipient) the reaction is fast, as described by Lloyd and Johnson (1979). The two stage nature of the RecF pathway is important. First an intermediate product is formed during a short time interval. This product is resistant to the degrading exonuclease V. Afterward the intermediate product is slowly integrated into the recipient chromosome. Autoradiography of this intermediate product, extracted from exconjugants, shows that it consists of closed DNA circles. Their length is within the limits 2--15 min on the E. coli map. Their average value is in fair agreement with genetic estimations of the integrated DNA fragments. Taking into consideration the similarity between genetic determinations of the fre-effects and the heterogeneity of the progeny, we conclude that the intermediate structures formed contribute to this heterogeneity.