RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.     

RecA protein-promoted homologous pairing and strand exchange between intact and partially single-stranded duplex DNA.

In the pairing reaction between circular gapped and fully duplex DNA, RecA protein first polymerizes on the gapped DNA to form a nucleoprotein filament. Conditions that removed the formation of secondary structure in the gapped DNA, such as addition of Escherichia coli single-stranded DNA binding protein or preincubation in 1 mM-MgCl2, optimized the binding of RecA protein and increased the formation of joint molecules. The gapped duplex formed stable joints with fully duplex DNA that had a 5' or 3' terminus complementary to the single-stranded region of the gapped molecule. However, the joints formed had distinct properties and structures depending on whether the complementary terminus was at the 5' or 3' end. Pairing between gapped DNA and fully duplex linear DNA with a 3' complementary terminus resulted in strand displacement, symmetric strand exchange and formation of complete strand exchange products. By contrast, pairing between gapped and fully duplex DNA with a 5' complementary terminus produced a joint that was restricted to the gapped region; there was no strand displacement or symmetric strand exchange. The joint formed in the latter reaction was likely a three-stranded intermediate rather than a heteroduplex with the classical Watson-Crick structure. We conclude that, as in the three-strand reaction, the process of strand exchange in the four-strand reaction is polar and progresses in a 5' to 3' direction with respect to the initiating strand. The present study provides further evidence that in both three-strand and four-strand systems the pairing and strand exchange reactions share a common mechanism.     

Construction and analysis of viable deletion mutants of polyoma virus.

Viable mutants of polyoma with small deletions ranging in size from 2 to 75 base pairs were obtained by infecting 3T3 cells with polyoma DNA that had been cleaved once with HaeII endonuclease or with DNase-Mn2+ digestion. The HaeII endonuclease-cleaved DNA yielded mutants with deletions at map position 72--73, whereas the mutants generated by DNase I-Mn2+ digestion had deletions either at map position 72--73 or within the map coordinates 92 and 99. Both groups of mutants appeared to grow as well as wild-type virus in 3T3 cells. The deletions at map position 72--73 did not alter the virus's ability to transform rat cells. Hence, the region just to the early side of the origin of DNA replication is not essential for vegetative growth or transformation. But the mutants with deletions in the region between map coordinates 92 and 99, a segment thought to code for polyoma large and middle T antigens (Hutchinson et al., Cell 15:65--77, 1978; Smart and Ito, Cell 15:1427--1437, 1978; Soeda et al., Cell 17:357--370, 1979), transformed rat cells at 0.2 to 0.05 the efficiency of wild-type virus.     

Oligonucleotide directed mutagenesis: Selection of mutants by hemimethylation of GATC-sequences

Summary We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost, solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an ATCG transversion in the nut L region of phage . Under optimal conditions, about 50–60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.Abbreviations bp base pairs - RF replicative form - ssDNA single stranded DNA - Ap gene carbenicillin resistance gene - EtBr ethidium bromide - O.D. optical density - Kb kilobases - P_L major leftward promoter of phage      

Homologous pairing in duplex DNA regions and the formation of four-stranded paranemic joints promoted by RecA protein. Effects of gap length and negative superhelicity

RecA protein catalyzes homologous pairing of partially single-stranded duplex DNA and fully duplex DNA to form stable joint molecules. We constructed circular duplex DNA with various defined gap lengths and studied the pairing reaction between the gapped substrate with fully double-stranded DNA. The reaction required a stoichiometric amount of RecA protein, and the optimal reaction was achieved at a ratio of 1 RecA monomer per 4 base pairs. The length of the gap, ranging from 141 to 1158 nucleotides, had little effect on the efficiency of homologous pairing. By using a circular gapped duplex DNA prepared from the chimeric phage M13Gori1, we were able to show the formation of nonintertwined or paranemic joints in duplex regions between the gapped and fully duplex molecules. The formation of such paranemic joints occurred efficiently and included nearly all of the DNA in the reaction mixture. The reaction required negative superhelicity, and pairing was greatly reduced with linear or nicked circular DNA. We conclude that one functional role of the single-stranded gap is for facilitating the binding of RecA protein to the duplex region of the gapped DNA. Once the nucleoprotein filament is formed, homologous pairing between the gapped and fully duplex DNA can take place anywhere along the length of the nucleoprotein complex.     

T-antigen expression by polyoma mutants with modified RNA splicing

Polyoma virus mutants were constructed that could not express all the three T-antigens. The mutagenesis was directed to the two 5' splice sites utilized in the maturation of early RNA. The mutant bc1051 had a base change at the splice site of large T-antigen mRNA, and the mutants dl1061 and dl1062 had deletions at the corresponding splice point of small and middle T-antigen mRNA. The site was removed in mutant dl1061 and altered by fusion to upstream sequences in mutant dl1062. Analysis of viral RNA showed that dl1061 and dl1062 formed only large T-antigen mRNA, whereas bc1051 did not produce this RNA-species. However, the biological properties of dl1062 suggested that it also produced mRNA directing the synthesis of a small T-antigen-related polypeptide, at least in low amounts. Only mutant bc1051 could induce transformation of rat cells. In mouse 3T3 cells dl1062 multiplied to a limited extent, while bc1051 and dl1061 failed to produce virus. However, dl1061 DNA was synthesized at a low rate which could be increased to normal levels by co-transfection with mutant bc1051. This result suggests that polyoma small and middle T-antigen have a previously unrecognized function in the early phase of the infection process, or in viral DNA-synthesis.     

Effect of deletions in adenovirus early region 1 genes upon replication of adeno-associated virus.

The growth of adeno-associated virus (AAV) is dependent upon helper functions provided by adenovirus. We investigated the role of adenovirus early gene region 1 in the AAV helper function by using six adenovirus type 5 (Ad5) host range mutants having deletions in early region 1. These mutants do not grow in human KB cells but are complemented by and grow in a line of adenovirus-transformed human embryonic kidney cells (293 cells); 293 cells contain and express the Ad5 early region 1 genes. Mutants having extensive deletions of adenovirus early region 1a (dl312) or regions 1a and 1b (dl313) helped AAV as efficiently as wild-type adenovirus in 293 cells, but neither mutant helped in KB cells. No AAV DNA, RNA, or protein synthesis was detected in KB cells in the presence of the mutant adenoviruses. Quantitative blotting experiments showed that at 20 h after infection with AAV and either dl312 or dl313 there was less than one AAV genome per cell. In KB cells infected with AAV alone, the unreplicated AAV genomes were detected readily. Apparently, infection with adenovirus mutant dl312 or dl313 results in degradation of most of the infecting AAV genomes. We suggest that at least an adenovirus region 1b product (and perhaps a region 1a product also) is required for AAV DNA replication. This putative region 1b function appears to protect AAV DNA from degradation by an adenovirus-induced DNase. We also tested additional Ad5 mutants (dl311, dl314, sub315, and sub316). All of these mutants were inefficient helpers, and they showed varying degrees of multiplicity leakiness. dl312 and dl313 complemented each other for the AAV helper function, and each was complemented by Ad5ts125 at the nonpermissive temperature. The defect in region 1 mutants for AAV helper function acts at a different stage of the AAV growth cycle than the defect in the region 2 mutant ts125.     

Intermolecular disintegration and intramolecular strand transfer activities of wild-type and mutant HIV-1 integrase.

We report the activities of HIV integrase protein on a novel DNA substrate, consisting of a pair of gapped duplex molecules. Integrase catalyzed an intermolecular disintegration reaction that requires positioning of a pair of the gapped duplexes in a configuration that resembles the intgration intermediate. However, the major reaction resulted from an intramolecular reaction involving a single gapped duplex, giving rise to a hairpin. Surprisingly, a deletion mutant of integrase that lacks both the amino and carboxyl terminal regions still catalyzed the intermolecular disintegration reaction, but supported only a very low level of the intramolecular reaction. The central core region of integrase is therefore sufficient to both bind the gapped duplex DNA and juxtapose a pair of such molecules through protein-protein interactions. We suggest that the branched DNA structures of the previously reported disintegration substrate, and the intermolecular disintegration substrate described here, assist in stabilizing protein-protein interactions that otherwise require the amino and carboxy terminal regions of integrase.     

Regulatory mutants of polyoma virus defective in DNA replication and the synthesis of early proteins

In polyoma virus the origin of replication, the 5′ ends of early mRNAs, and the initiation codon for early protein synthesis map within an approximately 200 bp region of the genome. We have previously reported the isolation and partial characterization of viable mutants of polyoma virus with deletions in this important regulatory region of the genome. Three of the mutants with large deletions, one of which had significantly altered growth properties, have been further characterized with respect to their nucleotide sequence alterations and their levels of viral DNA replication and of early protein synthesis. The nearly coincident deletions in mutants 17 and 2–19 reduce the capacity of these viruses to replicate, even in the presence of a coinfecting virus; thus they help define one boundary of the origin of DNA replication. The deletion in mutant 75 appears to remove sequences that are essential for efficient expression of early genes, but has little or no effect upon DNA replication. Its defect is complemented in trans by wild-type virus. All three mutants eliminate sequences which are candidates for RNA polymerase and ribosome binding sites near the initiation codon for early proteins.     

Can recA protein promote homologous pairing between duplex regions of DNA?

RecA protein has been shown to promote the formation of joint molecules between intact duplex DNA and homologous gapped DNA. When examined by electron microscopy, such joint molecules display a junction that is, in most cases, distant from the site of the gap. This led us to test whether the observed location of the joint was due to pairing at the gap followed by branch migration, or whether recA-promoted pairing could also take place between duplex homologous regions away from the gap. To test the latter possibility, intact duplex DNA was incubated with DNA which contained a gap in a region of non-homology. Joint molecules were detected by filter binding assay and by electron microscopy at about one-third of the yield observed for fully homologous molecules. These results indicate that initial homologous pairing promoted by recA protein is not restricted to the single-stranded region in the gap but can also take place in regions where both molecules are duplex.     

Characterization of polyoma mutants with altered middle and large T-antigens.

The viable polyoma mutants dl1013, dl1014, and dl1015 produced shortened middle and large T-antigens. In mouse 3T3 cells, dl1013 and dl1014 grew at normal rates, and dl1015 grew at a reduced rate. dl1015 behaved phenotypically as a double mutant, with deficiencies both in the stimulation of the host cell and the replication of viral DNA. Only the former defect could be complemented by the ts-a mutant, which produced a normal middle T-antigen and a temperature-sensitive large T-antigen. This result suggests that middle T-antigen is involved in the induction of cellular DNA synthesis. Of the three mutants, dl1015 alone failed to transform rat fibroblasts to growth in semisolid medium. This defect could not be complemented by the ts-a mutant. Determination of the base sequences of the mutant DNAs showed that dl1013 and dl1014 had overlapping deletions of 21 and 9 base pairs, respectively, and that the dl1015 deletion of 30 base pairs was contiguous to the other mutations on their 3' sides. Analyses of the mutant t-antigens showed that all three mutants produced shortened middle T-antigens, whereas only dl1015 large T-antigen was detectably reduced in size.     

Nucleotide sequence changes in polyoma ts-a mutants: correlation with protein structure.

The mutations in three polyoma ts-a mutants have been determined. Two mutants, ts-25 and ts-52, have different single-base changes at the same position (2883) in the early region corresponding to a conserved glycine residue very near the C-terminus of the polyoma large T antigen. Mutant ts-48 has a single-base change at position 2341, as well as a second change at position 1228, in the region of large T antigen shared with medium T antigen.     

Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding.

The recA protein of Escherichia coli promotes pairing in vitro between covalent circular duplex DNA and homologous circular duplex DNA containing a single stranded region. We have used a filter binding assay to investigate the frequency of homologous pairing between gapped and intact duplex DNA when unwinding of the free 3' and 5' ends of the gapped molecules was blocked. In order to obtain DNA without free ends, the gapped DNA was treated with trimethylpsoralen and 360 nm light so as to introduce about 6 crosslinks per DNA molecule and the double stranded regions on either side of the gaps were then digested up to the first crosslinks with exonuclease III and lambda exonuclease. This treatment did not diminish the frequency of homologous pairing, an observation which is difficult to reconcile with models for recombination requiring strand unwinding before pairing.     

Nonviable mutants of simian virus 40 with deletions near the 3'' end of gene A define a function for large T antigen required after onset of viral DNA replication

Deletion mutants of simian virus 40 (SV40) with lesions at the three DdeI sites near the 3' end of the early region were constructed. Mutants with deletions at 0.203 and 0.219 map units (mu) which did not change the large T antigen reading frame were viable. This extends slightly the upstream boundary for the location of viable mutants with deletions in the 3' end of the A gene. Mutants with frameshift deletions at 0.193 and 0.219 mu were nonviable. These are the first nonviable mutants with deletions in this portion of the A gene. None of the three nonviable mutants with deletions at 0.219 mu produced progeny viral DNA. These three mutants all used the alternate reading frame located in this portion of the SV40 early region. The mutant with a deletion at 0.193 mu, dlA2459, was positive for viral DNA replication and was defective for adenovirus helper function. All of these mutations were located in the portion of the SV40 large T antigen which has no homology to the polyoma T antigens. These results indicate that this portion of large T antigen is required for some late step in the viral growth cycle and suggest that adenovirus helper function is required for productive infection by SV40.     

Immunization against the polyoma virus-induced tumor-specific transplantation antigen by early region mutants of the virus

To investigate the relation between the polyoma tumor-specific transplantation antigen and the virus-coded proteins, mice were immunized by inoculation of a variety of viable polyoma virus mutants and then challenged with polyoma virus-induced tumors. Two classes of early region mutants were used. One class produces a normal small T-antigen and truncated middle and large T-antigens. The second class (hr-t mutants) forms a normal large T-antigen together with N-terminal fragments of small and middle T-antigens. All mutants, transforming as well as nontransforming, induced protection against polyoma virus tumors. However, there were quantitive differences between the mutants. The finding that an hr-t mutant could induce tumor rejection suggests that full-length middle and small T-antigens are not necessary for the induction of this response. Since intact middle T-antigen is the only virus-coded protein known to associate with the plasma membrane, the possibility must be considered that the polyoma virus tumor-specific transplantation antigen consists of cellular components.     

Isolation and characterization of simian virus 40 early region deletion mutants.

We constructed a tsB4/dl884 double-mutant helper virus and used it to isolate two simian virus 40 early region deletion mutants that lack about half of the DNA sequences normally used to encode the large tumor antigen (T). Both mutants make a normal-sized small t antigen, but neither mutant can replicate its DNA in the absence of a T+ helper.