Herpes simplex virus type 1 DNA replication is specifically required for high-frequency homologous recombination between repeated sequences.

Using an assay for recombination that measures deletion of a beta-galactosidase gene positioned between two directly repeated 350-bp sequences in plasmids transiently maintained in COS cells, we have found that replication from a simian virus 40 origin produces a high frequency of nonhomologous recombination. In contrast, plasmids replicating from a herpesvirus origin (oris) in COS cells superinfected with herpes simplex virus type 1 (HSV-1) show high levels of homologous recombination between the repeats and an enhanced recombinogenicity of the HSV-1 a sequence that is not seen during simian virus 40 replication. When the same assay was used to study recombination between 120- to 150-bp repeats in uninfected Vero cells, the level of recombination was extremely low or undetectable (< 0.03%), consistent with the fact that these repeats are smaller than the minimal efficient processing sequence for homologous recombination in mammalian cells. Recombination between these short repeats was easily measurable (0.5 to 0.8%) following HSV-1 infection, suggesting that there is an alteration of the recombination machinery. The frequency of recombination between repeats of the Uc-DR1 region, previously identified as the only segment of the HSV-1 a sequence indispensable for enhanced a-sequence recombination, was not significantly higher than that measured for other short sequences.     

Mutant of simian virus 40 large T-antigen that is defective for viral DNA synthesis, but competent for transformation of cultured rat cells.

A mutant was isolated which demonstrates that the transforming activity of simian virus 40 large T-antigen is separable from its function in viral DNA replication. The mutant, SVR9D, is nonconditionally defective for viral DNA synthesis, but competent at wild-type level for morphological transformation of cultured rat cells. The lytic growth defect in SVR9D is complemented by the simian virus 40 A gene product present in the transformed CV1 cell line, COS1. The lesion in SVR9D DNA was mapped genetically by marker rescue of plaque formation and localized to a 214-base-pair segment of the viral genome bounded by nucleotide numbers 4100 and 4314. DNA sequence analysis showed the mutation to be an adenine-to-guanine transition at nucleotide number 4178. This change predicts a lysine-to-glutamic acid amino acid change at residue number 214 of the mutant large T-antigen polypeptide.     

Formation of a cruciform structure at the simian virus 40 replication origin abolishes T-antigen binding to the origin in vitro.

Heteroduplex DNA molecules were formed by annealing an intact simian virus replication origin-containing fragment to a mutant derivative lacking the indigenous wild-type 27-base-pair (bp) inverted repeat within this structure and containing a nonhomologous 26-bp inverted repeat sequence in its place. Results of restriction enzyme and S1 endonuclease cleavage analyses strongly suggested that a 13-bp stem-loop structure formed at the site of nonhomology between these two DNAs. This structure lies within the boundary of simian virus 40 T-antigen-binding site 2, and its presence inhibited T-antigen binding to that sequence but not to an adjacent higher-affinity binding site (site 1). Therefore, the conformation of sequences within an otherwise intact T-antigen-binding site can have major effects upon T-antigen binding there.     

Induction of Duplication Reversion in Human Fibroblasts, by Wild-Type and Mutated Sv40 T Antigen, Covaries with the Ability to Induce Host DNA Synthesis

Intrachromosomal homologous recombination, manifest as reversion of a 14-kbp duplication in the hypoxanthine phosphoribosyl transferase (HPRT) gene, is elevated in human cells either stably transformed or transiently transfected by the SV40 (simian virus 40) large T antigen gene. Following introduction of wild-type SV40, or any of several T-antigen point mutations in a constant SV40 background, we observed a strong correlation between the stimulation of chromosomal recombination and induction of host-cell DNA synthesis. Moreover, inhibitors of DNA replication (aphidicolin and hydroxyurea) suppress SV40-induced homologous recombination to the extent that they suppress DNA synthesis. Stable integration of plasmids encoding T antigen also augments homologous recombination, which is suppressed by aphidicolin. We infer that the mechanism by which T antigen stimulates homologous recombination in human fibroblasts involves DNA replicative synthesis.     

A primer vector system that allows temperature dependent gene amplification and expression in mammalian cells: regulation of the influenza virus NS1 gene expression.

Influenza virus RNA segment 8 has been cloned into primer-vector pSLts1. This vector was designed to replicate in simian cells in a temperature dependent fashion by use of the SV40 tsA209 T-antigen gene. The oriented synthesis of cDNA on dT-tailed pSLts1 was performed on in vitro synthesized mRNA, and the second DNA strand was primed with an influenza-specific terminal oligodeoxynucleotide. Recombinant pSLVa232 contained the RNA segment 8 sequence directly fused to the SV40 late promoter contained in pSLts1, and followed by the SV40 polyadenylation signal. Expression of NS1 gene in transfected COS cells took place at a level comparable to that found in infected cells. When VERO cell cultures were transfected with recombinant pSLVa232, expression of the NS1 gene was temperature dependent. Close to one hundred fold increase in the amplification and expression of the cloned gene was observed after shift down of the transfected cells to permissive temperature. Vector pSLts1 and the cloning strategy described may be useful for the specific cloning and regulated expression of mRNAs of known 5'-terminal sequence.     

A novel selection system for recombinational and mutational events within an intron of a eucaryotic gene.

In order to identify a poison sequence that might be useful in studying illegitimate recombination of mammalian cell chromosomes, several DNA segments were tested for their ability to interfere with gene expression when placed in an intron. A tRNA gene and its flanking sequences (267 bp) were shown to inhibit SV40 plaque formation 100-fold, when inserted into the intron in the T-antigen gene. Similarly, when the same DNA segment was placed in the second intron of the adenosine phosphoribosyl transferase (APRT) gene from CHO cells, it inhibited transformation of APRT-CHO cells 500-fold. These two tests indicated that the 267-bp DNA segment contained a poison sequence. The poison sequence did not affect replication since the replication of poisoned SV40 genomes was complemented by viable SV40 genomes and poisoned APRT genes were stably integrated into cell chromosomes. Cleavage of the poison sequence in the SV40 T-antigen intron by restriction enzymes indicated that the tRNA structural sequences and the 5' flanking sequences were not required for inhibition of SV40 plaque formation. Sequence analysis of viable mutant SV40, which arose after transfection of poisoned genomes, localized the poison sequence to a 35 bp segment immediately 3' of the tRNA structural sequences.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

Mechanism of interferon action. Expression of vesicular stomatitis virus G gene in transfected COS cells is inhibited by interferon at the level of protein synthesis

The effect of interferon on the expression of the vesicular stomatitis virus glycoprotein G gene was examined in simian COS cells transfected with the expression vector pSVGL containing the G gene under the control of the SV40 late promoter. When COS cells were treated with interferon 24 h after transfection, the synthesis of vesicular stomatitis virus G protein was inhibited by about 80% as compared to that in untreated controls. By contrast, under the same conditions, neither the plasmid copy number nor the G gene mRNA levels were detectably affected by interferon treatment. Likewise, the synthesis of simian virus 40 large T-antigen was not inhibited by interferon treatment of transfected COS cells even though the synthesis of vesicular stomatitis virus G protein was markedly inhibited. The residual G protein synthesized in transfected, interferon-treated COS cells appeared to be normally glycosylated.     

DNA-mediated gene transfer in Friend leukemia cells by cotransfection of simian virus 40 DNA with herpes simplex virus thymidine kinase DNA.

Thymidine kinase-negative Friend leukemia cells were cotransfected with simian virus 40 (SV40) DNA and thymidine kinase gene DNA of herpes simplex virus type 1. The transfected thymidine kinase-positive cells were selected in HAT medium, and SV40 T-antigen expression was observed over many months in cells cultured under selective conditions, and after adaptation to normal growth medium under nonselective conditions. It was shown by Southern blot hybridization that SV40 DNA was integrated in multiple copies in the chromosomal DNA of several clones. All SV40 DNA-containing Friend leukemia cell clones analyzed were able to undergo induced erythroid differentiation. Induced cultures still expressed SV40 T-antigen to the same extent that untreated control cultures did.     

Homologous recombination of monkey alpha-satellite repeats in an in vitro simian virus 40 replication system: possible association of recombination with DNA replication.

To study homologous recombination between repeated sequences in an in vitro simian virus 40 (SV40) replication system, we constructed a series of substrate DNAs that contain two identical fragments of monkey alpha-satellite repeats. Together with the SV40-pBR322 composite vector encoding Apr and Kmr, the DNAs also contain the Escherichia coli galactokinase gene (galK) positioned between two alpha-satellite fragments. The alpha-satellite sequence used consists of multiple units of tandem 172-bp sequences which differ by microheterogeneity. The substrate DNAs were incubated in an in vitro SV40 DNA replication system and used to transform the E. coli galK strain DH10B after digestion with DpnI. The number of E. coli galK Apr Kmr colonies which contain recombinant DNAs were determined, and their structures were analyzed. Products of equal and unequal crossovers between identical 172-bp sequences and between similar but not identical (homeologous) 172-bp sequences, respectively, were detected, although those of the equal crossover were predominant among all of the galK mutant recombinants. Similar products were also observed in the in vivo experiments with COS1 cells. The in vitro experiments showed that these recombinations were dependent on the presence of both the SV40 origin of DNA replication and SV40 large T antigen. Most of the recombinant DNAs were generated from newly synthesized DpnI-resistant DNAs. These results suggest that the homologous recombination observed in this SV40 system is associated with DNA replication and is suppressed by mismatches in heteroduplexes formed between similar but not identical sequences.     

Recombination between poly[d(GT).d(CA)] sequences in simian virus 40-infected cultured cells.

CVI cells were transfected with oversized simian virus 40 (SV40) genomes that could be reduced to packageable size by alternative homologous recombination pathways involving either two polydeoxyguanylic-thymidylic acid X polydeoxycytidylic-adenylic acid (poly[d(GT).d(CA)]; abbreviated hereafter as poly(GT)] tracts or two tracts of homologous SV40 sequence. Plaque-forming viruses rescued by this procedure were found to contain genomes formed by homologous and nonhomologous recombination events. Half of the viable viral DNA molecules recovered were the result of recombination between two tracts of poly(GT). Approximately 20% of the rescued viral genomes were produced by homologous recombination between tracts of SV40 DNA. Nonhomologous recombination involving SV40 sequences was also a major pathway of deletion, producing ca. 30% of the viral plaques. Tracts of poly(GT) generated by recombination were variable in length, suggesting that recombination between poly(GT) tracts was usually unequal. On a per-nucleotide basis, poly(GT) recombination occurred eight times more frequently than did recombination between homologous SV40 DNA. This eightfold difference is the maximum recombinatory enhancement attributable to poly(GT) sequences. Although DNA sequence analysis showed that tracts of poly(GT) generated by recombination retained the alternating G-T repeat motif throughout their length, the contribution of the nonhomologous pathway to poly(GT) recombination cannot be ruled out, and the relative proclivity of a given length of d(GT).d(CA) sequence to undergo homologous recombination is probably less than eight times greater than that of an SV40 sequence of the same length.     

Recombinational resolution in primate cells of two homologous human DNA segments with a gradient of sequence divergence.

Human alpha-thalassemia-2 genotype -alpha 4.2 is the result of meiotic recombination between two 1.3 kb long, homologous DNA segments, X(alpha 2) and X(alpha 1), located in the adult alpha globin locus. The two segments can also undergo intramolecular recombination on extrachromosomal vectors transfected into mitotically dividing primate cells (COS 7). The existence of a gradient of sequence divergence between X(alpha 2) and X(alpha 1) makes them an interesting system to study the relationship between efficiencies of homologous DNA recombination and the extent of dispersed and localized base mismatches. By partial restriction mapping and DNA sequencing of plasmids recombined in COS 7 cells and rescued from bacteria HB 101, we have determined the distribution of recombinational resolution sites along the two X blocks. Most, if not all, of the homologous recombination events between the two X blocks appear to be single crossing-over without efficient gene correction or repair of base mismatches. The distribution of the sites of recombinational resolution is inversely correlated with that of the gradient of sequence divergence, with only approximately 7% of the X recombinants resolved within the 3' third of the X blocks where two diverged Alu family repeats reside. The Alu sequence within which one of the X recombinants resolved is homologous to a previously characterized alpha thalassemia deletion point.     

DNA binding properties of simian virus 40 T-antigens synthesized in vivo and in vitro.

Simian virus 40 large T- and small t-antigens have been shown previously to share immunological determinants and common sequences and to have roles in virus-induced cell transformation. However, only large T-antigen is a DNA binding protein. Under all conditions tested, small t-antigen did not interact with DNA. Large T-antigen synthesized in infected cells bound to both native calf thymus and simian virus 40 DNAs. As its binding efficiency was less than 100%, it is likely that there are different forms of T-antigen which vary in their affinity for DNA. Large T-antigen synthesized in cell-free protein-synthesizing systems primed by simian virus 40 mRNA also bound to DNA-cellulose, whereas small t-antigen similarly synthesized in vitro did not. An 82,000-molecular-weight T-antigen polypeptide synthesized in cell-free protein-synthesizing systems primed by simian virus 40 complementary RNA transcribed in vitro from simian virus 40 DNA by Escherichia coli RNA polymerase bound efficiently to simian virus 40 DNA. As this product did not share sequences with the small t-antigen, it can be concluded that the amino-terminal portion of the T-antigen is not required for some of its specific DNA binding properties.     

Construction of a specific amber codon in the simian virus 40 T-antigen gene by site-directed mutagenesis.

The site-directed bisulfite mutagenesis technique has been used to construct a specific mutation, am404, at nucleotide position 3124 in the simian virus 40 genome. The mutation was contained within a PstI restriction site (map position 0.27) and prevented cleavage by PstI at that position. Nucleotide sequence analysis of the mutagenized region indicated that only a single base pair change had occurred: a guanosine x cytosine leads to adenine x thymine transition. Comparison of the nucleotide sequence of am404 with the known DNA sequence of simian virus 40 indicted that the mutation in am404 resulted in the conversion of a glutamine codon to an amber codon. am404 could not replicate autonomously when transfected into monkey cells (BSC-40) but did replicate when it was cotransfected with the late deletion helper virus dl1007. On the basis of its position in the T-antigen, gene am404 should produce a T-antigen 24% shorter than the wild-type protein.     

Homologous recombination of SV40 DNA in COS7 cells occurs with high frequency in a gene dose independent fashion.

Homologous recombination between microinjected SV40 DNA fragments and endogenous SV40 DNA in COS7 cells was analysed by immunofluorescence staining and DNA blotting. Time course experiments revealed that recombination between the transferred (trans) DNA and the chromosomal DNA occurred about 8 hours after microinjection with high efficiency in a gene dose independent fashion. Deletions of up to 1018 basepairs (bp) within the early or the late SV40 region were efficiently repaired after the transfer of linear but not of circular DNA molecules. A 22 bp homology between the trans DNA and the endogenous DNA was sufficient to initiate recombination but 14 nonhomologous bp at one open end of the SV40 DNA fragments hindered gap repair.     

Extrachromosomal and chromosomal gene conversion in mammalian cells.

We constructed substrates to study gene conversion in mammalian cells specifically without the complication of reciprocal recombination events. These substrates contain both an insertion mutation of the neomycin resistance gene (neoX) and an internal, homologous fragment of the neo gene (neo-526), such that gene conversion from neo-526 to neoX restores a functional neo gene. Although two reciprocal recombination events can also produce an intact neo gene, these double recombination events occur much less frequently that gene conversion in mammalian cells, We used our substrates to characterize extrachromosomal gene conversion in recombination-deficient bacteria and in monkey COS cells. Chromosomal recombination was also studied after stable integration of these substrates into the genome of mouse 3T6 cells. All extrachromosomal and chromosomal recombination events analyzed in mammalian cells resulted from gene conversion. Chromosomal gene conversion events occurred at frequencies of about 10(-6) per cell generation and restored a functional neo gene without overall effects on sequence organization.     

Patterns of integration of DNA microinjected into cultured mammalian cells: evidence for homologous recombination between injected plasmid DNA molecules.

We examined the fate of DNA microinjected into nuclei of cultured mammalian cells. The sequence composition and the physical form of the vector carrying the selectable gene affected the efficiency of DNA-mediated transformation. Introduction of sequences near the simian virus 40 origin of DNA replication or in the long terminal repeat of avian sarcoma provirus into a recombinant plasmid containing the herpes simplex virus thymidine kinase gene. (pBR322/HSV-tk) enhanced the frequency of transformation of LMtk- and RAT-2tk- cells to the TK+ phenotype 20- to 40-fold. In cells receiving injections of only a few plasmid DNA molecules, the transformation frequency was 40-fold higher after injection of linear molecules than after injection of supercoiled molecules. By controlling the number of gene copies injected into a recipient cell, we could obtain transformants containing a single copy or as many as 50 to 100 copies of the selectable gene. Multiple copies of the transforming gene were not scattered throughout the host genome but were integrated as a concatemer at one or a very few sites in the host chromosome. Independent transformants contained the donated genes in different chromosomes. The orientation of the gene copies within the concatemer was not random; rather, the copies were organized as tandem head-to-tail arrays. By analyzing transformants obtained by coinjecting two vectors which were identical except that in one a portion of the vector was inverted, we were able to conclude that the head-to-tail concatemers were generated predominantly by homologous recombination. Surprisingly, these head-to-tail concatemers were found in transformants obtained by injecting either supercoiled or linear plasmid DNA. Even though we demonstrated that cultured mammalian cells contain the enzymes for ligating two DNA molecules very efficiently irrespective of the sequences or topology at their ends, we found that even linear plasmid DNA was recruited into the concatemer by homologous recombination.     

Intramolecular recombination between transfected repeated sequences in mammalian cells is nonconservative.

When plasmids carrying a fragmented gene with segments present as direct repeats are introduced into mammalian cells, recombination or gene conversion between the repeated sequences can reconstruct the gene. Intramolecular recombination leads to the deletion of the intervening sequences and the loss of one copy of the repeat. This process is known to be stimulated by double-strand breaks. Two current models for recombination in eucaryotic cells propose that the reaction is initiated by double-strand breaks, but differ in their predictions as to the fate of the intervening sequences. One model suggests that these sequences are always lost, while the other indicates that the reaction will be conservative as a function of the position of the double-strand break. We have constructed a plasmid in which two overlapping portions of the simian virus 40 early region, which contains the origin and T-antigen gene, are present as direct repeats separated by sequences containing a plasmid with a simian virus 40 origin of replication. Recombination across the repeated segments could produce a plasmid with an origin of replication and/or a plasmid with a gene for a functional T-antigen which would drive the replication of both. Introduction of this construction into African green monkey kidney cells, without coinfection, establishes a condition in which the products of the recombination or gene conversion can be interpreted unambiguously. We find that the majority of the reconstruction reactions are nonconservative.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.