RecA independent recombination of poly[d(GT)-d(CA)] in pBR322.

Short sequence tracts composed of alternating guanosine and thymidine nucleotide residues poly[d(GT)-d(CA)] carried in a derivative of pBR322 were recombinogenic in a recA host. Recombination brought about by poly[d(GT)-d(CA)] tracts displayed two interesting properties: (i) the reaction was quasi-sequence-specific in that while recombination usually occurred between two poly[d(GT)-d(CA)] tracts, recombination also occurred between sequences bordering the dinucleotide repeats. (ii) recombination was enhanced when two poly[d(GT)-d(CA)] tracts were clustered within 250 base pairs of each other, but not when the repeats were separated by 3 kilobase pairs. The mechanism by which poly[d(GT)-d(CA)] stimulated recombination remains to be determined, but the behavior of these sequences is consistent with the idea that general recombination in E. coli may involve formation of Z-DNA.     

Homologous and nonhomologous recombination in monkey cells.

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.     

Characterization of flat revertant cells isolated from simian virus 40-transformed mouse and rat cells which contain multiple copies of viral genomes.

Eight clones of flat revertants were isolated by negative selection from simian virus 40 (SV40)-transformed mouse and rat cell lines in which two and six viral genome equivalents per cell were integrated, respectively. These revertants showed either a normal cell phenotype or a phenotype intermediate between normal and transformed cells as to cellular morphology and saturation density and were unable to grow in soft agar medium. One revertant derived from SV40-transformed mouse cells was T antigen positive, whereas the other seven revertants were T antigen negative. SV40 could be rescued only from the T-antigen-positive revertant by fusion with permissive monkey cells. The susceptibility of the revertants to retransformation by wild-type SV40 was variable among these revertants. T-antigen-negative revertants from SV40-transformed mouse cells were retransformed at a frequency of 3 to 10 times higher than their grandparental untransformed cells. In contrast, T-antigen-negative revertants from SV40-transformed rat cells could not be retransformed. The arrangement of viral genomes was analyzed by digestion of cellular DNA with restriction enzymes of different specificity, followed by detection of DNA fragments containing a viral sequence and rat cells were serially arranged within the length of about 30 kilobases, with at least two intervening cellular sequences. A head-to-tail tandem array of unit length viral genomes was present in at least one insertion site in the transformed rat cells. All of the revertants had undergone a deletion(s), and only a part of the viral genome was retained in T-antigen-negative revertants. A relatively high frequency of reversion in the transformed rat cells suggests that reversion occurs by homologous recombination between the integrated viral genomes.     

In vivo assessment of the Z-DNA-forming potential of d(CA.GT)n and d(CG.GC)n sequences cloned into SV40 minichromosomes

Alternating repeated d(CA.GT)n and d(CG.GC)n sequences constitute a significant proportion of the simple repeating elements found in eukaryotic genomic DNA. These sequences are known to form left-handed Z-DNA in vitro. In this paper, we have addressed the question of the in vivo determination of the Z-DNA-forming potential of such sequences in eukaryotic chromatin. For this purpose, we have investigated the ability of a d(CA.GT)30 sequence and a d(CG.GC)5 sequence to form left-handed Z-DNA when cloned into simian virus 40 (SV40) minichromosomes at two different positions: the TaqI site, which occurs in the intron of the T-antigen gene, and the HpaII site, which is located in the late promoter region within the SV40 control region. Formation of Z-DNA at the inserted repeated sequences was analyzed through the change in DNA linkage associated with the B to Z transition. Our results indicate that regardless of: (1) the site of insertion (either TaqI or HpaII), (2) the precise moment of the viral lytic cycle (from 12 h to 48 h postinfection) and (3) the condition of incorporation of the SV40 recombinants to the host cells (either as minichromosomes or as naked DNA, relaxed or negatively supercoiled), neither the d(CA.GT)30 nor the d(CG.GC)5 sequence are stable in the left-handed Z-DNA conformation in the SV40 minichromosome. The biological relevance of these results is discussed.     

Construction of a viable simian virus 40 variant that carries a poly[d(GT) . d(CA)] insertion.

A 90-base-pair tract of a simple sequence composed of alternating guanosine and thymidine nucleotide residues (poly[d(GT) . d(CA)]) was inserted into the simian virus 40 genome at nucleotide 2666 (0.17 map units). The poly[d(GT) . d(CA)] insertion was stably maintained in the viral genome, but the variant virus grew more slowly than simian virus 40.     

The effect of the simple repeating d(CG.GC)n, d(CA.GT)n, and d(A.T)n DNA sequences on the nucleosomal organization of SV40 minichromosomes.

The effect of several simple repeating DNA sequences--d(CG.GC)5, d(CA.GT)30, and d(A.T)60--on the nucleosomal organization of the SV40 minichromosome is analyzed. These three different sequences were cloned at the Hpa II site of SV40 (position 346) which occurs at the 3' border of the nucleosome-free SV40 control region. Our results show that neither the d(A.T)60 sequence nor the d(CG.GC)5 sequence appear to have any relevant effect on the nucleosomal organization of the region of the minichromosome surrounding the inserted repeated sequence. Both sequences are hypersensitive to micrococcal nuclease cleavage in the minichromosome, indicating that they are not organized into nucleosomes. On the other hand, the d(CA.GT)30 sequence is found organized as nucleosomes and causes the delocation of nucleosomes in the minichromosomal region close to the inserted repeated sequence.     

Quantitation of a simian virus 40 nonhomologous recombination pathway.

We describe an infectious-center in situ plaque hybridization procedure which quantitates simian virus 40 (SV40) nonhomologous recombination in terms of the number of recombinant-producing cells in the DNA transfected cell population. Using this assay to measure the efficiency of recombination with SV40 DNA in permissive monkey BSC-1 cells, we found that: (i) over a range of DNA concentrations, polyomavirus DNA (which is partially homologous to SV40 DNA) cannot be distinguished from nonhomologous phi X174 RF1 DNA with respect to its ability to recombine with SV40 DNA; (ii) at defined DNA concentrations, polyomavirus and phi X174 RF1 DNA compete with each other for recombination with SV40 DNA; (iii) virtually all segments of the phi X174 genome recombine, apparently at random, with SV40 DNA; (iv) the frequency of recombinant-producing cells, among the successfully transfected (virion-producing) cells, depends upon the input SV40 DNA concentration in the transfection solution; and (v) replication-defective SV40 mutant DNAs compete with wild-type SV40 DNA for recombination with phi X174 RF1 DNA. From these observations, we conclude that the efficiency of recombination with SV40, in the system under study, is unaffected by nucleotide sequence homology and that a limiting stage in the recombination pathway occurs before SV40 DNA replication. Comparison of the dependency of recombination on initial SV40 DNA concentration with the dependency on initial phi X174 RF1 DNA concentration indicates that SV40 DNA sequences are a controlling factor in the nonhomologous recombination pathway.     

SV40 recombinants carrying a d(CT.GA)22 sequence show increased genomic instability.

Repetitive d(CT.GA)n sequences are commonly found in eukaryotic genomic DNA. They are frequently located in sites involved in genetic recombination or in promoter regions. To test for their possible biological function, a d(CT.GA)22 synthetic sequence was introduced into the genome of SV40, since it constitutes an appropriate model system for eukaryotic chromatin. When SV40 infects permissive cells, it proliferates in the form of a minichromosome. The simple repetitive sequence indicated above was inserted at the unique HpaII site of SV40 (at nt 346), and the genomic stability of SV40 recombinants carrying the d(CT.GA)22 sequence (SV/CT22 viruses) was analyzed. Upon serial passage through permissive CV1 cells, SV/CT22 recombinants show an increased production of defective viruses. Generation of SV/CT22 variants is likely to take place via recombination between and within viral molecules. The enhancement of the rate of recombination induced by the repetitive sequence is likely to be related to its known propensity to form triple-stranded structures. Many different variants coexist in the same viral population indicating that the mechanism by which they are produced is not unique. One variant (SV/X), showing a replicative advantage, was characterized in detail. Variant SV/X accounts for a large proportion of the total viral population. Its genomic organization corresponds to a tandem duplication of an early SV40 DNA fragment spanning from approx. nt 3200-nt 160. Variant SV/X contains a duplicated SV40 ori.     

Evolutionary relationships of the primate papovaviruses: base sequence homology among the genomes of simian virus 40, stump-tailed macaque virus, and SA12 virus.

Physical maps of the genomes of the two newly discovered primate papovaviruses, SA12 and stump-tailed macaque virus (STMV), were generated by restriction endonuclease analysis. The base sequence homologies among the genomes of SA12, stump-tailed macaque virus, and simian virus 40 (SV40) were studied by heteroduplex analysis. Heteroduplexes between SA12 and SV40 DNAs and stump-tailed macaque virus and SV40 DNAs were constructed and mounted for electron microscopy in various amounts of formamide to achieve a range of effective temperatures. At each effective temperature, the regions of duplex DNA in the heteroduplexes were measured and localized on the SV40 physical and functional maps. By analyzing the data from this study and rom our previous study (N. Newell, C. J. Lai, G. Khoury, and T. J. Kelly Jr., J. Virol. 25:193-201, 1978) on the base sequence homology between the genomes of BK virus and SV40, some general conclusions have been drawn concerning the evolutionary relationships among the genomes of the primate papovaviruses. The extent of homology among the viral genomes does not reflect the phylogenetic relationships of their hosts. At comparable effective temperatures Tm - 33 degrees C), the heteroduplexes between the DNAs of BK virus and SV40 contained the largest amount of duplex (about 90%). The heteroduplexes made between SA12 and SV40 DNAs were slightly less homologous, containing about 80% duplex. The heteroduplexes made between SV40 and stump-tailed macaque virus DNAs were only 20% duplex under the same conditions. When the various heteroduplexes were mounted for microscopy at effective temperatures greater than Tm - 33 degrees C, the fraction of the duplex DNA decreased in each case, indicating the existence of considerable base mismatching in the homologous regions. When specific coding or noncoding regions of the viral genomes were compared, the data indicated that the extent of sequence divergence differed markedly from one region to another. In all the heteroduplexes studied, there were two regions, located near the junctions between early and late regions on the SV40 map, which were essentially nonhomologous. All of the heteroduplexes studied showed significantly greater homology in the late region than in early region. Within the late region, the sequences coding for the major capsid polypeptide, VP1, were the most highly conserved.     

Characterization of a multisubunit human protein which selectively binds single stranded d(GA)n and d(GT)n sequence repeats in DNA.

A protein which selectively binds d(GA)n and d(GT)n sequence repeats in single stranded DNA has been identified in human fibroblasts. This protein, designated PGB, has been purified at least 500-fold by ammonium sulfate precipitation followed by DEAE-Sepharose column chromatography and affinity chromatography in a column of d(GA)-Sepharose. Electrophoretic mobility shift assays revealed that the PGB protein bound most avidly d(GA)n and d(GT)n tracts of n > 5. It also bound other G-rich DNA sequence repeats, including dGn tracts, with lower affinities. It did not manifest significant binding affinities to single stranded M13 DNA, or to the homopolynucleotides poly dA, poly dC and poly dT, or to various DNA sequence repeats which do not contain G residues, such as d(A-C)n and d(TC)n. It did not bind double stranded d(T-C)n.d(GA)n tracts or other double stranded DNA sequences. In glycerol gradient centrifugation assays the d(GA)n- and the d(GT)n-binding activities cosedimented as a homogeneous protein species having an S20,w = 9.4 +/- 0.7 and an estimated native molecular weight of 190,000 +/- 7,000. UV crosslinking assays revealed that the protein contains 33.6 +/- 2.1 kd subunits which bind d(GA)n and d(GT)n sequences. However, SDS-polyacrylamide gel electrophoresis of the purified protein followed by silver staining indicated that it may also contain other subunits that do not contact the DNA. It is proposed that binding of the PGB protein to single stranded d(GA)n or d(GT)n tracts in double stranded topologically restricted DNA may stimulate strand separation and formation of triple helices or other unusual DNA structures.     

Effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on homologous recombination in somatic cells.

Sequencing studies have shown that in somatic cells alternating runs of purines and pyrimidines are frequently associated with recombination crossover points. To test whether such sequences actually promote recombination, we have examined the effects of poly[d(pGpT).d(pApC)] and poly[d(pCpG).d(pCpG)] repeats on a homologous recombination event. The parental molecule used in this study, pSVLD, is capable of generating wild-type simian virus 40 DNA via recombination across two 751-base-pair regions of homology and has been described previously (Miller et al., Proc. Natl. Acad. Sci. USA 81:7534-7538, 1984). Single inserts of either a poly[d(pGpT).d(pApC)] repeat or a poly[d(pCpG).d(pCpG)] repeat were positioned adjacent to one region of homology in such a way that the recombination product, wild-type simian virus 40 DNA, could be formed only by recombination within the homologies and not by recombination across the alternating purine-pyrimidine repeats. We have found that upon transfection of test DNAs into simian cells, a poly[d(pCpG).d(pCpG)] repeat enhanced homologous recombination 10- to 15-fold, whereas a poly[d(pGpT).d(pApC)] repeat had less effect. These results are discussed in terms of the features of these repeats that might be responsible for promoting homologous recombination.     

Electron microscope study of the base sequence homology between simian virus 40 and human papovavirus BK.

The base sequence homology between the genomes of simian virus 40 (SV40) and human papovavirus BK (BKV) was studied by the heteroduplex method of Ferguson and Davis (J. Mol. Biol. 94:135-149, 1975). When mounted for microscopy in 30% formamide (Tm-35 degrees C), BKV/SV40 heteroduplexes were an average of 92% double-stranded and contained only two small nonhomologous regions that mapped near the junctions between the early and late regions of the SV40 Genome. At higher formamide concentrations, the fraction of duplex DNA in the BKV/SV40 heteroduplexes decreased, indicating significant base mismatching in the homologous regions. The strongest regions of homology were located in the late region.     

Somatic cells efficiently join unrelated DNA segments end-to-end.

Molecular substrates for probing nonhomologous recombination in somatic cells were constructed by inserting pBR322 sequences at selected sites on the simian virus 40 (SV40) genome. The chimeric products are too large to be packaged into an SV40 capsid. Therefore, production of viable progeny requires that most of the pBR322 sequences be deleted without altering any SV40 sequences that are essential for lytic infection. As judged by plaque assay, these recombination events occur at readily detectable frequencies after transfection into CV1 monkey kidney cells. Depending on the site of pBR322 insertion, the infectivities of the full-length circular or linear chimeras ranged from 0.02 to 2% of the infectivity of linear wild-type SV40 DNA. Nucleotide sequence analysis of several recombinant progeny revealed three distinct classes of recombination junction and indicated that the causative recombination events were minimally dependent on sequence homology. Potential mechanisms involving recombination at internal sites or at ends were distinguished by measuring the infectivity of chimeric molecules from which various lengths of pBR322 had been removed. These data support end-to-end joining as the primary mechanism by which DNA segments recombine nonhomologously in somatic cells. This end joining appears to be very efficient, since SV40 genomes with complementary single-stranded tails or with short non-complementary pBR322 tails were comparably infectious. Overall, this study indicates that mammalian somatic cells are quite efficient at the willy-nilly end-to-end joining of unrelated DNA segments.     

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells.

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.     

Two types of deletion within integrated viral sequences mediate reversion of simian virus 40-transformed mouse cells.

Simian virus 40 (SV40) DNA insertions from SV40-transformed mouse cell line W-2K-11 and its revertants M18, M31, and M42 were cloned. W-2K-11 cells contain 1.5 copies of the SV40 sequences in a partially tandem duplicated form. The endpoints of the viral sequences at the virus-host junctions are located very close to those reported by others, indicating that there are some preferred sites for integration and rearrangement in SV40 sequences. One flanking cellular sequence is a long stretch of adenine and thymine with repeated AAAT, and the other is a stretch of guanine and cytosine with repeated CCG. There are patchy homologies between the flanking cellular sequences and the corresponding parental SV40 sequences. The sequences around both junctions were retained in all the revertants, whereas most of the internal SV40 sequences coding for large T antigen were deleted. The coding sequences for small T antigen are intact, and small T antigen was expressed in all the revertants. The fragments cloned from M18 and M42 were identical and 3.9 kilobases of SV40 sequences were deleted. The parental SV40 sequences around the deletion site have sequences capable of forming a secondary structure which might reduce the effective distance between the two regions. The SV40 DNA retained in M31 is colinear with SV40 virion DNA, and a unit length of SV40 DNA was deleted within the SV40 sequences present in W-2K-11 cells. These results indicated that two types of deletion occurred during the reversion, one between homologous sequences and the other between nonhomologous sequences.     

Cloning and sequencing of viral integration site in human fibroblasts immortalized by simian virus 40.

We have analyzed cellular DNA sequences at the viral genome integration site in a human fibroblast cell line VA13 immortalized by simian virus 40 (SV40). The computer analysis of the junctional cellular DNA sequences did not show any homology to the DNA sequences previously reported. This suggests that immortalization by SV40 was not induced by the destruction of any known oncogene or anti-oncogene at the integration site. We did not find the precise substantial sequence homology at the junctional site between the cellular DNA and SV40 DNA, indicating that the recombination mechanism involved does not require precise sequence homology and therefore, SV40 genome was probably not integrated by homologous recombination. Short direct and inverted repeats of 5 to 29 nucleotides were found in the junctional cellular and SV40 DNA. Cellular DNA abutting SV40 DNA was found by the Northern blot analysis to be expressed in diploid human fibroblasts and SV40-transformed cells. The nature of this RNA is now under study.     

Instability of simple sequence DNA in Saccharomyces cerevisiae.

All eukaryotic genomes thus far examined contain simple sequence repeats. A particularly common simple sequence in many organisms (including humans) consists of tracts of alternating GT residues on one strand. Allelic poly(GT) tracts are often of different lengths in different individuals, indicating that they are likely to be unstable. We examined the instability of poly(GT) and poly(G) tracts in the yeast Saccharomyces cerevisiae. We found that these tracts were dramatically unstable, altering length at a minimal rate of 10(-4) events per division. Most of the changes involved one or two repeat unit additions or deletions, although one alteration involved an interaction with the yeast telomeres.     

In vivo assay of p53 function in homologous recombination between simian virus 40 chromosomes.

To investigate a possible role of p53 in DNA exchange mechanisms, we have developed a model system which allows us to quantify homologous recombination rates in eukaryotic cells. We generated two types of simian virus 40 (SV40) whose genomes were mutated in such a way that upon double infection of monkey cells, virus particles can be released only after interchromosomal exchange of genetic material. This test system allowed us to determine recombination rates in the order of 10(-4) to 10(-6) for chromatin-associated SV40 genomes. To study the role of p53-T-antigen (T-Ag) complexes in this process, we designed viral test genomes with an additional mutation leading to a single amino acid exchange in T-Ag (D402H) and specifically blocking T-Ag-p53 interactions. Analysis of primary rhesus monkey cells endogenously expressing wild-type p53 showed a decreased recombination rate upon loss of efficient T-Ag-p53 complex formation. However, cells expressing mutant p53 (LLC-MK2 cells), the introduction of mutant T-Ag did not affect the DNA exchange rates. Our data are interpreted to indicate an inhibitory role of wild-type p53 in recombination. In agreement with this hypothesis, p53-T-Ag complex formation alleviates the inhibitory effect of wild-type p53.