Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.     

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.     

Rescue of chromosomal T-antigen sequences onto extrachromosomally replicating, defective simian virus 40 DNA by homologous recombination.

Recombination between chromosomal and extrachromosomal DNA sequences was analyzed by investigation of the recombinational rescue of a 1,018-base-pair (bp) segment of the T-antigen gene of simian virus 40 from the chromosome of monkey COS cells to two different, extrachromosomally replicating, simian virus 40 DNA molecules lacking this 1,018-bp sequence. The ratio of rescued to unrecombined virus was as high as 10(-3). The rescued molecules, detected optimally 5 to 9 days after transfection of COS cells, had completely recovered the 1,018-bp DNA segment from the chromosome. The recombination event is proposed to occur either by double reciprocal recombination or by gene conversion between the chromosomal T-antigen gene and the extrachromosomal molecules missing the 1,018-bp sequence.     

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.     

Evidence for Conservative (Two-Progeny) DNA Double-Strand Break Repair

The double-strand break repair models for homologous recombination propose that a double-strand break in a duplex DNA segment is repaired by gene conversion copying a homologous DNA segment. This is a type of conservative recombination, or two-progeny recombination, which generates two duplex DNA segments from two duplex DNA segments. Transformation with a plasmid carrying a double-strand gap and an intact homologous DNA segment resulted in products expected from such conservative (two-progeny) repair in Escherichia coli cells with active E. coli RecE pathway (recBC sbcA) or with active bacteriophage λ Red pathway. Apparently conservative double-strand break repair, however, might result from successive events of nonconservative recombination, or one-progeny recombination, which generates only one recombinant duplex DNA segment from two segments, involving multiple plasmid molecules. Contribution of such intermolecular recombination was evaluated by transformation with a mixture of two isogenic parental plasmids marked with a restriction site polymorphism. Most of the gap repair products were from intramolecular and, therefore, conservative (two-progeny) reaction under the conditions chosen. Most were conservative even in the absence of RecA protein. The double-strand gap repair reaction was not affected by inversion of the unidirectional replication origin on the plasmid. These results demonstrate the presence of the conservative (two-progeny) double-strand break repair mechanism. These experiments do not rule out the occurrence of nonconservative (one-progeny) recombination since we set up experimental conditions that should favor detection of conservative (two-progeny) recombination.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.     

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.     

Molecular analysis of DNA junctions produced by illegitimate recombination in human cells.

In a human HeLa derived-cell line carrying permanently a single integrated copy of an SV40 shuttle vector, the transient expression of the SV40 T-antigen led to the production of heterogeneous populations of circular DNA molecules which retained both integrated vector and its surrounding cellular sequences. Comparison between the integrated copy and the linear maps of 80 different plasmids rescued in bacteria suggested that the formation of circular DNA was the result of bidirectional replication from the SV40 origin of replication followed by a single intramolecular joining leading to the cyclization of the replicated molecules. Sequence analysis of 45 recombinational junctions demonstrated that the cyclization occurred via illegitimate recombination process which did not require preferential nucleotide sequence at the joining sites. However, extensive characterization of recombination junctions revealed that the sequences involved in the recombination at each side of the SV40 origin of replication were not randomly distributed, suggesting the presence of regions which were more prone to be involved in the illegitimate recombination process in human cells. Search of common features usually implied in illegitimate recombination in mammalian cells revealed some association of these regions with palindromes, A + T-rich DNA segments, alternating purine/pyrimidine sequences and Alu family repeats.     

Requirement for double-strand breaks but not for specific DNA sequences in herpes simplex virus type 1 genome isomerization events.

Herpes simplex virus type 1 (HSV-1) genome isomerization occurs as a result of DNA replication-mediated homologous recombination between several sets of inverted repeat sequences present in the viral DNA. The frequency with which this recombination occurs has been demonstrated to be dependent upon DNA homology length rather than specific sequences. However, the smallest of the viral inverted repeats, the alpha sequence, has been shown to function as a recombinational hot spot, leading to speculation that this sequence may represent a specific element through which genome isomerization is mediated. To investigate this apparent paradox, a quantitative transient recombination assay system was developed and used to examine the recombinogenic properties of a panel of alpha sequence mutants. This analysis revealed that the presence of both the pac1 and pac2 elements was both necessary and sufficient for the induction of high-frequency recombination events by the alpha sequence. However, it was the double-strand break promoted by pac1 and pac2 during cleavage and packaging at the alpha sequence, and not the DNA sequences of the elements themselves, which appeared to be critical for recombination. This was illustrated (i) by the inability of the same pac1 and pac2 sequences to mediate inversion events in cells infected with an HSV-1 mutant which was competent for DNA replication-dependent recombination but defective for the cleavage and packaging process and (ii) by the ability of double-strand breaks generated in non-HSV-1 DNA by an in vivo-expressed restriction endonuclease to significantly stimulate the initiation of recombination events in virus-infected cells. Thus, the alpha sequence appears to act as a hot spot for homologous recombination simply because it happens to coincide with the site of the double-strand break which is generated during the cleavage and packaging process, not because it contains discrete sequences which are required for this activity. However, it was found that this enhanced recombinogenicity disappeared when the element was flanked by regions of extensive sequence homology, particularly that of the large inverted repeats which flank the alpha sequence at its natural site in the HSV-1 genome. These findings are consistent with a model for HSV-1 genome isomerization in which recombination is initiated primarily by multiple random double-strand breaks which arise during DNA replication across the inverted repeats of the genome, rather than by a single specific break which occurs at the alpha sequence during the cleavage and packaging process.     

An Examination of the Effects of Double-Strand Breaks on Extrachromosomal Recombination in Mammalian Cells

We studied the effects of double-strand breaks on intramolecular extrachromosomal homologous recombination in mammalian cells. Pairs of defective herpes thymidine kinase (tk) sequences were introduced into mouse Ltk- cells on a DNA molecule that also contained a neo gene under control of the SV40 early promoter/enhancer. With the majority of the constructs used, gene conversions or double crossovers, but not single crossovers, were recoverable. DNA was linearized with various restriction enzymes prior to transfection. Recombination events producing a functional tk gene were monitored by selecting for tk-positive colonies. For double-strand breaks placed outside of the region of homology, maximal recombination frequencies were measured when a break placed the two tk sequences downstream from the SV40 early promoter/enhancer. We observed no relationship between recombination frequency and either the distance between a break and the tk sequences or the distance between the tk sequences. The quantitative effects of the breaks appeared to depend on the degree of homology between the tk sequences. We also observed that inverted repeats recombined as efficiently as direct repeats. The data indicated that the breaks influenced recombination indirectly, perhaps by affecting the binding of a factor(s) to the SV40 promoter region which in turn stimulated or inhibited recombination of the tk sequences. Taken together, we believe that our results provide strong evidence for the existence of a pathway for extrachromosomal homologous recombination in mammalian cells that is distinct from single-strand annealing. We discuss the possibility that intrachromosomal and extrachromosomal recombination have mechanisms in common.     

Recombination during early herpes simplex virus type 1 infection is mediated by cellular proteins

Homologous recombination was examined in cells infected with herpes simplex virus type I. Circular and linear DNA with directly repeated sequences was introduced as recombination substrates into cells. Recombination was measured either by origin-dependent amplification of recombination products or by recombination-dependent expression of luciferase from a disrupted gene. Homologous recombination in baby hamster kidney cells converted linear DNA to circular templates for DNA replication and luciferase expression in the complete absence of virus. The products of homologous recombination were efficiently amplified by the viral replication apparatus. The efficiency of recombination was dependent on the structure of the substrate as well as the cell type. Linear DNA with the direct repeats at internal positions failed to recombine in Balb/c 3T3 cells and induced p53-dependent apoptosis. In contrast, linear DNA with directly repeated sequences precisely at the ends recombined and replicated in 3T3 cells. Homologous recombination in baby hamster kidney cells did not depend on the position of the repeated sequences. We conclude that homologous recombination is independent of viral gene functions and that it is likely to be carried out by cellular proteins. We suggest that homologous recombination between directly repeated sequences in the linear herpes simplex virus type 1 chromosome may help to avoid p53-dependent apoptosis and to promote viral DNA replication.     

Characterization of RAD51-independent break-induced replication that acts preferentially with short homologous sequences

Repair of double-strand breaks by gene conversions between homologous sequences located on different Saccharomyces cerevisiae chromosomes or plasmids requires RAD51. When repair occurs between inverted repeats of the same plasmid, both RAD51-dependent and RAD51-independent repairs are found. Completion of RAD51-independent plasmid repair events requires RAD52, RAD50, RAD59, TID1 (RDH54), and SRS2 and appears to involve break-induced replication coupled to single-strand annealing. Surprisingly, RAD51-independent recombination requires much less homology (30 bp) for strand invasion than does RAD51-dependent repair (approximately 100 bp); in fact, the presence of Rad51p impairs recombination with short homology. The differences between the RAD51- and RAD50/RAD59-dependent pathways account for the distinct ways that two different recombination processes maintain yeast telomeres in the absence of telomerase.     

High-frequency homologous recombination in vaccinia virus DNA.

A recombinant vaccinia virus genome was constructed in which the viral thymidine kinase (tk) gene was placed between direct repeats of a 1.5-kilobase-pair DNA sequence of heterologous origin. When forced to replicate in tk- cells in the presence of methotrexate (i.e., under tk+-selective conditions), the recombinant maintained its tk+ phenotype. Under nonselective conditions, however, the tk gene was frequently excised by both inter- and intramolecular recombination events because the repeated sequences provided substantial targets for homologous DNA recombination. Unique DNA products of intramolecular recombination were detected in the cytoplasm of infected cells soon after the onset of viral DNA replication, and their appearance was blocked by inhibitors of DNA synthesis. During repeated passage of the virus under nonselective conditions, the tk+ fraction decreased with first-order kinetics at a rate that reflected the frequency of recombination per cycle of virus replication. Eventually, a residual population of stable tk+ viruses remained, and analyses of the genome structures of individual members of this population showed that some of them appeared to be the products of nonhomologous DNA recombination.     

Preferred crossover sites on polyomavirus DNA.

RmI is a hybrid replicon consisting of polyomavirus (Py) and mouse sequences that yields unit-length polyomavirus DNA via recombination between two directly repeated viral sequences of 182 base pairs (S repeats). To define the contribution of the S repeats in this intramolecular recombination, we derived from RmI a series of replicons containing the original S repeats as well as additional direct viral repeats which were 1 to 2 kilobases in length (L repeats). After mouse 3T6 cells were transfected with these constructs, recombination products that displayed the physical properties of homologous recombinants were detected. The structures of these recombinants indicated that whereas repeat length influences the likelihood of recombination, crossover occurs preferentially near the S repeats, provided that one of them is proximal to the viral origin of replication. This finding suggests that recombination near the S repeats depends on a process initiated near the viral origin of replication.     

Circular and linear simian virus 40 DNAs differ in recombination.

Linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and phi X174 RFI DNAs, enhanced the frequency of SV40/phi X174 recombination, as measured by infectious center in situ plaque hybridization in monkey BSC-1 cells. The sequences required for the enhancement of recombination by linear DNA reside within the SV40 replication origin/regulatory region (nucleotides 5,171 to 5,243/0 to 128). Linearization of phi X174 RFI DNA did not increase the recombination frequency. The SV40/phi X174 recombinant structures arising from transfections supplemented with linear forms of origin-containing SV40 DNA contained phi X174 DNA sequences interspersed within tandem head-to-tail repeats derived from the recombination-enhancing linear DNA. Evidence is presented that the tandem repeats are not formed by homologous recombination and that linear forms of SV40 DNA must compete with circular SV40 DNA for the available T antigen to enhance recombination. We propose that the enhancement of recombination by linear SV40 DNA results from the entry of that DNA into a rolling circle type of replication pathway which generates highly recombinogenic intermediates.