The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

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.     

The rate of extrachromosomal homologous recombination within a novel reporter plasmid is elevated in cells lacking functional ATM protein

Homologous recombination between identical stretches of DNA depends on the coordinated action of many tightly regulated proteins. Cellular defects in homologous recombination are strongly associated with increased genomic instability and tumorigenesis. In cells of the cancer-prone syndrome ataxia telangiectasia (A-T), increased intrachromosomal recombination has been demonstrated, while extrachromosomal recombination has been discussed controversially. We constructed a novel, episomally replicating pGrec recombination vector containing two mutated alleles of the enhanced green fluorescent protein (eGFP) gene. Homologous recombination can reconstitute functional wildtype eGFP, thus allowing detection of recombination events based on cellular eGFP fluorescence. Using an isogenic cell pair of A-T fibroblasts and derivatives complemented by an ATM expression vector, we were able to demonstrate in A-T cells high extrachromosomal recombination rates, which are suppressed upon ectopic ATM expression. We thus found that ATM deficiency increases spontaneous recombination not only in intrachromosomal but also in extrachromosomal substrates, suggesting that lack of ATM increases homologous recombination independent of the chromatin structure.     

Mismatch repair of heteroduplex DNA intermediates of extrachromosomal recombination in mammalian cells.

Previous work indicated that extrachromosomal recombination in mammalian cells could be explained by the single-strand annealing (SSA) model. This model predicts that extrachromosomal recombination leads to nonconservative crossover products and that heteroduplex DNA (hDNA) is formed by annealing of complementary single strands. Mismatched bases in hDNA may subsequently be repaired to wild-type or mutant sequences, or they may remain unrepaired and segregate following DNA replication. We describe a system to examine the formation and mismatch repair of hDNA in recombination intermediates. Our results are consistent with extrachromosomal recombination occurring via SSA and producing crossover recombinant products. As predicted by the SSA model, hDNA was present in double-strand break-induced recombination intermediates. By placing either silent or frameshift mutations in the predicted hDNA region, we have shown that mismatches are efficiently repaired prior to DNA replication.     

DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens.     

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.     

Epstein–Barr Virus Plasmid Model System for Analyzing Recombination in Human Cells

Homologous recombination stimulated by a double-strand break at a desired target site offers a method to achieve site-specific integration useful for gene therapy and other genetic engineering. To test parameters needed for this strategy, we developed an Epstein-Barr virus shuttle vector model system as a genetic tool. This extrachromosomal plasmid assay system has several advantages over a chromosomal assay. The system detects all classes of recombination events without selection and allows rapid analysis of the frequency and nature of recombination events. We found that a double-strand break at the target site stimulated a large increase in recombination frequency. The resulting recombinants included one-sided insertion events, as well as two-sided or gene conversion events. A circular donor substrate was more effective in recombination than linearized donor DNA.     

Unequal access of chromosomal regions to each other in Salmonella: probing chromosome structure with phage lambda integrase-mediated long-range rearrangements

We have investigated the fluidity of the Salmonella chromosome architecture using the phage lambda site-specific recombination system as a probe. We determined how chromosome position affects the extent of integrase-mediated recombination between pairs of inversely oriented att sites at various loci. We also investigated the accessibility of each chromosomal att site to an extrachromosomal partner carried on a low-copy plasmid. Recombination events were assayed by semi-quantitative polymerase chain reaction of the attP product. The extent of recombination between the chromosome and the plasmid was generally higher than intrachromosomal recombination except for two loci, araA::attL and galT::attL, which gave no detectable recombination with any other locus. Based on 20 intervals, we found that chromosomal locations are not equally accessible to each other. Although multiple factors probably affect accessibility, the most important is the specific combination of the end-points used. Neither the size of the intervals nor the accessibility of individual end-points to extrachromosomal sequences is as important. These results suggest that the chromosome is not completely fluid but rather organized in some way, with barriers that limit the movement of DNA within the cell. The nature of the barriers involved in chromosomal organization remains to be determined.     

'Illegitimate' recombination events in polyoma-transformed rat cells

In the LPT line of polyoma (Py)-transformed rat cells, amplification of the integrated viral DNA and of cell nucleotide sequences flanking the viral integration site, can be induced either spontaneously or by treatment with carcinogens. We show here that the amplified DNA includes interspersed viral and cellular sequences generated by 'illegitimate' recombination events. Genomic libraries have been prepared in phage lambda vectors from LPT cells treated with the inducing agent mitomycin C and from untreated LPT cells. Four phages, including viral-cell DNA recombinants, have been isolated from these libraries. Sequencing through the recombination sites revealed the following characteristics: (i) The crossover points map at four different positions in the viral DNA and at four different positions in the flanking cell DNA. (ii) There are very short homologous sequences of 1, 2, or 4 bp, at the recombination sites. (iii) Aside from the exchanges between the viral and the cellular DNA, no further rearrangements occurred around the new viral-cellular DNA junctions. (iv) Next to the recombination sites, there are blocks of homopurine-homopyrimidine sequences, which may assume a structure that differs from the Watson-Crick double helix. (v) Clustered homologous sequence blocks of up to 10 bp are present less than 200 bp away from the recombination sites. These homologies are not in register. Based on these results, we propose a model that may account for these recombination events and, more generally, for recombination events that occur during gene amplification in mammalian cells.     

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.     

A model of the replication fork blocking protein Fob1p based on the catalytic core domain of retroviral integrases

The replication fork blocks are common in both prokaryotes and eukaryotes. In most cases, these blocks are associated with increased levels of mitotic recombination. One of the best-characterized replication fork blocks in eukaryotes is found in ribosomal DNA (rDNA) repeats of Saccharomyces cerevisiae. It has been shown that the replication fork blocking protein Fob1p regulates the recombination rate and the number of rDNA copies in S. cerevisiae, but the mechanistic aspects of these events are still poorly understood. Sequence profile searches revealed that Fob1p is related to retroviral integrases. Subsequently, the catalytic domain of HIV-1 integrase was used as a template to build a reliable three-dimensional model of Fob1p. Structural insights from this study may be useful in explaining Fob1p-mediated formation of extrachromosomal rDNA circles that accelerate aging in yeast and recombination events that lead to expansion or contraction of rDNA.     

Sister chromatid gene conversion is a prominent double-strand break repair pathway in mammalian cells

In mammalian cells, repair of DNA double-strand breaks (DSBs) occurs by both homologous and non-homologous mechanisms. By definition, homologous recombination requires a template with sufficient sequence identity to the damaged molecule in order to direct repair. We now show that the sister chromatid acts as a repair template in a substantial proportion of DSB repair events. The outcome of sister chromatid repair is primarily gene conversion unassociated with reciprocal exchange. This contrasts with expectations from the classical DSB repair model originally proposed for yeast meiotic recombination, but is consistent with models in which recombination is coupled intimately with replication. These results may explain why cytologically observable sister chromatid exchanges are induced only weakly by DNA-damaging agents that cause strand breaks, since most homologous repair events would not be observed. A preference for non-crossover events between sister chromatids suggests that crossovers, although genetically silent, may be disfavored for other reasons. Possibly, a general bias against crossing over in mitotic cells exists to reduce the potential for genome alterations when other homologous repair templates are utilized.     

A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae

In most eukaryotic organisms, recombination events leading to exchanges between homologous chromosomes link the homologs in a manner that allows their proper attachment to the meiotic spindle. In the yeast Saccharomyces cerevisiae these exchanges are initiated in early prophase as double-strand breaks in the DNA. These breaks are processed through a series of intermediates to yield mature crossovers late in prophase. The following experiments were designed to monitor the appearance of the earliest recombinant DNA strands formed in this process. A polymerase chain reaction assay was devised that allows the detection of recombinant strands at a known initiation site for meiotic recombination. The time and rate of appearance of recombinant strands was found to coincide with commitment to recombination, demonstrating that DNA strands bearing sequences from both parental chromosomes are rapidly formed after the initiation of meiotic recombination.     

Inhibitors of topoisomerase II enzymes: a unique group of environmental mutagens and carcinogens

Many inhibitors of topoisomerase II enzymes are potent mutagens, leading to major chromosomal deletions, illegitimate recombination and aneuploidy. There is increasing evidence that they are also human carcinogens. However, their lack of chemical reactivity means that they may give weak or negative results in commonly used mutagenicity tests, or may give data with characteristics quite distinct from chemicals that alkylate DNA. They do not form DNA adducts and assays such as ³²P-postlabelling will not detect their presence in the body. They are generally not point mutagens and may fail to provide distinctive fingerprints in mutation spectra. These characteristics may be limiting a realistic evaluation of their role in human carcinogenesis using current methodologies.     

Frequent occurrence of UVB-induced sister chromatid exchanges in telomere regions and its implication to telomere maintenance

Sister chromatid exchanges (SCEs) are symmetrical exchanges between newly replicated chromatids and their sisters. While homologous recombination may be one of the principal mechanisms responsible for SCEs, the full details of their molecular basis and biological significance remain to be elucidated. Following exposure to ultraviolet light B (UVB), mitomycin C (MMC) and cisplatin, we analyzed the location of SCEs on metaphase chromosomes in Chinese hamster CHO cells. The frequency of SCEs increased over the spontaneous level in proportion to the agent's dose. UVB-induced SCEs occurred frequently in telomere regions, as cisplatin-induced SCEs did, differing from MMC-induced ones. The remarkable difference of intrachromosomal distribution among the three mutagens may be attributed to the specificity of induced DNA lesions and structures of different chromosome regions. Telomeric DNA at the end of chromosomes is composed of multiple copies of a repeated motif, 5'-TTAGGG-3' in mammalian cells. Telomeric repeats may be potential targets for UVB and cisplatin, which mainly form pyrimidine dimers and intrastrand d(GpG) cross-links, respectively, resulting in SCE formation. UVB irradiation shortened telomeres and augmented the telomerase activity. The possible implications of the frequent occurrence of SCEs in telomere regions are discussed in connection with the maintenance of telomere integrity.     

Repair of double-stranded DNA breaks by homologous DNA fragments during transfer of DNA into mouse L cells.

To test the validity of various models for recombination between extrachromosomal DNAs in mammalian cells, we measured recombination between a plasmid containing a herpesvirus thymidine kinase (tk) gene with an internal BamHI linker insertion mutation (ptkB8) and a tk gene deleted at both ends (tk delta 3' delta 5'). The two DNAs shared 885 base pairs of perfect tk homology except for the interruption at the linker insertion site. Recombination events that restored the mutated insertion site to wild type were monitored by the generation of hypoxanthine-aminopterine-thymidine-resistant colonies after cotransformation of Ltk- cells with the two DNAs. We found that cleavage of the ptkB8 DNA at the linker insertion site was essential for gene restoration. If the tk delta 3' delta 5' DNA was ligated into mp10 vector DNA, then recombination with the cleaved ptkB8 DNA was inefficient. In contrast, if it was excised from that vector by cleavage at flanking restriction sites, then recombination was stimulated about 150-fold. Using restriction site polymorphisms, we showed that most of the recombination events leading to restoration of the tk gene with the excised tk delta 3' delta 5' fragment involved three double-strand duplexes: two ptkB8 DNAs and one tk delta 3' delta 5' fragment. These results are much more readily explained by the single-strand annealing model of recombination than by the double-strand break repair model, and they suggest that the deficiency of the latter pathway for extrachromosomal mammalian recombination may be due, at least in part, to the obligate tripartite nature of the reaction. Finally, we measured the effect of DNA homology on the efficiency of the ptkB8-tk delta 3' delta 5' reaction. Our results showed a near-linear relationship between the efficiency of recombination and the amount of homology flanking either side of the linker insertion site. Moreover, we could detect thymidine kinase-positive transformants with as little as 10 base pairs of homology.     

Ataxia telangiectasia: the effects of chemical mutagens and x-rays on sister chromatid exchanges in blood lymphocytes

It is now possible to examine in detail exchanges between sister chromatids (SCEs) and to attempt to investigate the relationships of such exchanges to aberration formation and DNA-repair mechanisms. The frequency of SCEs is dramatically increased by chemical mutagens and may reflect the level of DNA damage. Lymphocytes from patients with ataxia telangiectasis (AT) show high levels of spontaneous chromosome damage and are hypersentive to ionising radiations and it was of interest to examine the levels of SCE induced in these cells by various mutagens. The frequencies of SCE after treatment with X=rays or three chemical mutagens were equivalent to those in normal cells. The effects of fluorodeoxyuridine and deoxycytidine on SCE frequencies were also tested.     

Induction of sister chromatid exchanges by chemical mutagens and its possible relevance to DNA repair

Several chemical mutagens were found to induce sister chromatid exchanges in Chinese hamster chromosomes. Among them, effects of 4NQO and MMC were very similar to those of UV light in that the exchange frequency increased with increasing dose of chemicals and that it was markedly lowered in the presence of 1 mM caffeine during a post-treatment period. The frequency of proflavin-induced sister chromatid exchanges was also found to be dose dependent, but it was insensitive to the caffeine post-treatment. On the other hand, no appreciable increase was detected in the incidence of sister chromatid exchanges in MNNG-treated cells over a 100-fold range of variation in chemical dose. Caffeine by itself raised the exchange frequency only slightly over a control level. It was found that 4NQO and MMC exerted remarkable delayed effects on the exchange induction, whereas proflavin did not. This seems to suggest that the lesions caused by the former mutagens would be long-lived and repeatedly provoke sister chromatid exchanges. These data imply that there are several possible ways in which the initial DNA lesions ultimately lead to the formation of sister chromatid exchanges, and that at least UV-, 4NQO- and MMC-induced sister chromatid exchanges would have evolved through a caffeine sensitive repair process, probably related to a post-replication repair of DNA damage.     

Molecular mechanisms of sister-chromatid exchange

Sister-chromatid exchange (SCE) is the process whereby, during DNA replication, two sister chromatids break and rejoin with one another, physically exchanging regions of the parental strands in the duplicated chromosomes. This process is considered to be conservative and error-free, since no information is generally altered during reciprocal interchange by homologous recombination. Upon the advent of non-radiolabel detection methods for SCE, such events were used as genetic indicators for potential genotoxins/mutagens in laboratory toxicology tests, since, as we now know, most forms of DNA damage induce chromatid exchange upon replication fork collapse. Much of our present understanding of the mechanisms of SCE stems from studies involving nonhuman vertebrate cell lines that are defective in processes of DNA repair and/or recombination. In this article, we present a historical perspective of studies spearheaded by Dr. Anthony V. Carrano and colleagues focusing on SCE as a genetic outcome, and the role of the single-strand break DNA repair protein XRCC1 in suppressing SCE. A more general overview of the cellular processes and key protein "effectors" that regulate the manifestation of SCE is also presented.     

Mitotic inter-homologue junctions accumulate at damaged DNA replication forks in recQ mutants

Mitotic homologous recombination is utilised to repair DNA breaks using either sister chromatids or homologous chromosomes as templates. Because sister chromatids are identical, exchanges between sister chromatids have no consequences for the maintenance of genomic integrity unless they involve repetitive DNA sequences. Conversely, homologous chromosomes might differ in genetic content, and exchanges between homologues might lead to loss of heterozygosity and subsequent inactivation of functional genes. Genomic instability, caused by unscheduled recombination events between homologous chromosomes, is enhanced in the absence of RecQ DNA helicases, as observed in Bloom's cancer-prone syndrome. Here, we used two-dimensional gel electrophoresis to analyse budding yeast diploid cells that were modified to distinguish replication intermediates originating from each homologous chromosome. Therefore, these cells were suitable for analysing the formation of inter-homologue junctions. We found that Rad51-dependent DNA structures resembling inter-homologue junctions accumulate together with sister chromatid junctions at damaged DNA replication forks in recQ mutants, but not in the absence of Srs2 or Mph1 DNA recombination helicases. Inter-homologue joint molecules in recQ mutants are less abundant than sister chromatid junctions, but they accumulate with similar kinetics after origin firing under conditions of DNA damage. We propose that unscheduled accumulation of inter-homologue junctions during DNA replication might account for allelic recombination defects in recQ mutants.