Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination

Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be transferred from A. tumefaciens to Saccharomyces cerevisiae and integrates into the yeast genome via homologous recombination. We show here that when the T-DNA lacks homology with the S. cerevisiae genome, it integrates at random positions via illegitimate recombination. From 11 lines the integrated T-DNA was cloned back to Escherichia coli along with yeast flanking sequences. The T-DNA borders and yeast DNA flanking the T-DNA were sequenced and characterized. It was found that T-DNA integration had resulted in target DNA deletions and sometimes T-DNA truncations or filler DNA formation. Therefore, the molecular mechanism of illegitimate recombination by which T-DNA integrates in higher and lower eukaryotes seems conserved.

Recombination-dependent deletion formation in mammalian cells deficient in the nucleotide excision repair gene ERCC1

Nucleotide excision repair proteins have been implicated in genetic recombination by experiments in Saccharomyces cerevisiae and Drosophila melanogaster, but their role, if any, in mammalian cells is undefined. To investigate the role of the nucleotide excision repair gene ERCC1, the hamster homologue to the S. cerevisiae RAD10 gene, we disabled the gene by targeted knockout. Partial tandem duplications of the adenine phosphoribosyltransferase (APRT) gene then were constructed at the endogenous APRT locus in ERCC1− and ERCC1+ cells. To detect the full spectrum of gene-altering events, we used a loss-of-function assay in which the parental APRT+ tandem duplication could give rise to APRT− cells by homologous recombination, gene rearrangement, or point mutation. Measurement of rates and analysis of individual APRT− products indicated that gene rearrangements (principally deletions) were increased at least 50-fold, whereas homologous recombination was affected little. The formation of deletions is not caused by a general effect of the ERCC1 deficiency on gene stability, because ERCC1− cell lines with a single wild-type copy of the APRT gene yielded no increase in deletions. Thus, deletion formation is dependent on the tandem duplication, and presumably the process of homologous recombination. Recombination-dependent deletion formation in ERCC1− cells is supported by a significant decrease in a particular class of crossover products that are thought to arise by repair of a heteroduplex intermediate in recombination. We suggest that the ERCC1 gene product in mammalian cells is involved in the processing of heteroduplex intermediates in recombination and that the misprocessed intermediates in ERCC1− cells are repaired by illegitimate recombination.

Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants

Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of ≈103. Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution.

Molecular origin of the mosaic sequence arrangements of higher primate α-globin duplication units

The human adult α-globin locus consists of three pairs of homology blocks (X, Y, and Z) interspersed with three nonhomology blocks (I, II, and III), and three Alu family repeats, Alu1, Alu2, and Alu3. It has been suggested that an ancient primate α-globin-containing unit was ancestral to the X, Y, and Z and the Alu1/Alu2 repeats. However, the evolutionary origin of the three nonhomologous blocks has remained obscure. We have now analyzed the sequence organization of the entire adult α-globin locus of gibbon (Hylobates lar). DNA segments homologous to human block I occur in both duplication units of the gibbon α-globin locus. Detailed interspecies sequence comparisons suggest that nonhomologous blocks I and II, as well as another sequence, IV, were all part of the ancestral α-globin-containing unit prior to its tandem duplication. However, sometime thereafter, block I was deleted from the human α1-globin-containing unit, and block II was also deleted from the α2-globin-containing unit in both human and gibbon. These were probably independent events both mediated by independent illegitimate recombination processes. Interestingly, the end points of these deletions coincide with potential insertion sites of Alu family repeats. These results suggest that the shaping of DNA segments in eukaryotic genomes involved the retroposition of repetitive DNA elements in conjunction with simple DNA recombination processes.

The evolution of plant nuclear genes

We analyze the evolutionary dynamics of three of the best-studied plant nuclear multigene families. The data analyzed derive from the genes that encode the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS), the gene family that encodes the enzyme chalcone synthase (Chs), and the gene family that encodes alcohol dehydrogenases (Adh). In addition, we consider the limited evolutionary data available on plant transposable elements. New Chs and rbcS genes appear to be recruited at about 10 times the rate estimated for Adh genes, and this is correlated with a much smaller average gene family size for Adh genes. In addition, duplication and divergence in function appears to be relatively common for Chs genes in flowering plant evolution. Analyses of synonymous nucleotide substitution rates for Adh genes in monocots reject a linear relationship with clock time. Replacement substitution rates vary with time in a complex fashion, which suggests that adaptive evolution has played an important role in driving divergence following gene duplication events. Molecular population genetic studies of Adh and Chs genes reveal high levels of molecular diversity within species. These studies also reveal that inter- and intralocus recombination are important forces in the generation allelic novelties. Moreover, illegitimate recombination events appear to be an important factor in transposable element loss in plants. When we consider the recruitment and loss of new gene copies, the generation of allelic diversity within plant species, and ectopic exchange among transposable elements, we conclude that recombination is a pervasive force at all levels of plant evolution.

Transposons and genome evolution in plants

Although it is known today that transposons comprise a significant fraction of the genomes of many organisms, they eluded discovery through the first half century of genetic analysis and even once discovered, their ubiquity and abundance were not recognized for some time. This genetic invisibility of transposons focuses attention on the mechanisms that control not only transposition, but illegitimate recombination. The thesis is developed that the mechanisms that control transposition are a reflection of the more general capacity of eukaryotic organisms to detect, mark, and retain duplicated DNA through repressive chromatin structures.

Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Functional copies of a human gene can be directly isolated by transformation-associated recombination cloning with a small 3′ end target sequence

Unique, small sequences (sequence tag sites) have been identified at the 3′ ends of most human genes that serve as landmarks in genome mapping. We investigated whether a single copy gene could be isolated directly from total human DNA by transformation-associated recombination (TAR) cloning in yeast using a short, 3′ unique target. A TAR cloning vector was constructed that, when linearized, contained a small amount (381 bp) of 3′ hypoxanthine phosphoribosyltransferase (HPRT) sequence at one end and an 189-bp Alu repeat at the other end. Transformation with this vector along with human DNA led to selective isolations of the entire HPRT gene as yeast artificial chromosomes (YACs) that extended from the 3′ end sequence to various Alu positions as much as 600 kb upstream. These YACs were retrofitted with a NeoR and a bacterial artificial chromosome (BAC) sequence to transfer the YACs to bacteria and subsequently the BACs to mouse cells by using a Neo selection. Most of the HPRT isolates were functional, demonstrating that TAR cloning retains the functional integrity of the isolated material. Thus, this modified version of TAR cloning, which we refer to as radial TAR cloning, can be used to isolate large segments of the human genome accurately and directly with only a small amount of sequence information.

The high mobility group protein Abf2p influences the level of yeast mitochondrial DNA recombination intermediates in vivo

Abf2p is a high mobility group (HMG) protein found in yeast mitochondria that is required for the maintenance of wild-type (ρ+) mtDNA in cells grown on fermentable carbon sources, and for efficient recombination of mtDNA markers in crosses. Here, we show by two-dimensional gel electrophoresis that Abf2p promotes or stabilizes Holliday recombination junction intermediates in ρ+ mtDNA in vivo but does not influence the high levels of recombination intermediates readily detected in the mtDNA of petite mutants (ρ−). mtDNA recombination junctions are not observed in ρ+ mtDNA of wild-type cells but are elevated to detectable levels in cells with a null allele of the MGT1 gene (Δmgt1), which codes for a mitochondrial cruciform-cutting endonuclease. The level of recombination intermediates in ρ+ mtDNA of Δmgt1 cells is decreased about 10-fold if those cells contain a null allele of the ABF2 gene. Overproduction of Abf2p by ≥ 10-fold in wild-type ρ+ cells, which leads to mtDNA instability, results in a dramatic increase in mtDNA recombination intermediates. Specific mutations in the two Abf2p HMG boxes required for DNA binding diminishes these responses. We conclude that Abf2p functions in the recombination of ρ+ mtDNA.

Heteroduplex joint formation in Escherichia coli recombination is initiated by pairing of a 3′-ending strand

The formation of heteroduplex joints in Escherichia coli recombination is initiated by invasion of double-stranded DNA by a single-stranded homologue. To determine the polarity of the invasive strand, linear molecules with direct terminal repeats were released by in vivo restriction of infecting chimeric phage DNA and heteroduplex products of intramolecular recombination were analyzed. With this substrate, the invasive strand is expected to be incorporated into the circular crossover product and the complementary strand is expected to be incorporated into the reciprocal linear product. Strands of both polarities were incorporated into heteroduplex structures, but only strands ending 3′ at the break were incorporated into circular products. This result indicates that invasion of the 3′-ending strand initiates the heteroduplex joint formation and that the complementary 5′-ending strand is incorporated into heteroduplex structures in the process of reciprocal strand exchange. The polarity of the invasive strand was not affected by recD, recJ, or xonA mutations. However, xonA and recJ mutations increased the proportion of heteroduplexes containing 5′-ending strands. This observation suggests that RecJ exonuclease and exonuclease I may enhance recombination by degrading the displaced strands during branch migration and thereby causing strand exchange to be unidirectional.

Free recombination within Helicobacter pylori

Sequences of three gene fragments (flaA, flaB, and vacA) from Helicobacter pylori strains isolated from patients in Germany, Canada, and South Africa were analyzed for diversity and for linkage equilibrium by using the Homoplasy Test and compatibility matrices. Horizontal genetic exchange in H. pylori is so frequent that different loci and polymorphisms within each locus are all at linkage equilibrium. These results indicate that H. pylori is panmictic. Comparisons with sequences from Escherichia coli, Neisseria meningitidis, and Drosophila melanogaster showed that recombination in H. pylori was much more frequent than in other species. In contrast, when multiple family members infected with H. pylori were investigated, some strains were indistinguishable at all three loci. Thus, H. pylori is clonal over short time periods after natural transmission.

Role of Saccharomyces cerevisiae Msh2 and Msh3 repair proteins in double-strand break-induced recombination

When gene conversion is initiated by a double-strand break (DSB), any nonhomologous DNA that may be present at the ends must be removed before new DNA synthesis can be initiated. In Saccharomyces cerevisiae, removal of nonhomologous ends depends not only on the nucleotide excision repair endonuclease Rad1/Rad10 but also on Msh2 and Msh3, two proteins that are required to correct mismatched bp. These proteins have no effect when DSB ends are homologous to the donor, either in the kinetics of recombination or in the proportion of gene conversions associated with crossing-over. A second DSB repair pathway, single-strand annealing also requires Rad1/Rad10 and Msh2/Msh3, but reveals a difference in their roles. When the flanking homologous regions that anneal are 205 bp, the requirement for Msh2/Msh3 is as great as for Rad1/Rad10; but when the annealing partners are 1,170 bp, Msh2/Msh3 have little effect, while Rad1/Rad10 are still required. Mismatch repair proteins Msh6, Pms1, and Mlh1 are not required. We suggest Msh2 and Msh3 recognize not only heteroduplex loops and mismatched bp, but also branched DNA structures with a free 3′ tail.

Barriers to recombination between closely related bacteria: MutS and RecBCD inhibit recombination between Salmonella typhimurium and Salmonella typhi

Previous studies have shown that inactivation of the MutS or MutL mismatch repair enzymes increases the efficiency of homeologous recombination between Escherichia coli and Salmonella typhimurium and between S. typhimurium and Salmonella typhi. However, even in mutants defective for mismatch repair the recombination frequencies are 102- to 103-fold less than observed during homologous recombination between a donor and recipient of the same species. In addition, the length of DNA exchanged during transduction between S. typhimurium and S. typhi is less than in transductions between strains of S. typhimurium. In homeologous transductions, mutations in the recD gene increased the frequency of transduction and the length of DNA exchanged. Furthermore, in mutS recD double mutants the frequency of homeologous recombination was nearly as high as that seen during homologous recombination. The phenotypes of the mutants indicate that the gene products of mutS and recD act independently. Because S. typhimurium and S. typhi are ≈98–99% identical at the DNA sequence level, the inhibition of recombination is probably not due to a failure of RecA to initiate strand exchange. Instead, these results suggest that mismatches act at a subsequent step, possibly by slowing the rate of branch migration. Slowing the rate of branch migration may stimulate helicase proteins to unwind rather than extend the heteroduplex and leave uncomplexed donor DNA susceptible to further degradation by RecBCD exonuclease.

Recombination activities of HsDmc1 protein, the meiotic human homolog of RecA protein

Meiosis-specific homologs of RecA protein have been identified in Saccharomyces cerevisiae and higher eukaryotes including mammals, but their enzymatic activities have not been described. We have purified the human protein HsDmc1 produced in Escherichia coli from a cloned copy of the cDNA. The recombinant enzyme had DNA-dependent ATPase activity with an estimated kcat of 1.5 min−1. DNase protection experiments with oligonucleotides as substrates indicated that HsDmc1 protein binds preferentially to single-stranded DNA with a stoichiometry of approximately one molecule of protein per three nucleotide residues. HsDmc1 protein catalyzed the formation of D-loops in superhelical DNA, as well as strand exchange between single-stranded and double-stranded oligonucleotides. The requirements for strand exchange catalyzed by HsDmc1 were similar to those of RecA protein, but exchange caused by HsDmc1 was not supported by ATPγS.

Arabidopsis thaliana mutants altered in homologous recombination

Homologous recombination contributes both to the generation of allelic diversity and to the preservation of genetic information. In plants, a lack of suitable experimental material has prevented studies of the regulatory and enzymatic aspects of recombination in somatic and meiotic cells. We have isolated nine Arabidopsis thaliana mutants hypersensitive to x-ray irradiation (xrs) and examined their recombination properties. For the three xrs loci described here, single recessive mutations were found to confer simultaneous hypersensitivities to the DNA-damaging chemicals mitomycin C (MMCs) and/or methyl methanesulfonate (MMSs) and alterations in homologous recombination. Mutant xrs9 (Xrays, MMSs) is reduced in both somatic and meiotic recombination and resembles yeast mutants of the rad52 epistatic group. xrs11 (Xrays, MMCs) is deficient in the x-ray-mediated stimulation of homologous recombination in somatic cells in a manner suggesting a specific signaling defect. xrs4 (Xrays, MMSs, MMCs) has a significant deficiency in somatic recombination, but this is accompanied by meiotic hyper-recombination. A corresponding phenotype has not been reported in other systems and thus this indicates a novel, plant-specific regulatory circuit linking mitotic and meiotic recombination.