Homologous recombination between transfected DNAs.

An extensive analysis of the fate and structure of polyomavirus-plasmid recombinant molecules transfected into Rat-1 cells has revealed that the DNA often becomes integrated within transformed cell DNA in a head-to-tail tandem arrangement. This occurs independently of the replicative capacity of the transforming DNA and is facilitated by the use of large quantities of DNA during transfection. These observations have led us to suggest that head-to-tail tandems are formed by homologous recombination between transfected DNAs either before or after integration within cellular DNA. To test this hypothesis, we have measured the transforming activity of pairs of mutant, nontransforming, recombinant plasmid DNAs that carry different lesions in the transforming gene of polyomavirus. The results show that, although the individual mutant DNAs are incapable of transformation, transfection with pairs of mutant DNAs leads to the formation of transformed cells at high frequency. Moreover, there is a direct relationship between the distance between the lesions in pairs of mutant DNAs and their transforming activity. Finally, analyses of the structures of integrated recombinant plasmid DNAs and the viral proteins within independent transformed cells prove that recombination occurs between the mutant genomes to generate a wild-type transforming gene.     

Homologous recombination and the repair of double-strand breaks during cotransformation of Dictyostelium discoideum.

We examined the ability of unlinked nonreplicating plasmid molecules to undergo homologous recombination during cotransformation of Dictyostelium amoebae. The transformation vector B10S confers resistance to the antibiotic G418 and was always presented to amoebae as a closed circle. Cotransforming DNA, containing a slime mold cDNA and sequences homologous to the primary vector, was presented either as a closed circle or as a linear molecule after digestion with restriction endonucleases which cut within one of three distinct regions of the plasmid. Remarkably, homologous recombination occurred in every clone examined. Moreover, the products of recombination were identical in all instances, irrespective of the presence or position of linearized ends. The ends of the linear templates were not recombinogenic. Repair of the introduced double-strand break occurred frequently during recombination. The repair could occur intermolecularly or, more likely, intramolecularly, i.e., by recircularization. Many of the recombination events were of a nonreciprocal nature. Despite the startlingly frequent level of homologous recombination, the use of cotransforming DNA which contains no homology to the selected vector established that such recombination was not required for cotransformation.     

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.     

Homologous recombination catalyzed by human cell extracts.

Two plasmids containing noncomplementing and nonreverting deletions in a bacterial phosphotransferase gene conferring resistance to neomycin (Neor) were incubated with human cell extracts, and the mixtures were used to transform recombination-deficient (recA-) Escherichia coli cells. We were able to obtain Neor colonies at a frequency of 2 X 10(-3). This frequency was 100 to 1,000 times higher than that obtained with no extracts. The removal of riboadenosine 5'-triphosphate, Mg2+, or deoxynucleoside triphosphates from the reaction mixture severely reduced the yield of Neor colonies. Examination of plasmid DNA from the Neor colonies revealed that they resulted from gene conversion and reciprocal recombination. On the basis of these results, we conclude that mammalian somatic cells in culture have the enzymatic machinery to catalyze homologous recombination in vitro.     

Homologous recombination and its regulation

Homologous recombination (HR) is critical both for repairing DNA lesions in mitosis and for chromosomal pairing and exchange during meiosis. However, some forms of HR can also lead to undesirable DNA rearrangements. Multiple regulatory mechanisms have evolved to ensure that HR takes place at the right time, place and manner. Several of these impinge on the control of Rad51 nucleofilaments that play a central role in HR. Some factors promote the formation of these structures while others lead to their disassembly or the use of alternative repair pathways. In this article, we review these mechanisms in both mitotic and meiotic environments and in different eukaryotic taxa, with an emphasis on yeast and mammal systems. Since mutations in several proteins that regulate Rad51 nucleofilaments are associated with cancer and cancer-prone syndromes, we discuss how understanding their functions can lead to the development of better tools for cancer diagnosis and therapy.     

Homologous RNA recombination allows efficient introduction of site-specific mutations into the genome of coronavirus MHV-A59 via synthetic co-replicating RNAs.

We describe a novel strategy to site-specifically mutagenize the genome of an RNA virus by exploiting homologous RNA recombination between synthetic defective interfering (DI) RNA and the viral RNA. The construction of a full-length cDNA clone, pMIDI, of a DI RNA of coronavirus MHV strain A59 was reported previously (R.G. Van der Most, P.J. Bredenbeek, and W.J.M. Spaan (1991). J. Virol. 65, 3219-3226). RNA transcribed from this construct, is replicated efficiently in MHV-infected cells. Marker mutations introduced in MIDI RNA were replaced by the wild-type residues during replication. More importantly, however, these genetic markers were introduced into viral genome: even in the absence of positive selection MHV recombinants could be isolated. This finding provides new prospects for the study of coronavirus replication using recombinant DNA techniques. As a first application, we describe the rescue of the temperature sensitive mutant MHV Albany-4 using DI-directed mutagenesis. Possibilities and limitations of this strategy are discussed.     

Homologous recombination in plant cells after Agrobacterium-mediated transformation.

A single amino-acid change in the acetolactate synthase (ALS) protein of tobacco confers resistance to the herbicide chlorsulfuron. A deleted, nonfunctional fragment from the acetolactate synthase gene, carrying the mutant site specifying chlorsulfuron resistance plus a closely linked novel restriction site marker, was cloned into a binary vector. Tobacco protoplasts transformed with Agrobacterium tumefaciens carrying this vector yielded chlorsulfuron-resistant colonies. DNA gel blot analysis of DNA from these colonies suggested that in three transformants homologous recombination had occurred between the endogenous ALS gene and the deleted ALS gene present in the incoming T-DNA. Plants were regenerated from these chlorsulfuron-resistant colonies, and in two of the transformants, genetic analysis of their progeny showed that the novel gene segregated as a single Mendelian locus. Possible models for the generation of these recombinant plants are discussed.