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PubMed1995

Homologous recombination proteins in prokaryotes and eukaryotes.

Genetic recombination is common to all forms of life and involves the exchange of DNA sequences between two chromosomes or DNA molecules. Such exchanges contribute to the generation of genetic diversity and the repair of damaged DNA. There are two major classes of recombination, site-specific recombination and general or homologous recombination. In homologous recombination the joining of the DNA duplexes exhibits a similar degree of precision or fidelity but, generally speaking, does not take place at specific sites. Since exchange can occur anywhere along the length of two homologous chromosomes, it follows that the proteins that catalyze homologous recombination are not sequence- or site-specific binding proteins. This review focuses on genetic and biochemical analyses of homologous recombination proteins that carry out conjugational recombination in E. coli and meiotic recombination in eukaryotes.

Amino Acid Sequence↗
PubMed2005

High-frequency homologous recombination in plants mediated by zinc-finger nucleases.

Homologous recombination offers great promise for plant genome engineering. This promise has not been realized, however, because when DNA enters plant cells homologous recombination occurs infrequently and random integration predominates. Using a tobacco test system, we demonstrate that chromosome breaks created by zinc-finger nucleases greatly enhance the frequency of localized recombination. Homologous recombination was measured by restoring function to a defective GUS:NPTII reporter gene integrated at various chromosomal sites in 10 different transgenic tobacco lines. The reporter gene carried a recognition site for a zinc-finger nuclease, and protoplasts from each tobacco line were electroporated with both DNA encoding the nuclease and donor DNA to effect repair of the reporter. Homologous recombination occurred in more than 10% of the transformed protoplasts regardless of the reporter's chromosomal position. Approximately 20% of the GUS:NPTII reporter genes were repaired solely by homologous recombination, whereas the remainder had associated DNA insertions or deletions consistent with repair by both homologous recombination and non-homologous end joining. The DNA-binding domain encoded by zinc-finger nucleases can be engineered to recognize a variety of chromosomal target sequences. This flexibility, coupled with the enhancement in homologous recombination conferred by double-strand breaks, suggests that plant genome engineering through homologous recombination can now be reliably accomplished using zinc-finger nucleases.

Base Sequence↗
PubMed2002

Wild-type p53 inhibits replication-associated homologous recombination.

In mammalian cells homologous recombination is stimulated, when the replication fork stalls at DNA breaks or unrepaired lesions. The tumor suppressor p53 downregulates homologous recombination independently of its transcriptional transactivation function and has been linked to enzymes of DNA recombination and replication. To study recombination with respect to replication, we utilized a SV40 virus based assay, to follow the synchronous events after primate cell infection. gamma-ray treatment at different times after viral entry unveiled an increase of interchromosomal exchange frequencies, when the damage was introduced during DNA synthesis. Elevated recombination frequencies were fully suppressed by p53. With respect to the downregulation of spontaneous recombination, we noticed a requirement for active p53 molecules, when replication started. After a transient treatment with replication inhibitors, we observed inhibition of the drug induced recombination by p53, particularly for the elongation inhibitor aphidicolin. Consequently, we propose that p53 is a surveillance factor of homologous recombination at replication forks, when they stall as a consequence of endogenous or of exogenously introduced damage.

Animals↗
PubMed2004

Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display.

Homologous recombination in yeast can be exploited to reliably generate libraries of >10(7) transformants from a pool of PCR products and a linearized plasmid vector. Homology in the PCR insertion products drives shuffling of these genes in vivo by yeast homologous recombination. Two scFvs that share 89.8% homology were shuffled in vivo by homologous recombination, and chimeric genes were generated regardless of whether or not one of the scFv PCR products lacked 5' homology to the cut vector and the second scFv PCR product lacked 3' homology to the cut vector, or both PCR products had both 5' and 3' homology to the cut vector. A majority of the chimeras had single crossovers; however, double and triple crossovers were isolated. Crossover points were evenly distributed in the hybrids created and homology of as little as two nucleotides was able to produce a chimeric clone. The numbers of clones isolated with a given number of crossovers was approximated well by a Poisson distribution. Transformation efficiencies for the chimeric libraries were of the order of 10(4)-10(5) transformants per microgram of insert, which is the same order of magnitude as when a single PCR product is inserted alone into the display vector by homologous recombination. This method eliminates ligation and Escherichia coli transformation steps of previous methods for generating yeast-displayed libraries, requires fewer PCR cycles than in vitro DNA shuffling and, unlike site-specific recombination methods, allows for recombination anywhere that homology exists between the genes to be recombined. This simple technique should prove useful for protein engineering in general and antibody engineering, specifically in yeast.

DNA Shuffling↗
PubMed2001

RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination.

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60-70 degrees C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.

Adenosine Diphosphate↗
PubMed1991

Characterization of recombination intermediates from DNA injected into Xenopus laevis oocytes: evidence for a nonconservative mechanism of homologous recombination.

Homologous recombination between DNA molecules injected into Xenopus laevis oocyte nuclei is extremely efficient if injected molecules have overlapping homologous ends. Earlier work demonstrated that ends of linear molecules are degraded by a 5'----3' exonuclease activity, yielding 3' tails that participate in recombination. Here, we have characterized intermediates further advanced along the recombination pathway. The intermediates were identified by their unique electrophoretic and kinetic properties. Two-dimensional gel electrophoresis and hybridization with oligonucleotide probes showed that the intermediates had heteroduplex junctions within their homologous overlaps in which strands ending 3' were full length and those ending 5' were shortened. Additional characterization suggested that these intermediates had formed by the annealing of complementary 3' tails. Annealed junctions made in vitro were rapidly processed to products, indicating that they are on the normal recombination pathway. These results support a nonconservative, single-strand annealing mode of recombination. This recombination mechanism appears to be shared by many organisms, including bacteria, fungi, plants, and mammals.

Animals↗
PubMed1994

Chimerization of antitumor antibodies via homologous recombination conversion vectors.

Homologous recombination vectors were designed to convert murine hybridoma cell lines expressing IgG3, IgG1, or IgG2a heavy chains into chimeric human IgG1 producers. These conversion vectors included homology both upstream and downstream of the target sequences and consistently resulted in a higher frequency of successful gene targeting than an insertion vector bearing a single region of homology. A human kappa light chain conversion vector was also constructed and used to complete chimerization of the anticarcinoma hybridoma cell line BR96. The resulting cell line expressed antigen-specific chimeric antibody at comparable levels to those found in the murine parental cell line. Southern blots confirm that recombination occurred within the upstream and downstream regions of homology for both vectors, resulting in the loss of murine constant region sequences.

Animals↗
PubMed1992

A mathematical model of homologous recombination in cultured cells.

This work presents a model describing the rate of recombination between homologous segments of DNA stably integrated into the genome of cultured cells. The model has been applied to rat cell lines carrying the polyomavirus middle T oncogene and a functional origin of viral DNA replication. Introduction of the gene coding for the polyoma large T antigen or the SV40 large T antigen into cells by DNA transfection promotes homologous recombination in the resident viral inserts with rates varying between 0.1 x 10(-3) and 3.7 x 10(-1) per cell generation.

Animals↗
PubMed2002

Nuclear dynamics of RAD52 group homologous recombination proteins in response to DNA damage.

Recombination between homologous DNA molecules is essential for the proper maintenance and duplication of the genome, and for the repair of exogenously induced DNA damage such as double-strand breaks. Homologous recombination requires the RAD52 group proteins, including Rad51, Rad52 and Rad54. Upon treatment of mammalian cells with ionizing radiation, these proteins accumulate into foci at sites of DNA damage induction. We show that these foci are dynamic structures of which Rad51 is a stably associated core component, whereas Rad52 and Rad54 rapidly and reversibly interact with the structure. Furthermore, we show that the majority of the proteins are not part of the same multi-protein complex in the absence of DNA damage. Executing DNA transactions through dynamic multi-protein complexes, rather than stable holo-complexes, allows flexibility. In the case of DNA repair, for example, it will facilitate cross-talk between different DNA repair pathways and coupling to other DNA transactions, such as replication.

Adenosine Triphosphatases↗
PubMed2004

How homologous recombination is initiated: unexpected evidence for single-strand nicks from v(d)j site-specific recombination.

Views of how homologous recombination is initiated have changed over the past several decades: in the 1960s and 1970s, single-strand DNA lesions (nicks) were the leading contenders, but in the last decade, double-strand DNA breaks (DSBs) have reigned. In this issue of Cell, evidence is presented that nicks can stimulate homologous recombination and, in uncontrolled situations, may lead to translocations and other potentially dangerous genome rearrangements.

Animals↗
PubMed1994

Homologous recombination and gene replacement at the dihydrofolate reductase-thymidylate synthase locus in Toxoplasma gondii.

To investigate the feasibility of genomic transgene expression and gene targeting in Toxoplasma gondii, parasites have been transfected with constructs differing in the length of contiguous genomic sequence spanning the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene. We have previously reported that vectors derived from a DHFR-TS cDNA 'minigene' containing mutations in the DHFR coding sequence confer pyrimethamine resistance to transfected parasites (Donald and Roos, 1993). Stably resistant parasite clones arise at high frequency, generally by virtue of transgene integration into parasite chromosomes at locations scattered throughout the genome. In contrast, using a vector which contains 8 kb of contiguous genomic sequence (vs. < 2 kb for the cDNA-derived vectors), approximately half of the integration events occur by homologous recombination. Homologous recombination appears to occur at even higher frequency when a 16 kb genomic clone is used. Circular plasmids were more efficient than linearized molecules at producing homologous recombination in this system, integrating by reciprocal crossing-over to produce a duplication of the DHFR-TS locus. Double crossing-over (or gene conversion) was also observed at low frequency, resulting in complete allelic replacement in this haploid stage of the parasite. The ability to produce either homologous or non-homologous recombinants, by the selection of appropriate transformation constructs, has considerable genetic potential.

Alleles↗
PubMed2004

Discriminatory suppression of homologous recombination by p53.

Homologous recombination (HR) is used in vertebrate somatic cells for essential, RAD51-dependent, repair of DNA double-strand-breaks (DSBs), but inappropriate HR can cause genome instability. A transcriptional transactivation-independent role for p53 in suppressing HR has been established, but is not detected in all HR assays. To address the basis of such exceptions, and the possibility that suppression by p53 may be discriminatory, we have conducted a controlled comparison of the effects of p53 depletion on three different kinds of HR. We show that, within the same cells, p53 depletion promotes both intra-chromosomal HR (ICHR) and extra-chromosomal HR (ECHR), but not homologous DNA integration (gene targeting; GT). This conclusion holds true for both spontaneous and DSB-induced ICHR and GT. We show further that non-conservative ICHR is more susceptible than conservative ICHR to inhibition by p53. These results provide strong evidence that p53 can discriminate between different forms of HR and, despite the fact that GT is used experimentally for gene disruption, is consistent with the possibility that p53 preferentially suppresses genome-destabilizing forms of HR. While the mechanism of suppression by p53 remains unclear, our data suggest that it is independent of mismatch repair and of changes in RAD51 protein levels.

Base Pair Mismatch↗
PubMed2003

Construction of a retroviral vector production system with the minimum possibility of a homologous recombination.

A recombination between the short homologous regions of nucleotide sequences in the retroviral vector and packaging cell line has been thought to be a major cause of the production of replication-competent retrovirus (RCR). Therefore, the removal of overlapping sequences between the vector and the packaging constructs is crucial for minimizing the possibility of homologous recombination, and therefore, the production of RCR. We have recently constructed a series of retroviral vectors that contain no viral coding sequences, but still produce high viral titer and high-level gene expression. However, many previously constructed murine leukemia virus (MLV)-based packaging constructs contained significantly long 5' and/or 3' untranslated regions of MLV, which are also present in the retroviral vector, and as such could possibly lead to homologous recombination. To make a retroviral production system that is free from homologous recombination, we constructed expression plasmids for gag-pol and env, precisely starting from the start codon and ending at the stop codon of respective open reading frames. When the packaging function was provided from one plasmid, a vector containing bits of all three viral coding sequences produced RCR at a significant frequency, while our vector remained free of any RCR. Our retrovirus production system is anticipated to have the minimum possible frequency of RCR production due to the elimination of potential sites for homologous recombination. Based on these results, a highly efficient new packaging line Vamp that contains no overlapping sequences with our retroviral vector was also developed.

Base Sequence↗
PubMed2005

Srs2 and RecQ homologs cooperate in mei-3-mediated homologous recombination repair of Neurospora crassa.

Homologous recombination and post-replication repair facilitate restart of stalled or collapsed replication forks. The SRS2 gene of Saccharomyces cerevisiae encodes a 3'-5' DNA helicase that functions both in homologous recombination repair and in post-replication repair. This study identifies and characterizes the SRS2 homolog in Neurospora crassa, which we call mus-50. A knockout mutant of N.crassa, mus-50, is sensitive to several DNA-damaging agents and genetic analyses indicate that it is epistatic with mei-3 (RAD51 homolog), mus-11 (RAD52 homolog), mus-48 (RAD55 homolog) and mus-49 (RAD57 homolog), suggesting a role for mus-50 in homologous recombination repair. However, epistasis evidence has presented that MUS50 does not participate in post-replication repair in N.crassa. Also, the N.crassa mus-25 (RAD54 homolog) mus-50 double mutant is viable, which is in contrast to the lethal phenotype of the equivalent rad54 srs2 mutant in S.cerevisiae. Tetrad analysis revealed that mus-50 in combination with mutations in two RecQ homologs, qde-3 and recQ2, is lethal, and this lethality is suppressed by mutation in mei-3, mus-11 or mus-25. Evidence is also presented for the two independent pathways for recovery from camptothecin-induced replication fork arrest: one pathway is dependent on QDE3 and MUS50 and the other pathway is dependent on MUS25 and RECQ2.

DNA Helicases↗
PubMed2003

Homologous recombination in human embryonic stem cells.

Homologous recombination applied to mouse embryonic stem (ES) cells has revolutionized the study of gene function in mammals. Although most often used to generate knockout mice, homologous recombination has also been applied in mouse ES cells allowed to differentiate in vitro. Homologous recombination is an essential technique if human ES cells are to fulfill their promise as a basic research tool. It also has important implications for ES cell-based transplantation and gene therapies. Significant differences between mouse and human ES cells have hampered the development of homologous recombination in human ES cells. High, stable transfection efficiencies in human ES cells have been difficult to achieve, and, in particular, electroporation protocols established for mouse ES cells work poorly in human ES cells. Also, in contrast to their murine counterparts, human ES cells cannot be cloned efficiently from single cells, making it difficult to screen for rare recombination events. Here we report an electroporation approach, based on the physical characteristics of human ES cells, that we used to successfully target HPRT1, the gene encoding hypoxanthine phosphoribosyltransferase-1 (HPRT1), and POU5F1, the gene encoding octamer-binding transcription factor 4 (Oct4; also known as POU domain, class 5, transcription factor 1 (POU5F1)).

DNA-Binding Proteins↗
PubMed2005

The dual nature of homologous recombination in plants.

Homologous recombination creates covalent linkages between DNA in regions of highly similar or identical sequence. Recent results from several laboratories, many of them based on forward and reverse genetics in Arabidopsis, give insights into the mechanisms of the enzymatic machinery and the involvement of chromatin in somatic and meiotic DNA recombination. Also, signaling pathways and interconnections between repair pathways are being discovered. In addition, recent work shows that biotic and abiotic influences from the environment can dramatically affect plant genomes. The resulting changes in the DNA sequence, exerted at the level of somatic or meiotic tissue, might contribute to evolution.

Arabidopsis↗
PubMed2006

Disparate requirements for the Walker A and B ATPase motifs of human RAD51D in homologous recombination.

In vertebrates, homologous recombinational repair (HRR) requires RAD51 and five RAD51 paralogs (XRCC2, XRCC3, RAD51B, RAD51C and RAD51D) that all contain conserved Walker A and B ATPase motifs. In human RAD51D we examined the requirement for these motifs in interactions with XRCC2 and RAD51C, and for survival of cells in response to DNA interstrand crosslinks (ICLs). Ectopic expression of wild-type human RAD51D or mutants having a non-functional A or B motif was used to test for complementation of a rad51d knockout hamster CHO cell line. Although A-motif mutants complement very efficiently, B-motif mutants do not. Consistent with these results, experiments using the yeast two- and three-hybrid systems show that the interactions between RAD51D and its XRCC2 and RAD51C partners also require a functional RAD51D B motif, but not motif A. Similarly, hamster Xrcc2 is unable to bind to the non-complementing human RAD51D B-motif mutants in co-immunoprecipitation assays. We conclude that a functional Walker B motif, but not A motif, is necessary for RAD51D's interactions with other paralogs and for efficient HRR. We present a model in which ATPase sites are formed in a bipartite manner between RAD51D and other RAD51 paralogs.

Adenosine Triphosphatases↗
PubMed1987

Disruption of the Dictyostelium myosin heavy chain gene by homologous recombination.

The phenomenon of homologous recombination, which allows specific gene conversion and gene insertion, can be a powerful system for the study of eukaryotic cell biology. Data are presented demonstrating that integration of a transfected plasmid by homologous recombination occurs in the motile eukaryotic cell Dictyostelium discoideum. A plasmid carrying a G418 resistance gene and the amino terminal half of the myosin heavy chain gene was used to transfect Dictyostelium. A large fraction of the resultant G418-resistant cells had the plasmid integrated into the single genomic copy of the heavy chain gene. These cells, which fail to express the native myosin but express the myosin fragment, are defective in cytokinesis and become large and multinucleate. In spite of the absence of native myosin, these cells, termed hmm cells, exhibit many forms of cell movement, including membrane ruffling, phagocytosis, and chemotaxis. The hmm cells can aggregate but are blocked at a later stage in the Dictyostelium developmental cycle. The hmm cells revert to the wild-type phenotype. Reversion of the hmm phenotype is due to excision and loss of the transforming plasmid. The revertant cells express native myosin, are G418 sensitive, and have a normal developmental cycle. These results constitute genetic proof that the intact myosin molecule is required for cytokinesis and not for karyokinesis.

Cell Division↗