RAD52-dependent and -independent homologous recombination initiated by Flp recombinase at a single FRT site flanked by direct repeats

We have devised a system for isolating yeast DNA sequences that are able to act as initiators of recombination leading to deletions in mitotically growing yeast cells. This system has allowed us to identify the FRT site of the 2μ site-specific recombinase Flp as such a sequence. We show that Flp is able to initiate recombination leading to deletions at a single FRT site in cir ^o strains. These results indicate that Flp is able to cleave a single FRT site, supporting the observation that the mechanism of cleavage by Flp is trans-horizontal. Interestingly, Flp can induce homologous recombination in both a RAD52-dependent and RAD52-independent manner. Our work provides a new system for the study of homologous recombination leading to deletions, in which the initiation step can be efficiently controlled. We discuss the possibility that Flp-induced, RAD52-independent events occur by single-strand annealing. Received: 20 July 1999 / Accepted: 27 October 1999     

The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts

Summary A mutant Gin recombinase of the phage Mu DNA inversion system was successfully expressed in Arabidopsis thaliana and tobacco protoplasts. Site-specific recombination was monitored both physically and biologically with the help of a recombination assay system in which expression of a -glucuronidase (gus) gene requires Gin-mediated recombination. We demonstrate that the wild-type Gin protein is not able to promote recombination in plant protoplasts, presumably because plant cells do not contain a protein that can substitute for the Escherichia coli FIS protein needed for full activity of wild-type Gin in E. coli. A FIS-independent Gin mutant protein on the other hand was efficient in promoting recombination on recombination substrates introduced transiently and on substrates stably integrated into the plant genome. We discuss the various advantages this system can provide for genetic manipulation of plant cells.     

The interaction profile of homologous recombination repair proteins RAD51C,RAD51D and XRCC2 as determined by proteomic analysis

The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co‐precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1‐D SDS‐PAGE of co‐precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co‐precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate HR repair mechanisms.     

Role of RAD51AP1 in homologous recombination DNA repair and carcinogenesis

Homologous recombination (HR) serves to repair DNA double-strand breaks and damaged replication forks and is essential for maintaining genome stability and tumor suppression. HR capacity also determines the efficacy of anticancer therapy. Hence, there is an urgent need to better understand all HR proteins and sub-pathways. An emerging protein that is critical for RAD51-mediated HR is RAD51-associated protein 1 (RAD51AP1). Although much has been learned about its biochemical attributes, the precise molecular role of RAD51AP1 in the HR reaction is not yet fully understood. The available literature also suggests that RAD51AP1 expression may be relevant for cancer development and progression. Here, we review the efforts that led to the discovery of RAD51AP1 and elaborate on our current understanding of its biochemical profile and biological function. We also discuss how RAD51AP1 may help to promote cancer development and why it could potentially represent a promising new target for therapeutic intervention.     

Over-expression of RAD51 or RAD54 but not RAD51/4 enhances extra-chromosomal homologous recombination in the human sarcoma (HT-1080) cell line

RAD51 and RAD54, members of the RAD52 epistasis group, play key roles in homologous recombination (HR). The efficiency of homologous recombination (HR) can be increased by over-expression of either of them. A vector that allows co-expression of RAD51 and RAD54 was constructed to investigate interactions between the two proteins during extra-chromosomal HR. The efficiency of extra-chromosomal HR evaluated by GFP extra-chromosomal HR was enhanced (110-245%) in different transfected Human sarcoma (HT-1080) cell colonies. We observed that RAD51 clearly promotes extra-chromosomal HR; however, the actions of RAD54 in extra-chromosomal HR were weak. Our data suggest that RAD51 may function as a universal factor during HR, whereas RAD54 mainly functions in other types of HR (gene targeting or intra-chromosomal HR), which involves interaction with chromosomal DNA.     

Regulation of homologous recombination at telomeres in budding yeast

Homologous recombination is suppressed at normal length telomere sequences. In contrast, telomere recombination is allowed when telomeres erode in the absence of telomerase activity or as a consequence of nucleolytic degradation or incomplete replication. Here, we review the mechanisms that contribute to regulating mitotic homologous recombination at telomeres and the role of these mechanisms in signalling short telomeres in the budding yeast Saccharomyces cerevisiae.     

RadB acts in homologous recombination in the archaeon Haloferax volcanii,consistent with a role as recombination mediator

Homologous recombination plays a central role in the repair of double-strand DNA breaks, the restart of stalled replication forks and the generation of genetic diversity. Regulation of recombination is essential since defects can lead to genome instability and chromosomal rearrangements. Strand exchange is a key step of recombination – it is catalysed by RecA in bacteria, Rad51/Dmc1 in eukaryotes and RadA in archaea. RadB, a paralogue of RadA, is present in many archaeal species. RadB has previously been proposed to function as a recombination mediator, assisting in RadA-mediated strand exchange. In this study, we use the archaeon Haloferax volcanii to provide evidence to support this hypothesis. We show that RadB is required for efficient recombination and survival following treatment with DNA-damaging agents, and we identify two point mutations in radA that suppress the ΔradB phenotype. Analysis of these point mutations leads us to propose that the role of RadB is to act as a recombination mediator, which it does by inducing a conformational change in RadA and thereby promoting its polymerisation on DNA.     

Construction of a novel kind of expression plasmid by homologous recombination in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Based on a previously used plasmid pHC11, a new plasmid pHC11R was constructed. Cutting plasmid pHC11R with proper restriction enzymes, the resulting larger DNA fragment pHC11R’ was co-transformed with a PCR amplified expression cassette of human IFNα2b into yeast. By means of the homologous sequences at both ends of two DNA fragments, a novel expression plasmid pHC11R-IFNα2b was formed via homologous recombination in the yeast. Compared with pHC11-IFNα2b, the expression plasmid pHC11R-IFNα2b was smaller in size and in absence of antibiotic resistant gene. The stability and copy number of pHC11R-IFNα2b were greatly increased and the expression level of heterologous protein was improved. As the derivatives of pHC11R, a series of recombination expression vectors pHRs containing different combination of expression elements were developed. This led to a rapid and powerful method for cloning and expressing of different genes in yeast.     

Homologous recombination and transposition generate chromosome I neopolymorphism during meiosis in Saccharomyces cerevisiae

We have studied the meiotic segregation of a chromosome length polymorphism (CLP) in the yeast Saccharomyces cerevisiae. The neopolymorphism frequently observed within the smallest chromosomes (I, VI, III and IX) is not completely understood. We focused on the analysis of the structure of chromosome I in 88 segregants from a cross between YNN295 and FL100trp. Strain FL100trp is known to carry a reciprocal translocation between the left arm of chromosome III and the right arm of chromosome I. PCR and Southern hybridization analyses were performed and a method for the rapid detection of chromosome I rearrangements was developed. Seven chromosome I types were identified among the 88 segregants. We detected 22 recombination events between homologous chromosomes I and seven ectopic recombination events between FL100trp chromosome III and YNN295 chromosome I. These recombination events occurred in 20 of the 22 tetrads studied (91%). Nine tetrads (41%) showed two recombination events. This showed that homologous recombination involving polymorphic homologues or heterologous chromosomes is the main source of neopolymorphism. Only one of the seven chromosome I variants resulted from a transposition event rather than a recombination event. We demonstrated that a Ty1 element had transposed within the translocated region of chromosome I, generating mutations in the 3′ LTR, at the border between U5 and PBS. Received: 7 May 1999 / Accepted: 14 February 2000     

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. Received: 22 July 1997 / Accepted: 25 February 1998     

Construction of a novel kind of expression plasmid by ho-mologous recombination in Saccharomyces cerevisiae

Saccharomyces cerevisiae is one of the most im- portant heterologous expression systems. The stability and copy number of expression plasmid in the host are the important factors to affect the expression levels of foreign genes[1―3]. pHC11 is a yeast episomal plasmid constructed by our laboratory[4]. It contains the entire sequence of the 2μ plasmid without disrupting its coding elements and other functional regions. The stability and copy number of pHC11 are relatively high. Making use of…     

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR.     

Extensive homologous recombination in classical swine fever virus: A re-evaluation of homologous recombination events in the strain AF407339

In this short report, the genome-wide homologous recombination events were re-evaluated for classical swine fever virus (CSFV) strain {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339. We challenged a previous study which suggested only one recombination event in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 based on 25 CSFV genomes. Through our re-analysis on the 25 genomes in the previous study and the 41 genomes used in the present study, we argued that there should be possibly at least two clear recombination events happening in {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339 through genome-wide scanning. The reasons for identifying only one recombination event in the previous study might be due to the limited number of available CSFV genome sequences at that time and the limited usage of detection methods. In contrast, as identified by most detection methods using all available CSFV genome sequences, two major recombination events were found at the starting and ending zones of the genome {"type":"entrez-nucleotide","attrs":{"text":"AF407339","term_id":"15488012","term_text":"AF407339"}}AF407339, respectively. The first one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF333000","term_id":"13383931","term_text":"AF333000"}}AF333000 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"AY554397","term_id":"49860681","term_text":"AY554397"}}AY554397 (major) with beginning and ending breakpoints located at 19 and 607 nt of the genome respectively. The second one has two parents {"type":"entrez-nucleotide","attrs":{"text":"AF531433","term_id":"22347661","term_text":"AF531433"}}AF531433 (minor) and {"type":"entrez-nucleotide","attrs":{"text":"GQ902941","term_id":"298113017","term_text":"GQ902941"}}GQ902941 (major) with beginning and ending breakpoints at 8397 and 11,078 nt of the genome respectively. Phylogenetic incongruence analysis using neighbor-joining algorithm with 1000 bootstrapping replicates further supported the existence of these two recombination events. In addition, we also identified additional 18 recombination events on the available CSFV strains. Some of them may be trivial and can be ignored. In conclusion, CSFV might have relatively high frequency of homologous recombination events. Genome-wide scanning of identifying recombination events should utilize multiple detection methods so as to reduce the risk of misidentification.     

Molecular cloning in yeast by in vivo homologous recombination of the yeast putative alpha1 subunit of the voltage-gated calcium channel

Saccharomyces cerevisiae has only one gene encoding a putative voltage-gated Ca2+ channel pore-forming subunit, CCH1, which is not possible to be cloned by conventional molecular cloning techniques using Escherichia coli. Here, we report the successful cloning of CCH1 in yeast by in vivo homologous recombination without using E. coli. Overexpression of the cloned CCH1 or MID1 alone, which encodes a putative stretch-activated Ca2+ channel component, does not increase Ca2+ uptake activity, but co-overexpression results in a 2- to 3-fold increase. Overexpression of CCH1 does not substantially complement the lethality and low Ca2+ uptake activity of a mid1 mutant and vice versa. These results indicate that co-overproduction of Cch1 and Mid1 is sufficient to increase Ca2+ uptake activity.     

Effects of XRCC2 and RAD51B mutations on somatic and meiotic recombination in Arabidopsis thaliana

Homologous recombination is key to the maintenance of genome integrity and the creation of genetic diversity. At the mechanistic level, recombination involves the invasion of a homologous DNA template by broken DNA ends, repair of the break and exchange of genetic information between the two DNA molecules. Invasion of the template in eukaryotic cells is catalysed by the RAD51 and DMC1 recombinases, assisted by a number of accessory proteins, including the RAD51 paralogues. Eukaryotic genomes encode a variable number of RAD51 paralogues, ranging from two in yeast to five in animals and plants. The RAD51 paralogues form at least two distinct protein complexes, believed to play roles in the assembly and stabilization of the RAD51‐DNA nucleofilament. Somatic recombination assays and immunocytology confirm that the three ‘non‐meiotic’ paralogues of Arabidopsis, RAD51B, RAD51D and XRCC2, are involved in somatic homologous recombination, and that they are not required for the formation of radioinduced RAD51 foci. Given the presence of all five proteins in meiotic cells, the apparent absence of a meiotic role for RAD51B, RAD51D and XRCC2 is surprising, and perhaps simply the result of a more subtle meiotic phenotype in the mutants. Analysis of meiotic recombination confirms this, showing that the absence of XRCC2, and to a lesser extent RAD51B, but not RAD51D, increases rates of meiotic crossing over. The roles of RAD51B and XRCC2 in recombination are thus not limited to mitotic cells.     

S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is an essential process in maintenance of chromosomal stability. A key player of HR is the strand exchange factor RAD51 whose assembly at sites of DNA damage is tightly regulated. We detected an endogenous complex of RAD51 with the calcium-binding protein S100A11, which is localized at sites of DNA repair in HaCaT cells as well as in normal human epidermal keratinocytes (NHEK) synchronized in S phase. In biochemical assays, we revealed that S100A11 enhanced the RAD51 strand exchange activity. When cells expressing a S100A11 mutant lacking the ability to bind Ca²⁺, a prolonged persistence of RAD51 in repair sites and nuclear γH2AX foci was observed suggesting an incomplete DNA repair. The same phenotype became apparent when S100A11 was depleted by RNA interference. Furthermore, down-regulation of S100A11 resulted in both reduced sister chromatid exchange confirming the restriction of the recombination capacity of the cells, and in an increase of chromosomal aberrations reflecting the functional requirement of S100A11 for the maintenance of genomic stability. Our data indicate that S100A11 is involved in homologous recombination by regulating the appearance of RAD51 in DSB repair sites. This function requires the calcium-binding activity of S100A11.