Identification and characterization of the RAD51 gene from the ciliate Tetrahymena thermophila.

The RAD51 gene is a eukaryotic homolog of rec A, a critical component in homologous recombination and DNA repair pathways in Escherichia coli . We have cloned the RAD51 homolog from Tetrahymena thermophila , a ciliated protozoan. Tetrahymena thermophila RAD51 encodes a 36.3 kDa protein whose amino acid sequence is highly similar to representative Rad51 homologs from other eukaryotic taxa. Recombinant Rad51 protein was purified to near homogeneity following overproduction in a bacterial expression system. The purified protein binds to both single- and double-stranded DNA, possesses a DNA-dependent ATPase activity and promotes intermolecular ligation of linearized plasmid DNA. While steady-state levels of Rad51 mRNA are low in normally growing cells, treatment with UV light resulted in a >100-fold increase in mRNA levels. This increase in mRNA was time dependent, but relatively independent of UV dose over a range of 1400-5200 J/m2. Western blot analysis confirmed that Rad51 protein levels increase upon UV irradiation. Exposure to the alkylating agent methyl methane sulfonate also resulted in substantially elevated Rad51 protein levels in treated cells, with pronounced localization in the macronucleus. These data are consistent with the hypothesis that ciliates such as T.thermophila utilize a Rad51-dependent pathway to repair damaged DNA.     

SUMO modification of Rad22, the Schizosaccharomyces pombe homologue of the recombination protein Rad52

The Schizosaccharomyces pombe rad31 and hus5 genes are required for the DNA damage response, as mutants defective in these genes are sensitive to DNA damaging agents, such as UV and ionising radiation and to the DNA synthesis inhibitor hydroxyurea (HU). Sequence analysis has suggested that rad31 and hus5 encode components of the Pmt3 (SUMO) modification process in S.pombe. We show here that the rad31 null and hus5.62 mutants display reduced levels of Pmt3 modification. We have initiated a search for proteins required for the DNA damage response, which may be modified by Pmt3 and have identified Rad22, the fission yeast homologue of the recombination protein Rad52. Purification of myc + His-tagged Rad22 protein from cells expressing HA-tagged Pmt3 identifies an 83 kDa species which cross-reacts with anti-HA antisera. We show here that Rad22 interacts with Rhp51 and Rpa70 (the fission yeast homologues of Rad51 and the large subunit of RPA, respectively), but that neither of these proteins appears to be responsible for the 83 kDa species. The 83 kDa species is observed when extracts are prepared under both native and denaturing conditions, and is also observed when myc + His-tagged Rad22 and Pmt3 are expressed at wild type levels, suggesting that Rad22 is modified by Pmt3 in vivo. We have established an S.pombe in vitro Pmt3 modification system and have shown that Rad22 and Rhp51 are modified in vitro, but that Rpa70 is not.     

An archaeal Rad54 protein remodels DNA and stimulates DNA strand exchange by RadA

Rad54 protein is a key member of the RAD52 epistasis group required for homologous recombination in eukaryotes. Rad54 is a duplex DNA translocase that remodels both DNA and protein–DNA complexes, and functions at multiple steps in the recombination process. Here we use biochemical criteria to demonstrate the existence of this important protein in a prokaryotic organism. The Sulfolobus solfataricus Rad54 (SsoRad54) protein is a double-strand DNA-dependent ATPase that can alter the topology of duplex DNA. Like its eukaryotic homolog, it interacts directly with the S. solfataricus Rad51 homologue, SsoRadA, to stimulate DNA strand exchange. Confirmation of this protein as an authentic Rad54 homolog establishes an essential phylogenetic bridge for identifying Rad54 homologs in the archaeal and bacterial domains.     

Roles of Rad51 paralogs for promoting homologous recombination in Leishmania infantum

To achieve drug resistance Leishmania parasite alters gene copy number by using its repeated sequences widely distributed through the genome. Even though homologous recombination (HR) is ascribed to maintain genome stability, this eukaryote exploits this potent mechanism driven by the Rad51 recombinase to form beneficial extrachromosomal circular amplicons. Here, we provide insights on the formation of these circular amplicons by analyzing the functions of the Rad51 paralogs. We purified three Leishmania infantum Rad51 paralogs homologs (LiRad51-3, LiRad51-4 and LiRad51-6) all of which directly interact with LiRad51. LiRad51-3, LiRad51-4 and LiRad51-6 show differences in DNA binding and annealing capacities. Moreover, it is also noteworthy that LiRad51-3 and LiRad51-4 are able to stimulate Rad51-mediated D-loop formation. In addition, we succeed to inactivate the LiRad51-4 gene and report a decrease of circular amplicons in this mutant. The LiRad51-3 gene was found to be essential for cell viability. Thus, we propose that the LiRad51 paralogs play crucial functions in extrachromosomal circular DNA amplification to circumvent drug actions and preserve survival.     

Human and Mouse Homologs of theSchizosaccharomyces pombe rad17+Cell Cycle Checkpoint Control Gene

TheSchizosaccharomyces pombe rad17⁺cell cycle checkpoint control gene is required for S-phase and G2/M arrest in response to both DNA damage and incomplete DNA replication. We isolated and characterized the putative human (RAD17Sp) and mouse (mRAD17Sp) homologs of theS. pombeRad17 (Rad17Sp) protein. The humanRAD17Spopen reading frame (ORF) encodes a protein of 681 amino acids; themRAD17SpORF codes for a protein of 688 amino acids. ThemRAD17Spmessenger is highly expressed in the testis as a single 3-kb mRNA species. The human RAD17Sp and mRAD17Sp proteins are 24% identical and 46% similar to theS.pombeRad17Sp protein. Sequence homology was also noted with theSaccharomyces cerevisiaeRad24Sc (which is the structural counterpart ofS.pombeRad17Sp) and structurally related polypeptides fromCaenorhabditis elegans, Arabidopsis thaliana, Pyrococcus horikoshii,andDrosophila melanogaster.The degree of conservation between the mammalian RAD17Sp proteins and those of the other species is consistent with the evolutionary distance between the species, indicating that these proteins are most likely true counterparts. In addition, homology was found between the Rad17Sp homologs and proteins identified as components of mammalian replication factor C (RF-C)/activator 1, especially in several highly conserved RF-C-like domains including a “Walker A” motif. Using FISH and analysis of a panel of rodent–human cell hybrids, the humanRAD17Spgene (HGMW-approved symbolRAD17could be localized on human chromosome 5q13–q14, a region implicated in the etiology of small cell lung carcinoma, non-small-cell lung carcinoma, duodenal adenocarcinoma, and head and neck squamous cell carcinoma. Our results suggest that the structure and function of the checkpoint “rad” genes in the G2/M checkpoint pathway are evolutionary conserved between yeast and higher eukaryotes.     

Coordinated response of mammalian Rad51 and Rad52 to DNA damage

Biochemical analysis has shown that mammalian Rad51 and Rad52 interact and synergize in DNA recombination reactions in vitro, but these proteins have not been shown to function together in response to DNA damage in vivo. By analysis of murine cells expressing murine Rad52 tagged with green fluorescent protein (GFP)–Rad52, we now show that DNA damage causes Rad51 and GFP–Rad52 to colocalize in distinct nuclear foci. Cells expressing GFP–Rad52 show both increased survival and an increased number of Rad51 foci, raising the possibility that Rad52 is limiting for repair. These observations provide evidence of coordinated function of Rad51 and Rad52 in vivo and support the hypothesis that Rad52 plays an important role in the DNA damage response in mammalian cells.     

RAD54 controls access to the invading 3′-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

Rad51 is a key protein in homologous recombination performing homology search and DNA strand invasion. After DNA strand exchange Rad51 protein is stuck on the double-stranded heteroduplex DNA product of DNA strand invasion. This is a problem, because DNA polymerase requires access to the invading 3′-OH end to initiate DNA synthesis. Here we show that, the Saccharomyces cerevisiae dsDNA motor protein Rad54 solves this problem by dissociating yeast Rad51 protein bound to the heteroduplex DNA after DNA strand invasion. The reaction required species-specific interaction between both proteins and the ATPase activity of Rad54 protein. This mechanism rationalizes the in vivo requirement of Rad54 protein for the turnover of Rad51 foci and explains the observed dependence of the transition from homologous pairing to DNA synthesis on Rad54 protein in vegetative and meiotic yeast cells.     

Rad10 exhibits lesion-dependent genetic requirements for recruitment to DNA double-strand breaks in Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, the Rad1–Rad10 protein complex participates in nucleotide excision repair (NER) and homologous recombination (HR). During HR, the Rad1–Rad10 endonuclease cleaves 3′ branches of DNA and aberrant 3′ DNA ends that are refractory to other 3′ processing enzymes. Here we show that yeast strains expressing fluorescently labeled Rad10 protein (Rad10-YFP) form foci in response to double-strand breaks (DSBs) induced by a site-specific restriction enzyme, I-SceI or by ionizing radiation (IR). Additionally, for endonuclease-induced DSBs, Rad10-YFP localization to DSB sites depends on both RAD51 and RAD52, but not MRE11 while IR-induced breaks do not require RAD51. Finally, Rad10-YFP colocalizes with Rad51-CFP and with Rad52-CFP at DSB sites, indicating a temporal overlap of Rad52, Rad51 and Rad10 functions at DSBs. These observations are consistent with a putative role of Rad10 protein in excising overhanging DNA ends after homology searching and refine the potential role(s) of the Rad1–Rad10 complex in DSB repair in yeast.     

Rad51 and Rad54 ATPase activities are both required to modulate Rad51-dsDNA filament dynamics

Rad51 and Rad54 are key proteins that collaborate during homologous recombination. Rad51 forms a presynaptic filament with ATP and ssDNA active in homology search and DNA strand exchange, but the precise role of its ATPase activity is poorly understood. Rad54 is an ATP-dependent dsDNA motor protein that can dissociate Rad51 from dsDNA, the product complex of DNA strand exchange. Kinetic analysis of the budding yeast proteins revealed that the catalytic efficiency of the Rad54 ATPase was stimulated by partial filaments of wild-type and Rad51-K191R mutant protein on dsDNA, unambiguously demonstrating that the Rad54 ATPase activity is stimulated under these conditions. Experiments with Rad51-K191R as well as with wild-type Rad51-dsDNA filaments formed in the presence of ATP, ADP or ATP-γ-S showed that efficient Rad51 turnover from dsDNA requires both the Rad51 ATPase and the Rad54 ATPase activities. The results with Rad51-K191R mutant protein also revealed an unexpected defect in binding to DNA. Once formed, Rad51-K191R-DNA filaments appeared normal upon electron microscopic inspection, but displayed significantly increased stability. These biochemical defects in the Rad51-K191R protein could lead to deficiencies in presynapsis (filament formation) and postsynapsis (filament disassembly) in vivo.     

Production and characterization of polyclonal antibody against recombinant ORF 049L of rock bream (Oplegnathus fasciatus) iridovirus

Rock bream iridovirus (RBIV) is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. The structure of the isolated RBIV was observed by an electron microscope, and the virus particles were icosahedral and 120–130 nm in diameter. From the complete genomic DNA sequence of RBIV, the protein encoded in ORF 049L (RBIV-049L) was selected and the property of protein was evaluated with the transmembrane sequence TMHMM 2.0 tool. The ORF 049L gene of RBIV (RBIV-049L) was cloned into pGEX-4T-1 expression vector. The recombinant RBIV-049L was overexpressed in Escherichia coli BL21 (DE3) as a fusion protein (GST-049L, 42 kDa) with a glutathione S-transferase. Antiserum against this recombinant GST-049L protein was prepared in mouse. Dot blot analysis was carried out to identify the reaction abilities and sensitivity of anti-RBIV-049L polyclonal antibody to RBIV-infected rock bream with enzyme linked immunosorbent assay (ELISA) and one-step PCR. These novel RBIV-049L protein and anti-RBIV-049L polyclonal antibody will facilitate the development of more specific and standardized diagnostic techniques.     

The Crithidia fasciculata RNH1 gene encodes both nuclear and mitochondrial isoforms of RNase H

The Crithidia fasciculata RNH1 gene encodes an RNase H, an enzyme that specifically degrades the RNA strand of RNA–DNA hybrids. The RNH1 gene is contained within an open reading frame (ORF) predicted to encode a protein of 53.7 kDa. Previous work has shown that RNH1 expresses two proteins: a 38 kDa protein and a 45 kDa protein which is enriched in kinetoplast extracts. Epitope tagging of the C-terminus of the RNH1 gene results in localization of the protein to both the kinetoplast and the nucleus. Translation of the ORF beginning at the second in-frame methionine codon predicts a protein of 38 kDa. Insertion of two tandem stop codons between the first ATG codon and the second in-frame ATG codon of the ORF results in expression of only the 38 kDa protein and the protein localizes specifically to the nucleus. Mutation of the second methionine codon to a valine codon prevents expression of the 38 kDa protein and results in exclusive production of the 45 kDa protein and localization of the protein only in the kinetoplast. These results suggest that the kinetoplast enzyme results from processing of the full-length 53.7 kDa protein. The nuclear enzyme appears to result from translation initiation at the second in-frame ATG codon. This is the first example in trypanosomatids of the production of nuclear and mitochondrial isoforms of a protein from a single gene and is the only eukaryotic gene in the RNase HI gene family shown to encode a mitochondrial RNase H.     

Rad51 gain-of-function mutants that exhibit high affinity DNA binding cause DNA damage sensitivity in the absence of Srs2

We previously identified several rad51 gain-of-function alleles that partially suppress the requirement for RAD55 and RAD57 in DNA repair. To gain further insight into the mechanism of action of these alleles, we compared the activities of Rad51-V328A, Rad51-P339S and Rad51-I345T with wild-type Rad51, for DNA binding, filament stability, strand exchange and interaction with the antirecombinase helicase, Srs2. These alleles were chosen because they show the highest activity in suppression of ionizing radiation sensitivity of the rad57 mutant, and Val 328 and Ile 345 are conserved in the human Rad51 protein. All three mutant proteins exhibited higher affinity for single-stranded DNA (ssDNA) and showed more robust strand exchange activity with oligonucleotide substrates than wild-type Rad51, with the Rad51-I345T and Rad51-V328A proteins displaying higher activity than Rad51-P339S. However, the Srs2 antirecombinase was able to disrupt Rad51–ssDNA complexes formed with all the mutant proteins. In vivo, the rad51-I345T mutant strain exhibited high resistance to methyl methane sulfonate that was dependent on functional SRS2. These results suggest the Srs2 translocase is able to disrupt Rad51–ssDNA complexes at stalled replication forks, but in the absence of Srs2 the enhanced DNA binding of the Rad51-I345T protein is detrimental to cell survival.     

Genetic re-engineering of Saccharomyces cerevisiae RAD51 leads to a significant increase in the frequency of gene repair in vivo

Oligonucleotides can be used to direct the alteration of single nucleotides in chromosomal genes in yeast. Rad51 protein appears to play a central role in catalyzing the reaction, most likely through its DNA pairing function. Here, we re-engineer the RAD51 gene in order to produce proteins bearing altered levels of known activities. Overexpression of wild-type ScRAD51 elevates the correction of an integrated, mutant hygromycin resistance gene ~3-fold. Overexpression of an altered RAD51 gene, which encodes a protein that has a higher affinity for ScRad54, enhances the targeting frequency nearly 100-fold. Another mutation which increases the affinity of Rad51 for DNA was also found to increase gene repair when overexpressed in the cell. Other mutations in the Rad51 protein, such as one that reduces interaction with Rad52, has little or no effect on the frequency of gene repair. These data provide the first evidence that the Rad51 protein can be modified so as to increase the frequency of gene repair in yeast.     

The Caenorhabditis elegansRAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes

In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule. Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms. We have cloned the homolog of RAD51 in Caenorhabditis elegans. The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively. We discuss the evolutionary implications of these findings.     

Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein

The Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.     

Role of the Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised amino-terminal DNA binding domain, is capable of Rad51 delivery to DNA but is deficient in DNA annealing. Results from chromatin immunoprecipitation experiments find that rad52-R70A associates with DNA double-strand breaks and promotes recruitment of Rad51 as efficiently as wild-type Rad52. Analysis of gene conversion intermediates reveals that rad52-R70A cells can mediate DNA strand invasion but are unable to complete the recombination event. These results provide evidence that DNA binding by the evolutionarily conserved amino terminus of Rad52 is needed for the capture of the second DNA end during homologous recombination.