C Rinaldo et al. (1999) International C. elegans Meeting "Ce-hcr-51 gene, the RAD51/DMC1 homolog in Caenorhabditis elegans: a functional characterization"

P Bazzicalupo, S Ederle, M Mastrogiacomo, M Hilliard, A La Volpe, C Rinaldo

[International C. elegans Meeting, 1999]

We have cloned a RAD51/DMC1 homolog in Caenorhabditis elegans , we named Ce-hcr-51 for h omolog of Saccharomyces c erevisiae R AD 51 . This locus, we mapped on chromosome IV on the left of the mec-3 gene on fosmid H36F17, is transcribed into two alternative mRNAs potentially coding for proteins of 395 (Hcr51L) and 357 (Hcr51S) aminoacids in length (Rinaldo et al. 1998). Experiments of RNA interference show that the inactivation of the locus results in adult hermaphrodite sterility of the F1. The F2 embryos show gross abnormality, develop to different stages, but they do not hatch, suggesting a meiotic defect leading to aneuploidy. We are also analysing the gene expression pattern by injecting the promoter sequences upstream the GFP/LacZ coding sequences, and by RNA analysis. We are using a two hybrid system assay to study the ability of the putative products of hrc-51 to form homodymers and heterodymers in vivo . We identified two regions of the Hrc51S protein involved in the homodymerization process, one at the aminoterminal corresponding at the homodymerization domain already mapped in the yeast Rad51 protein (Donovan et al. 1994), and another located in the carboxiterminal half of Hrc-51 that seems to play a role either in the self-association activity or in stabilizing the protein. We are also studying proteins interacting with Ce-hcr-51 products. Donovan J. W., Milne G. T., and Weaver D. T. (1994) Genes Dev 8 : 2552-62. Rinaldo C., Ederle S., Rocco V., and Volpe A. L. (1998) Mol. Gen. Genet. 260 : 289-294.

Rinaldo C et al. (1998) Worm Breeder's Gazette "The Polymerisation Domain of CeRAD51"

Rinaldo C, La Volpe A

[Worm Breeder's Gazette, 1998]

The RAD51 gene, first characterised in yeast, is the eukaryotic homolog of the Escherichia coli recA gene; it mediates, in yeast as in mammals, DNA strand transfer leading to recombination and therefore is a key molecule in meiotic and mitotic recombination, and in double strand break (DBS) repair. Yeast cells mutated in RAD51 do not perform normal meiosis but are viable, while in mouse loss of function mutations in RAD51 are lethal in early embryogenesis. Mammalian cells, homozygous for a RAD51 mutation, show also defect in cell proliferation suggesting a link with cell cycle control. Much is known in yeast about the nature, function of RAD51 and of the proteins acting synergetically with it. We decide to study the C. elegans homologous protein, CeRAD51, to understand how the mechanisms of DBS repair and homologous recombination work in multicellular organisms, in both germline and somatic cells. In more complex multicellular organisms, such as mammals, overlapping and redundant tissues-specific pathways may very well hinder an understanding of the basic regulation circuits. We have recently cloned the complete cDNA coding for the C. elegans homologue of RAD51 (CeRAD51). The sequence analysis has shown a level of similarity of 62.3% (47.7% of identity) between the C. elegans and yeast RAD51 protein and a level of similarity of 72%(59% of identity) between CeRAD51 and the human homologue HsRAD51. The deduced aminoacid sequence in C. elegans is 395 aminoacids long (very similar to the length of the yeast protein) while HsRAD51 is of 339 aminoacid only. The genomic sequence corresponding to the CeRAD51 locus now available from the C. elegans Genome Sequencing Consortium, shows the presence of eight exons and seven introns in such gene. RAD51 monomers are known in yeast to form long concatamers to perform the strand transfer activity. We tested the ability of the C. elegans protein, coded by the first cDNA isolated (lacking the first 35 aminoacids), to form dimers in vivo in a "Two hybrid" analysis, demonstrating that the 35 aminoacids at the aminoterminal are not essential for homodimerisation. The complete coding sequence of CeRAD51 has been subcloned in an appropriate vector in frame with the binding domain of GAL4 to be used as "bait" in a two hybrid screening in yeast. We found 9 independent "target" protein clones from our library corresponding to the CeRAD51 coding sequence. All of them begin around aminoacids 36/38 and we did not select shorter clones. Therefore we hypothesised that the part of the protein essential for polymerisation was likely to be contained in the sequence just downstream aminoacid 38. We have recently restricted, by "two hybrid" analysis the minimal region of interaction, between aminoacid 38 and aminoacid 95.

Rinaldo C et al. (1998) European Worm Meeting "Molecular analysis of the Caenorhabditis elegans RAD51 gene"

Ederle S, Rinaldo C, La Volpe A

[European Worm Meeting, 1998]

We have cloned the cDNA coding for the Caenorhabditis elegans homologue of RAD51 (CeRAD51) in order to study its regulation in a well characterized metazoan model system as well as to identify possible species-specific features of regulation of recombination. The CeRAD51 gene is located on chromosome IV on the left of the mec-3 gene. The entire genomic locus is composed of 8 exons and 7 introns. By transcriptional analysis and cDNA cloning we identified two alternative mRNAs, both of them including the entire coding region conserved throughout evolution. The shorter transcript does not contain the first two exons and presents the trans-spliced leader SL1 at the 5' end. Because of the mechanics of trans-splicing, it is likely that the two transcripts of CeRAD51 use different promoters. The transcription of the shorter mRNA should begin within the second intron so that, in absence of the donor site, SL1 can trans-splice without competition with cis- splicing. The longer hypothetical protein, that we call CeRad51L, differs from the shorter one, CeRad51S, solely for the first 38 aminoacids that are missing in the latter. CeRad51S approximates the size of most eukaryotic Rad51/Dmc1 proteins, while the only other protein of the group, beside CeRad51L with a long aminoterminal peptide is the budding yeast Rad51 protein. We are now trying to assign a role to the two anternative products. Furthermore, since the RAD51 monomers are known to form in yeast long concatamers to perform the strand transfer activity, we are studying, by "two hybrid" analysis, the protein domains of CeRad51 involved in this interaction.

Rinaldo C et al. (2002) Genetics "Roles for Caenorhabditis elegans rad-51 in meiosis and in resistance to ionizing radiation during development."

Rinaldo C, La Volpe A, Hilliard M, Bazzicalupo P, Ederle S

[Genetics, 2002]

We have investigated the role of Caenorhabditis elegans RAD-51 during meiotic prophase and embryogenesis, making use of the silencing effect of RNA interference (RNAi). rad-51 RNAi leads to severe defects in chromosome morphology in diakinesis oocytes. We have explored the effect of rad-51 RNAi in mutants lacking fundamental components of the recombination machinery. If double-strand breaks are prevented by spo-11 mutation, rad-51 RNAi does not affect chromosome appearance. This is consistent with a role for RAD-51 downstream of the initiation of recombination. In the absence of MRE-11, as in the absence of SPO-11, RAD-51 depletion has no effect on the chromosomes, which appear intact, thus indicating a role for MRE-11 in DSB induction. Intriguingly, rad-51 silencing in oocytes that lack MSH-5 leads to chromosome fragmentation, a novel trait that is distinct from that seen in msh-5 mutants and in rad-51 RNAi oocytes, suggesting new potential roles for the msh-5 gene. Silencing of the rad-51 gene also causes a reduction in fecundity, which is suppressed by mutation in the DNA damage checkpoint gene rad-5, but not in the cell death effector gene ced-3. Finally, RAD-51 depletion is also seen to affect the soma, resulting in hypersensitivity to ionizing radiation in late embryogenesis.

Rinaldo C et al. (1998) Mol Gen Genet "The Caenorhabditis elegans RAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes."

Ederle S, LaVolpe A, Rinaldo C, Rocco V

[Mol Gen Genet, 1998]

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.

Cinzia Rinaldo et al. (2001) International C. elegans Meeting "Roles and modes of action of rad-51"

Massimo Hilliard, Sara Ederle, Paolo Bazzicalupo, Adriana La Volpe, Cinzia Rinaldo

[International C. elegans Meeting, 2001]

Our understanding of genetic control of meiotic recombination comes from studies in the unicellular eukaryote Saccharomyces cerevisiae . Most gene involved in control of recombination and meiosis are conserved throughout evolution. However, metazoa have developed a germ cell line distinct from the somatic cell line requiring a different kind of regulation compared to unicellular eukaryotes. Furthermore, in distant species, in spite of evolutionary conservation, similar genes may be subject to different regulation and interactions. Accordingly, elimination of a conserved molecule or interference with a conserved pathway may lead to quite different results in different organisms. For this reason, genetic and biochemical studies in different organisms are likely to contribute to an understanding of the crucial features and fundamental components of recombination pathways and their evolution. Among eukaryotes, C. elegans is the only organism known so far provided with a single rec A like gene, the homolog of RAD51 . Surprisingly, the meiosis specific DMC1 gene present in fungi, plants and mammals, is absent in C. elegans. RNA interference of the rad-51 gene in C. elegans leads to a number of visible phenotypes such as i) high levels of embryonic lethality, ii) increase in the frequency of males, iii) reduced fertility, and iv) hypersensitivity to ionizing radiation in soma . We have analysed in details the mechanisms leading to the above described phenotypes, in order to understand the different functions and modes of action of rad-51 in somatic and germ line cells. We demonstrated that this gene is required at several steps of gametogenesis: gene inactivation, in fact, affects pre-meiotic repair, sister chromatid exchange and homologous recombination and triggers a meiotic checkpoint. Unlike what has been described in mammals, rad-51 expression is not required during embryogenesis, but is required and enhanced in soma in response to DNA damage induced by ionizing radiation.

Cinzia Rinaldo et al. (2001) International C. elegans Meeting "Genetic interplay between rad-51 and other genes involved in meiosis"

Massimo Hilliard, Cinzia Rinaldo, Adriana La Volpe, Paolo Bazzicalupo

[International C. elegans Meeting, 2001]

The Rad51 protein is the eukaryotic homolog of Escherichia coli RecA; it mediates DNA strand-transfer and is a key molecule in homologous recombination and in double strand breaks repair. RAD51 homologous genes have been found in several eukaryotes (fungi, plants, anfibes, birds, and mammals). However, the exact function and regulation of these genes in higher eukaryotes is still to be elucidated. In order to define the steps of gametogenesis in which the only C. elegans RAD51 homolog ( Cerad-51 ) is required, we decide to study epistasis relationships between this gene and other well characterized C. elegans genes in meiosis. Genes supposedly acting in closely spaced steps of meiotic recombination are expected to exhibit similar phenotypes. The appropriate choice of phenotypes to be followed and of mutants to be analysed is crucial in epistasis studies. Based on our observations we can outline a simple model of action of RAD-51 during gametogenesis: a) RAD-51 is required at the end of the replicative stage of oocyte maturation, in order to eliminate accidental DNA damages occurred before the onset of meiotic prophase b) RAD-51 performs its strand transfer activity after DSBs induction and it is required both for inter-homolog exchange and for sister chromosome exchange, failure of both these events leads to gross abnormalities in chromosome shape and triggers a meiotic check-point.

hydrolase activity

Catalysis of the hydrolysis of various bonds, e.g. C-O, C-N, C-C, phosphoric anhydride bonds, etc.

daf-11 : trx-1

At 25 deg C, but enhances the Daf-C at 15 deg C.

pyrimidine dimer repair

The repair of UV-induced T-T, C-T and C-C dimers.