Worm Breeder's Gazette 15(2): 39 (February 1, 1998)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

The Polymerisation Domain of CeRAD51

Cinzia Rinaldo, Adriana La Volpe

International Institute of Genetics and Biophysics - CNR- Naples - Italy

        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
        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.