Worm Breeder's Gazette 16(2): 39
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.
|1||International Institute of genetics and Biophysics -CNR - 10, Via Marconi 80125 Naples Italy|
|2||Dipartimento di Genetica e Biologia Generale e Molecolare, Facolta' di Scienze, Universita' di Napoli "Federico II"- 8, Via Mezzocannone 80134 Naples, Italy|
The eukaryotic Rad51 proteins are structurally and functionally related
to RecA of Escherichia coli and catalyse strand transfer between
homologous DNA molecules by forming helical nucleoprotein filaments around
double and single strand DNA and are therefore key molecules involved both
in double-strand breaks repair and in meiotic recombination. Beyond the
sequence similarity observed between the C. elegans gene and
the other eukaryotic RecA-like genes (Rinaldo et al. 1998)
, we attempted to assess the extent of conservation of functional domains.
Studying the ability of several deletion derivatives of CeRad51 to form dimers in vivo using a two hybrid system assay, we identify protein domains important for stable self-association measured as HIS3 prototrophy and LacZ expression. This study has shown that: i) the aminoterminal domain of CeRad51 is necessary, but not sufficient for self-association, ii) the aminoterminal domain of CeRad51 can be substituted by the GAL4 DNA-binding domain resulting in a chimerical protein with a restored ability to bind the wild-type CeRad51, iii) deletions at the carboxiterminus abolish the protein-protein interaction.
Our current model proposes that the aminoterminal portion of CeRad51 contains a DNA-binding domain; in order to homo-polymerize, each CeRad51 subunit needs to be bound to DNA; when the GAL4 DNA-binding domain is cloned in place of the amino-terminal portion of CeRad51, deletion derivative of CeRad51, that had lost their ability to self-associate, can recover their polymerizing capability. We also propose that, unlike what has been observed in the Saccharomyces cerevisiae Rad51 (Donovan et al. 1994) , the aminoterminal of CeRad51 cannot contain the only site of contact between two CeRad51 molecules, otherwise the GAL4 DNA-binding domain would not be able to suppress in cis the mutant phenotype of the deletion derivatives. The self-association domain itself is therefore located elsewhere in the protein probably in the carboxi-terminal region whose deletion leads to total loss of protein-protein binding activity.
Rinaldo C., Ederle S., Rocco V., and Volpe A. L. (1998) The Caenorhabditis
elegans RAD51 homolog is transcribed into two alternative mRNAs potentially
encoding proteins of different sizes. Mol. Gen. Genet. 260 : 289-294.
Donovan J. W., Milne G. T., and Weaver D. T. (1994) Homotypic and heterotypic protein associations control Rad51 function in double-strand break repair. Genes Dev 8 : 2552-62.