Aïllament i caracterització d'un nou tipus de repressor LexA pertanyent al grup alfa de les proteobacteries

Project Details

Description

The SOS genes, encoding a pool of proteins involved in DNA repair, are negatively controlled by the LexA repressor which binds to a specific sequence called the SOS box. The only two kinds of SOS boxes known until now are both palindromes (CTGTN\sub 8\nosub ACAG and GAACN\sub 4\nosub GTT) and belong to \i E.coli\i0 and \i B.subtilis\i0 , respectively. Our group has recently identified a new SOS box in the alpha group of proteobacteria, which is the direct repeat GAACN\sub 7\nosub GAAC. We have also shown that, in these bacteria, there is a repressor which binds to this sequence and its palindromic derivative GAACN\sub 7\nosub GTTC. This versatility of the alpha group LexA protein is a new feature and allows it to be distinguished from the other LexA characterized. Intending to go deeply into the knowledge and into the evolution of SOS system, the main goal of this project is the isolation of the gene codifying this new kind of LexA repressor and the indentification of aminoacids interacting with its corresponding SOS box. To achieve this,\i lexA\i0 genes from \i R.etli\i0 , \i R.sphaeroides\i0 , \i R.capsulatus\i0 and \i P.denitrificans\i0 will be cloned and sequenced. The \i R.etli lexA\i0 gene will be isolated from a genomic library in the \lambdaZAP vector by using the degenerated oligonucleotides designed from the N-terminal sequence of the LexA protein previously obtained as a probe. The other \i lexA\i0 genes will be obtained by hybridization, using the \i R.etli\i0 one as a probe. The comparison of \i lexA\i0 gene sequences will allow establishment of the specific and characteristic regions of the LexA repressor of the alpha group. The aminoacids of this repressor, involved in the binding to its SOS box, will be identified by the \i in vitro\i0 mutagenesis of \i R.etli lexA\i0 gene, analyzing then the repressing ability over its \i recA\i0 gene in the \i E.coli\i0 background. The ineraction \i in vitro\i0 between the purified \i R.etl
StatusFinished
Effective start/end date1/10/981/10/01

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