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ABSTRACT: In Escherichia coli, RecA protein catalyzes DNA pairing and strand exchange activities essential for genetic recombination. This is critical for normal cellular function under conditions that lead to altered DNA metabolism and DNA damage. The RecA proteins of E. coli and Bacillus halodurans both can bind to DNA and catalyze the specific proteolytic cleavage of LexA and lambda repressor which induces SOS response. At neutral pH self-cleavage of LexA depends exclusively on its binding to RecA filament, while at elevated pH (~10) it autodigests in the absence of RecA. We have shown in this work that the RecA-mediated cleavage and the binding of RecA to DNA promoted by B. halodurans are similar to those promoted by E. coli RecA, excepted that in the case of B. halodurans the rate of the cleavage reactions is increased at alkaline pH and that NaCl favors the binding of RecA to DNA. The results lead to two hypotheses for the pathway for RecA-mediated cleavage, in which we first suppose that the internal pH of the bacteria is neutral. Thus LexA cannot undergo autodigestion, the genes involving in DNA repair and replication are not transcribed, regulating the growth of the cell. The second hypothesis is that the external environment and the internal pH of the bacteria are alkaline; here also the bacteria may have developed strategies to maintain LexA not inactivated. These observations suggest that the LexA autodigestion in B. halodurans at high pH may be regulated at the transcriptional level and that B. halodurans may be haloalkaliphile bacterium.
AFRICAN JOURNAL OF BIOTECHNOLOGY 04/2009; 8:1827-1833. · 0.57 Impact Factor
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ABSTRACT: The SOS response to DNA damage in Escherichia coli involves at least 43 genes, all under the control of the LexA repressor. Activation of these genes occurs when the LexA repressor cleaves itself, a reaction catalyzed by an active, extended RecA filament formed on DNA. It has been shown that the LexA repressor binds within the deep groove of this nucleoprotein filament, and presumably, cleavage occurs in this groove. Bacteriophages, such as lambda, have repressors (cI) that are structural homologs of LexA and also undergo self-cleavage when SOS is induced. It has been puzzling that some mutations in RecA that affect the cleavage of repressors are in the C-terminal domain (CTD) far from the groove where cleavage is thought to occur. In addition, it has been shown that the rate of cleavage of cI by RecA is dependent upon both the substrate on which RecA is polymerized and the ATP analog used. Electron microscopy and three-dimensional reconstructions show that the conformation and dynamics of RecA's CTD are also modulated by the polynucleotide substrate and ATP analog. Under conditions where the repressor cleavage rates are the highest, cI is coordinated within the groove by contacts with RecA's CTD. These observations provide a framework for understanding previous genetic and biochemical observations.
Journal of Molecular Biology 12/2008; 385(3):779-87. · 4.00 Impact Factor
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ABSTRACT: The key event in the switch from lysogenic to lytic growth of phage lambda is the self-cleavage of lambda repressor, which is induced by the formation of a RecA-ssDNA-ATP filament at a site of DNA damage. Lambda repressor cleaves itself at the peptide bond between Ala111 and Gly112, but only when bound as a monomer to the RecA-ssDNA-ATP filament. Here we have designed a hyper-cleavable fragment of lambda repressor containing the hinge and C-terminal domain (residues 101-229), in which the monomer-monomer interface is disrupted by two point mutations and a deletion of seven residues at the C terminus. This fragment crystallizes as a monomer and its structure has been determined to 1.8 A resolution. The hinge region, which bears the cleavage site, is folded over the active site of the C-terminal oligomerization domain (CTD) but with the cleavage site flipped out and exposed to solvent. Thus, the structure represents a non-cleavable conformation of the repressor, but one that is poised for cleavage after modest rearrangements that are presumably stabilized by binding to RecA. The structure provides a unique snapshot of lambda repressor in a conformation that sheds light on how its self-cleavage is tempered in the absence of RecA, as well as a framework for interpreting previous genetic and biochemical data concerning the RecA-mediated cleavage reaction.
Journal of Molecular Biology 10/2006; 362(3):479-89. · 4.00 Impact Factor
