Cleavage of bacteriophage lambda cI repressor involves the RecA C-terminal domain.
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.
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ABSTRACT: Structured forms of DNA with intrastrand pairing are generated in several cellular processes and are involved in biological functions. These structures may arise on single-stranded DNA (ssDNA) produced during replication, bacterial conjugation, natural transformation, or viral infections. Furthermore, negatively supercoiled DNA can extrude inverted repeats as hairpins in structures called cruciforms. Whether they are on ssDNA or as cruciforms, hairpins can modify the access of proteins to DNA, and in some cases, they can be directly recognized by proteins. Folded DNAs have been found to play an important role in replication, transcription regulation, and recognition of the origins of transfer in conjugative elements. More recently, they were shown to be used as recombination sites. Many of these functions are found on mobile genetic elements likely to be single stranded, including viruses, plasmids, transposons, and integrons, thus giving some clues as to the manner in which they might have evolved. We review here, with special focus on prokaryotes, the functions in which DNA secondary structures play a role and the cellular processes giving rise to them. Finally, we attempt to shed light on the selective pressures leading to the acquisition of functions for DNA secondary structures.Microbiology and molecular biology reviews: MMBR 12/2010; 74(4):570-88. · 12.59 Impact Factor
Article: Interconversion between bound and free conformations of LexA orchestrates the bacterial SOS response.[show abstract] [hide abstract]
ABSTRACT: The bacterial SOS response is essential for the maintenance of genomes, and also modulates antibiotic resistance and controls multidrug tolerance in subpopulations of cells known as persisters. In Escherichia coli, the SOS system is controlled by the interplay of the dimeric LexA transcriptional repressor with an inducer, the active RecA filament, which forms at sites of DNA damage and activates LexA for self-cleavage. Our aim was to understand how RecA filament formation at any chromosomal location can induce the SOS system, which could explain the mechanism for precise timing of induction of SOS genes. Here, we show that stimulated self-cleavage of the LexA repressor is prevented by binding to specific DNA operator targets. Distance measurements using pulse electron paramagnetic resonance spectroscopy reveal that in unbound LexA, the DNA-binding domains sample different conformations. One of these conformations is captured when LexA is bound to operator targets and this precludes interaction by RecA. Hence, the conformational flexibility of unbound LexA is the key element in establishing a co-ordinated SOS response. We show that, while LexA exhibits diverse dissociation rates from operators, it interacts extremely rapidly with DNA target sites. Modulation of LexA activity changes the occurrence of persister cells in bacterial populations.Nucleic Acids Research 05/2011; 39(15):6546-57. · 8.03 Impact Factor
Article: Separation of recombination and SOS response in Escherichia coli RecA suggests LexA interaction sites.[show abstract] [hide abstract]
ABSTRACT: RecA plays a key role in homologous recombination, the induction of the DNA damage response through LexA cleavage and the activity of error-prone polymerase in Escherichia coli. RecA interacts with multiple partners to achieve this pleiotropic role, but the structural location and sequence determinants involved in these multiple interactions remain mostly unknown. Here, in a first application to prokaryotes, Evolutionary Trace (ET) analysis identifies clusters of evolutionarily important surface amino acids involved in RecA functions. Some of these clusters match the known ATP binding, DNA binding, and RecA-RecA homo-dimerization sites, but others are novel. Mutation analysis at these sites disrupted either recombination or LexA cleavage. This highlights distinct functional sites specific for recombination and DNA damage response induction. Finally, our analysis reveals a composite site for LexA binding and cleavage, which is formed only on the active RecA filament. These new sites can provide new drug targets to modulate one or more RecA functions, with the potential to address the problem of evolution of antibiotic resistance at its root.PLoS Genetics 09/2011; 7(9):e1002244. · 8.69 Impact Factor