No abstract

RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single-stranded DNA. The primary reaction of RecA with DNA is generally assumed to be unspecific. We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single-stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases. This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA. Furthermore, the enthalpy of interaction between the RecA . DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA. This result suggests a possibility for a rapid search mechanism by RecA . DNA filaments for homologous DNA molecules.Keywords : RecA ; recombination ; calorimetry ; protein-DNA interaction ; fluorescence.General genetic recombination is a process, common to all forms of life, by which new combinations of genetic material or nucleic acid sequences are generated. RecA is a key component of general genetic recombination in Escherichia coli [I -31, and related proteins with similar functions are found in a variety of organisms [3, 41. Genetic recombination consists of strand exchange between two homologous DNA molecules, and the reaction facilitates post-replicative DNA repair and is important for DNA segregation in cell division. Purified RecA in vitro promotes the strand-exchange reaction in the presence of cofactor ATP [5, 61, and has been extensively studied to understand the mechanism of homologous-recombination reactions (reviewed in [3,7-9]). RecA has a molecular mass of 38 kDa [lo], but it polymerises into very-high molecular-mass filaments. RecA binds co-operatively to any single-stranded DNA [9], and forms nucleofilaments by arranging itself in a helical manner around the DNA [ l l , 121. Each filament can bind, in the presence of a cofactor, a second DNA and thus direct two DNA molecules towards each other in preparation for strand exchange [13-161. Binding of a second DNA molecule only occurs after saturation of the first DNA-binding site in RecA; the stoichiometry is 3 DNA bases/RecA monomer for both interactions (3 base pairs/RecA monomer if the second DNA molecule is a duplex) [15]. Although structural details begin to accumulate, the physical nature of the DNA binding in RecA and the mechanisms for these protein-mediated DNA reactions are still largely unknown.To better understand the mechanism for RecA pairing of two homologous DNA molecules, we have studied the thermodynamics and kinetics of RecA-DNA interactions. Calorimetry directly measures the heat of interaction for a chemical reaction and gives thermodynamic parameters. We show here that iso- MATERIALS AND METHODSMaterials and experimental conditions. RecA was purified as described elsewhere [20] with HPLC (DEAE 5PW, Tosoh) as a final step. The HPLC-elu...

DNA repair is vital for both prokaryotic and eukaryotic cells. RecA is an ubiquitous and multi-functional enzyme involved in various steps of DNA repair: regulation of synthesis of DNA repair proteins (SOS induction), promotion of homologous recombination, and mutagenesis (For reviews, see Refs. 1-3). For these activities, RecA first binds to single-stranded DNA with a strong cooperativity and forms a filamentous complex in which RecA subunits are arranged in a helical manner around the DNA (4). This nucleofilament binds a second doublestranded DNA molecule for the strand exchange reaction (5) and interacts with repressors for the induction of the SOS system (6) and with UmuD and UmuDЈ proteins for mutagenesis (7,8).The molecular structure of the RecA filament in the absence of DNA has been determined by x-ray crystallography (9). A lower resolution structure of the nucleofilament (4) and of the DNA in the complex (5) has also been determined. These studies have shown that the DNA strands lie near the axis of the RecA-DNA filament. Determination of the DNA binding sites in RecA would allow us to build a higher resolution model of the RecA-DNA complex. Despite various studies, however, the DNA binding site(s) of RecA has not yet been determined. From the comparison of sequence with the DNA binding domain of ssDNA 1 binding protein of the Ike phage, GP5 protein, the domain comprising residues 240 -310 has been proposed as a DNA binding site (10 -12). But this region is in the subunitsubunit interface in the crystal of the RecA filament (9). Story et al. (9) rejected this possibility and proposed that two disordered loops that could not be traced in the x-ray analysis were the DNA binding domains because these loops line the cavity running down the middle of the RecA filament. In fact, mutations in L2 loop affect the activities of RecA (13), although modifications in L1 loop are easily tolerated (14).Limited proteolysis of RecA-DNA complexes in the presence of ATP␥S has shown that a peptide spanning loop L2 is protected from tryptic digestion (15). In addition, peptides derived from this region, including a 20-amino acid peptide from residues 193-212 can bind to ssDNA with a high affinity. Subsequent work has shown that such a peptide can also bind to dsDNA. In addition, a phenylalanine at position 203 in loop L2 that is highly conserved between prokaryotic RecAs and their eukaryotic homologues (16, 17) plays a central role in the binding. Only aromatic acid substitutions allow the peptides to bind to both ss-and dsDNA. 2 With the purpose of examining the possibility that this central aromatic residue in loop L2 interacts with DNA, we inserted a tryptophan residue in this loop in the place of phenylalanine. An interaction with DNA could then be detected by a change in the tryptophan fluorescence. Our results show that the modified protein is active, and its fluorescence is strongly decreased upon binding to DNA.

We review the locations of various functional domains of the RecA protein of Escherichiu coli, including how they have been assigned, and discuss the potential regulatory roles of spatial overlap between different domains. RecA is a multifunctional and ubiquitous protein involved both in general genetic recombination and in DNA repair: it regulates the synthesis and activity of DNA repair enzymes (SOS induction) and catalyses homologous recombination and mutagenesis. For these activities RecA interacts with a nucleotide cofactor, single-stranded and double-stranded DNAs, the LexA repressor, UmuD protein, the UmuD:C complex as well as with RecA itself in forming the catalytically active nucleofilament. Attempts to locate the respective interaction sites have been advanced in order to understand the various functions of RecA. An intriguing question is how these numerous functional sites are contained within this rather small protein (38 kDa). To assess more clearly the roles of the respective sites and to what extent the sites may be interacting with each other, we review and compare the results obtained from various biological, biochemical and physico-chemical approaches. From a three-dimensional model it is concluded that all sites are concentrated to one part of the protein. As a consequence there are significant overlaps between the sites and it is speculated that corresponding interactions may play important roles in regulating RecA activities.