RESUMEN
Homologous recombination is a fundamental biological process. Biochemical understanding of this process is most advanced for Escherichia coli. At least 25 gene products are involved in promoting genetic exchange. At present, this includes the RecA, RecBCD (exonuclease V), RecE (exonuclease VIII), RecF, RecG, RecJ, RecN, RecOR, RecQ, RecT, RuvAB, RuvC, SbcCD, and SSB proteins, as well as DNA polymerase I, DNA gyrase, DNA topoisomerase I, DNA ligase, and DNA helicases. The activities displayed by these enzymes include homologous DNA pairing and strand exchange, helicase, branch migration, Holliday junction binding and cleavage, nuclease, ATPase, topoisomerase, DNA binding, ATP binding, polymerase, and ligase, and, collectively, they define biochemical events that are essential for efficient recombination. In addition to these needed proteins, a cis-acting recombination hot spot known as Chi (chi: 5'-GCTGGTGG-3') plays a crucial regulatory function. The biochemical steps that comprise homologous recombination can be formally divided into four parts: (i) processing of DNA molecules into suitable recombination substrates, (ii) homologous pairing of the DNA partners and the exchange of DNA strands, (iii) extension of the nascent DNA heteroduplex; and (iv) resolution of the resulting crossover structure. This review focuses on the biochemical mechanisms underlying these steps, with particular emphases on the activities of the proteins involved and on the integration of these activities into likely biochemical pathways for recombination.
Asunto(s)
Escherichia coli/genética , Exodesoxirribonucleasas/metabolismo , Modelos Genéticos , Rec A Recombinasas/metabolismo , Recombinación Genética/genética , Escherichia coli/metabolismo , Exodesoxirribonucleasa V , Rec A Recombinasas/genéticaRESUMEN
We have investigated the biochemical properties of several Escherichia coli mutant recA proteins that display a null phenotype. These are the recA1, recA13 and recA56 proteins, each of which carries a single missense mutation. These proteins all share a common defect which is the inability to adopt the high affinity DNA binding state normally elicited by the nucleotide cofactor ATP. Consequently, other than the ability to bind ssDNA, they possess none of the in vitro enzymatic activities of recA protein. However, each protein has characteristics that are unique, leading to the conclusion that the observed mutant phenotypes arise through fundamentally different mechanisms. Despite the magnitude of these defects, the recA56 protein is able to differentially inhibit various activities of wild-type recA protein. Incorporation of recA56 protein into a presynaptic filament with the wild-type recA protein does not affect the ability of the wild-type protein to hydrolyze ATP, as judged by the turnover number (kcat), provided that the ssDNA concentration is not limiting; however, the affinity of wild-type recA protein for ATP is lowered by the presence of recA56 protein. Similarly, the ability to cleave lexA protein is only modestly inhibited. However, both the ability to compete with SSB protein for ssDNA binding sites and the DNA strand exchange activity of wild-type recA protein are severely inhibited by the presence of recA56 protein. These results suggest that individual monomeric components of the recA protein-DNA filament are translated through protein-protein contacts to become macroscopic properties of the filament.
Asunto(s)
Rec A Recombinasas/antagonistas & inhibidores , Serina Endopeptidasas , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli , Técnicas In Vitro , Sustancias Macromoleculares , Mutación Puntual , Unión Proteica , Rec A Recombinasas/genética , Rec A Recombinasas/metabolismo , Recombinación Genética , Relación Estructura-ActividadRESUMEN
The apparent DNA site size obtained from an assay monitoring the ATPase activity of Escherichia coli recA protein (n = 3.5) differs from that determined from a direct DNA binding assay (n = 7) done under identical conditions. Investigation of this discrepancy indicates that at a DNA:protein ratio of 3.5:1, one-half of the recA protein population is less sensitive to ATPase activity inhibition by the nonhydrolyzable ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), suggesting that the recA protein filament is asymmetric with respect to NTP affinity. This asymmetry does not depend on the presence of ATP gamma S since the apparent Km for ATP derived from single-stranded DNA-dependent ATP hydrolysis activity is dependent on the DNA:protein ratio. Three models are proposed to account for the observed site size discrepancy and the NTP binding affinity asymmetry. They differ mainly in the intrinsic site size for each recA protein monomer and in the number of DNA-binding sites/recA molecule. Gel filtration of recA-single-stranded DNA complexes at different DNA:protein ratios complements the enzymological data and provides an additional method of distinguishing among the proposed models. The phenomenon of subunit nonequivalence within the recA protein presynaptic filament may provide a molecular basis for understanding how recA protein can discriminate between different DNA molecules during homologous pairing.