RecA

RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA. A RecA structural and functional homolog has been found in every species in which one has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins. The homologous protein is called RAD51 in eukaryotes and RadA in archaea.

RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor.

RecA's association with DNA repair is based on its central role in homologous recombination. The RecA protein binds strongly and in long clusters to ssDNA to form a nucleoprotein filament. The protein has more than one DNA binding site, and thus can hold a single strand and double strand together. This feature makes it possible to catalyze a DNA synapsis reaction between a DNA double helix and a complementary region of single-stranded DNA. The RecA-ssDNA filament searches for sequence similarity along the dsDNA. A disordered DNA loop in RecA, Loop 2, contains the residues responsible for DNA homologous recombination. In some bacteria, RecA posttranslational modification via phosphorylation of a serine residue on Loop 2 can interfere with homologous recombination.

The search process induces stretching of the DNA duplex, which enhances sequence complementarity recognition (a mechanism termed conformational proofreading). The reaction initiates the exchange of strands between two recombining DNA double helices. After the synapsis event, in the heteroduplex region a process called branch migration begins. In branch migration an unpaired region of one of the single strands displaces a paired region of the other single strand, moving the branch point without changing the total number of base pairs. Spontaneous branch migration can occur, however, as it generally proceeds equally in both directions it is unlikely to complete recombination efficiently. The RecA protein catalyzes unidirectional branch migration and by doing so makes it possible to complete recombination, producing a region of heteroduplex DNA that is thousands of base pairs long.

Since it is a DNA-dependent ATPase, RecA contains an additional site for binding and hydrolyzing ATP. RecA associates more tightly with DNA when it has ATP bound than when it has ADP bound.

In Escherichia coli, homologous recombination events mediated by RecA can occur during the period after DNA replication when sister loci remain close. RecA can also mediate homology pairing, homologous recombination and DNA break repair between distant sister loci that had segregated to opposite halves of the E. coli cell.

E. coli strains deficient in RecA are useful for cloning procedures in molecular biology laboratories. E. coli strains are often genetically modified to contain a mutant recA allele and thereby ensure the stability of extrachromosomal segments of DNA, known as plasmids. In a process called transformation, plasmid DNA is taken up by the bacteria under a variety of conditions. Bacteria containing exogenous plasmids are called "transformants". Transformants retain the plasmid throughout cell divisions such that it can be recovered and used in other applications. Without functional RecA protein, the exogenous plasmid DNA is left unaltered by the bacteria. Purification of this plasmid from bacterial cultures can then allow high-fidelity PCR amplification of the original plasmid sequence.