All RecA-like recombinase enzymes catalyze DNA strand exchange as elongated filaments on DNA. Despite numerous biochemical and structural studies of RecA and the related Rad51 and RadA proteins, the unit oligomer(s) responsible for nucleoprotein filament assembly and coordinated filament activity remains undefined. We have created a RecA fused dimer protein and show that it maintains in vivo DNA repair and LexA co-protease activities, as well as in vitro ATPase and DNA strand exchange activities. Our results support the idea that dimeric RecA is an important functional unit both for assembly of nucleoprotein filaments as well as their coordinated activity during the catalysis of homologous recombination.The bacterial RecA protein is the founding member of a family of enzymes that catalyzes recombination between homologous DNA substrates, a family which includes the phage T4 UvsX, yeast Rad51, archael RadA and human Rad51 proteins (1,2). To perform this function these recombinase enzymes form a helical filament that assembles cooperatively on a singlestranded region of DNA resulting in the formation of an active nucleoprotein complex that subsequently interacts with a dsDNA substrate to catalyze a search for homology and DNA strand exchange (1,3,4). Early biochemical studies of RecA revealed that in the absence of substrate DNA the protein exists in solution as a complex heterogeneous mix of various oligomers (5-8) and later work showed that this mix included monomers, ring-shaped hexamers/heptamers, short filaments ranging in length from 0.03 to 0.15 μm as well as bundles of filaments (9-12).The RecA nucleoprotein filament assembles in an ATP-dependent, highly cooperative manner (13-16). Kinetic analysis of nucleoprotein filament assembly has shown that RecA polymerization onto ssDNA can be divided into two major steps, i. nucleation, which is rate limiting, and ii. filament extension (17). However, the oligomeric heterogeneity of RecA in these studies precluded identification of a specific RecA oligomer, e.g. monomers or dimers, as being an obligatory component of the assembly process, or whether various larger RecA oligomers, e.g. trimers or hexamers, could participate in filament assembly. It was also found that RecA oligomers formed in the absence of ATP or ssDNA are not directly interconvertible tel. -(508)
EXPERIMENTAL PROCEDURES Expression ConstructsThe gene encoding wild type recA was expressed using a previously described construct, pTRecA420, in which recA is regulated by the tac promoter (20,21). The gene encoding the fused RecA dimer (recA-FD) was constructed as follows. Two point mutations, Lys6Ala and Arg28Ala, were introduced into the wild type recA gene carried in plasmid pTRecA420 using the QuikChange protocol (Stratagene). This mutant recA gene was PCR amplified using a top strand primer that overlapped the NcoI restriction site (underlined) at the fMet initiation codon (5′-GG AGT GAT GCC ATG GCT ATC GAC G -3′) and a bottom strand primer that adds a five-residue linker (Gly 3 Ser 2 )...