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The RecA residues Lys 248 and Glu 96 are closely opposed across the RecA subunit-subunit interface in some recent models of the RecA nucleoprotein filament. The K248R and E96D single mutant proteins of the Escherichia coli RecA protein each bind to DNA and form nucleoprotein filaments but do not hydrolyze ATP or dATP. A mixture of K248R and E96D single mutant proteins restores dATP hydrolysis to 25% of the wild type rate, with maximum restoration seen when the proteins are present in a 1:1 ratio. The K248R/E96D double mutant RecA protein also hydrolyzes ATP and dATP at rates up to 10-fold higher than either single mutant, although at a reduced rate compared with the wild type protein. Thus, the K248R mutation partially complements the inactive E96D mutation and vice versa. The complementation is not sufficient to allow DNA strand exchange. The K248R and E96D mutations originate from opposite sides of the subunit-subunit interface. The functional complementation suggests that Lys 248 plays a significant role in ATP hydrolysis in trans across the subunit-subunit interface in the RecA nucleoprotein filament. This could be part of a mechanism for the long range coordination of hydrolytic cycles between subunits within the RecA filament.Homologous DNA recombination is a vital component of DNA metabolism, central to processes such as recombinational DNA repair of stalled replication forks and the exchange of genetic material during meiosis in eukaryotes and conjugation in prokaryotes. RecA protein is the central recombinase in Escherichia coli, and RecA homologues are present in nearly every organism. In E. coli, RecA participates not only in the restart of stalled replication forks but also the induction of the SOS response upon cellular DNA damage distress and translesion DNA synthesis via the error-prone DNA polymerase V (1-3).RecA protein functions as a nucleoprotein filament. When bound to DNA, RecA promotes the hydrolysis of ATP or dATP. Hydrolysis occurs uniformly throughout the filament. ATP hydrolysis is important for some RecA processes including net disassembly of the nucleoprotein filament, bypass of heterologous insertions during DNA three-strand exchange, complete DNA strand exchange with DNA substrates longer than ϳ3 kbp, and strand exchange with four DNA strands (1, 4 -8). The ATP hydrolytic cycles between adjacent subunits in the RecA filament bound to double-stranded DNA (dsDNA) 2 are coordinated such that waves of hydrolysis move sequentially through the filament with a

Proper formation of the bacteriophage T4 DNA polymerase holoenzyme requires a wide spectrum of protein-protein and protein-DNA interactions among the DNA polymerase gp43, the sliding clamp gp45, and gp44/62, the clamp loader complex (CLC). The 44 and 62 proteins associate to form a tight complex maintained in a 4:1 ratio. The 44 and 62 genes are adjacent to each other on the T4 genome, are cotranscribed, and are translationally coupled. It has been suggested that translational coupling may play a role in the formation of the clamp loader complex and may control its stoichiometry. To examine the effect of coupling on the assembly of the complex, expression in trans of genes 44 and 62 was accomplished by cotransforming Escherichia coli with compatible, inducible plasmid vectors. A gp44/62 complex could be purified from such cells. The complex assembled in trans exhibited stoichiometry and ATPase activity identical to native complex. Burst sizes were determined to gauge the efficiency of clamp loader complex formation. When gp44 was supplied by a plasmid and gp62 was supplied by the T4 genome, complex formation was as efficient as in wild-type virus. However, when gp62 was supplied by plasmid and gp44 was supplied by the T4 genome, efficiency of complex formation was decreased. This decrease in the efficiency of complex formation was temperature dependent, being more pronounced at higher temperatures. At higher temperatures, a larger proportion of gp62 expressed from the plasmid was found to be present in an insoluble form. The decrease in efficiency of complex formation correlated to a decrease in solubility of the gene 62 protein.