Defective Dissociation of a “Slow” RecA Mutant Protein Imparts an Escherichia coli Growth Defect

Abstract

The RecA and some related proteins possess a simple motif, called (KR)X(KR), that (in RecA) consists of two lysine residues at positions 248 and 250 at the subunit-subunit interface. This study and previous work implicate this RecA motif in the following: (a) catalyzing ATP hydrolysis in trans, (b) coordinating the ATP hydrolytic cycles of adjacent subunits, (c) governing the rate of ATP hydrolysis, and (d) coupling the ATP hydrolysis to work (in this case DNA strand exchange). The conservative K250R mutation leaves RecA nucleoprotein filament formation largely intact. However, ATP hydrolysis is slowed to less than 15% of the wild-type rate. DNA strand exchange is also slowed commensurate with the rate of ATP hydrolysis. The results reinforce the idea of a tight coupling between ATP hydrolysis and DNA strand exchange. When a plasmid-borne RecA K250R protein is expressed in a cell otherwise lacking RecA protein, the growth of the cells is severely curtailed. The slow growth defect is alleviated in cells lacking RecFOR function, suggesting that the defect reflects loading of RecA at stalled replication forks. Suppressors occur as recA gene alterations, and their properties indicate that limited dissociation by RecA K250R confers the slow growth phenotype. Overall, the results suggest that recombinational DNA repair is a common occurrence in cells. RecA protein plays a sufficiently intimate role in the bacterial cell cycle that its properties can limit the growth rate of a bacterial culture.Homologous DNA recombination is an important part of DNA metabolism. The primary function of bacterial recombination systems is the recombinational DNA repair of stalled replication forks (1-4). Recombination is also responsible for the exchange of genetic material during meiosis in eukaryotes and conjugation in bacteria. RecA protein is the central recombinase in Escherichia coli. RecA homologues are present in nearly every organism, and closely related RecA proteins are found in virtually all bacterial species with the exception of a few endosymbionts (5). 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, 6, 7).Replication forks are thought to stall often in bacteria under normal growth conditions, but the actual rate is a matter of some dispute. Some estimates suggest that forks stall often, up to once per cell cycle in bacteria (1, 4, 8 -11). At least some of these result in double strand breaks, with up to 50 double strand breaks per cell cycle occurring in humans (12). A direct measure of double strand break generation in bacteria suggests that fork stalling may be less frequent, or at least not always accompanied by double strand breaks (13). Notably, several pathways have been proposed for the reconstitution of replication forks that do not involve double strand breaks (1,4,11,14). However, the frequency with which these pathways are utilized ...

Disassembly of Escherichia coli RecA E38K/ΔC17 Nucleoprotein Filaments Is Required to Complete DNA Strand Exchange

“…In fact, dissociation of RecA filament subunits during DNA strand exchange has been previously documented (54). Whereas considerable evidence has been compiled linking ATP hydrolysis and DNA strand exchange (17,49,55,73,74), no evidence has appeared to indicate whether the role of the observed filament dissociation is determinant or incidental. This study begins to fill this void, presenting for the first time a set of conditions (the low pH, nonpermissive conditions with RecA E38K/⌬C17) in which ATP hydrolysis continues but dissociation of RecA filaments is suppressed.…”

Escherichia coli Genes and Pathways Involved in Surviving Extreme Exposure to Ionizing Radiation

“…Deletion or alteration of some genes involved in DNA repair are known to result in slow growth phenotypes in rich media (62)(63)(64)(65)(66)(67)(68)(69)(70). The otherwise-nonessential recA protein, clearly important for IR resistance, is not present in our list because strains with alterations resulting in recA gene inactivation grow somewhat slower and are unable to compete with the broader population during outgrowth.…”

Regulation of Deinococcus radiodurans RecA Protein Function via Modulation of Active and Inactive Nucleoprotein Filament States

“…This is likely to reflect a complex interplay of mobile amino acid residues and peptide segments that mediate the change in filament states, particularly at the subunit-subunit interface. The active site for ATP hydrolysis is at the interface between filament subunits, and amino acid residues from both flanking subunits cooperate in the hydrolytic reaction (26,27). Mutational changes in key active site resides produce large effects on EcRecA activities (26,27).…”

“…The active site for ATP hydrolysis is at the interface between filament subunits, and amino acid residues from both flanking subunits cooperate in the hydrolytic reaction (26,27). Mutational changes in key active site resides produce large effects on EcRecA activities (26,27). We speculate that the transition between active and inactive states in DrRecA similarly reflects changes in the position and ionization state of ATPase active site residues, although the changes are exaggerated in the case of the DrRecA such that they can bring about an inactive state.…”