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

Julia M Cox,Hao Li,Elizabeth A Wood,Sindhu Chitteni-Pattu,Ross B Inman,Michael M Cox

doi:10.1074/jbc.m803934200

Julia M Cox, Hao Li + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m803934200

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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.

Highlights

Replication forks are thought to stall often in bacteria under normal growth conditions, but the actual rate is a matter of some dispute
In E. coli RecA protein, the residues in the (KR)X(KR) motif are Lys-248 and Lys-250 [35]. These two residues may be important for ATP hydrolysis and potentially for communicating the conformation changes associated with ATP hydrolysis between adjacent subunits in the RecA nucleoprotein filament, based on the similarities to homologous proteins described above
In the first part, we first examined the involvement of the subunit-subunit interface residue Lys250 in (d)ATP hydrolysis catalyzed by the RecA protein by examining several purified proteins with single mutations at residue Lys-250 as well as a mutation at another subunit-subunit interface residue, Glu-96

Summary

Slow RecA Protein in Recombinational DNA Repair

The central core domain of RecA protein defines a structural motif ( called the RecA fold) found in a variety of proteins that hydrolyze ATP, and many of these proteins are motors [5, 18, 35]. In E. coli RecA protein, the residues in the (KR)X(KR) motif are Lys-248 and Lys-250 [35] These two residues may be important for ATP hydrolysis and potentially for communicating the conformation changes associated with ATP hydrolysis between adjacent subunits in the RecA nucleoprotein filament, based on the similarities to homologous proteins described above. Lys-248 and Lys-250 are not near the ATP-binding site of the RecA filament as resolved in the first RecA crystal structure [41, 42]. In the new reconstructions of the RecA nucleoprotein filament, the Lys-248 and Glu-96 residues are located at the subunit-subunit interface near the bound nucleotide but are opposed across the interface. RecA K250R is a slow RecA protein, promoting ATP hydrolysis and DNA strand exchange at rates that are 6-fold slower than the wild-type protein. When the protein is expressed in E. coli, a slow growth phenotype results

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION