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

Abstract

Disassembly of RecA protein subunits from a RecA filament has long been known to occur during DNA strand exchange, although its importance to this process has been controversial. An Escherichia coli RecA E38K/⌬C17 double mutant protein displays a unique and pH-dependent mutational separation of DNA pairing and extended DNA strand exchange. Single strand DNA-dependent ATP hydrolysis is catalyzed by this mutant protein nearly normally from pH 6 to 8.5. It will also form filaments on DNA and promote DNA pairing. However, below pH 7.3, ATP hydrolysis is completely uncoupled from extended DNA strand exchange. The products of extended DNA strand exchange do not form. At the lower pH values, disassembly of RecA E38K/⌬C17 filaments is strongly suppressed, even when homologous DNAs are paired and available for extended DNA strand exchange. Disassembly of RecA E38K/⌬C17 filaments improves at pH 8.5, whereas complete DNA strand exchange is also restored. Under these sets of conditions, a tight correlation between filament disassembly and completion of DNA strand exchange is observed. This correlation provides evidence that RecA filament disassembly plays a major role in, and may be required for, DNA strand exchange. A requirement for RecA filament disassembly in DNA strand exchange has a variety of ramifications for the current models linking ATP hydrolysis to DNA strand exchange.The RecA protein of Escherichia coli catalyzes homologous DNA pairing and strand exchange reactions that are central to recombination and recombinational DNA repair (1-4). RecA protein is found in virtually all bacteria. RecA homologs, such as RadA (5, 6) and Rad51 (7-10), are ubiquitous in archaea and eukaryotes, respectively.The DNA strand exchange reaction promoted by RecA protein occurs in several distinct phases. The first phase is binding of RecA protein to single-stranded DNA (ssDNA) 2 to form a helical nucleoprotein filament. Filament formation occurs in at least two steps, with a slow nucleation step preceding a rapid 5Ј to 3Ј extension of the filament to encompass the available DNA (11-13). During the second phase of DNA strand exchange, RecA aligns homologous DNA strands and catalyzes strand exchange over a short distance producing a length of hybrid DNA that typically consists of up to 1000 bp (2, 14). In the third phase of DNA strand exchange, RecA catalyzes extension of the hybrid DNA region via facilitated branch migration that occurs 5Ј to 3Ј with respect to the bound ssDNA (15).RecA protein subunits within a nucleoprotein filament hydrolyze ATP in a DNA-dependent manner. ATP is hydrolyzed uniformly throughout the RecA nucleoprotein filament (16) with a k cat of about 30 min Ϫ1 when RecA is bound to ssDNA. The k cat is reduced to 20 min Ϫ1 when RecA binds directly to double-stranded (ds) DNA (3) or when a RecA filament is catalyzing DNA strand exchange (17, 18). Homologous pairing and formation of short stretches of exchanged DNA require the binding of ATP but not ATP hydrolysis (19 -22). However, the efficient extension of this ne...

Purification and Characterization of the RecA Protein from Neisseria gonorrhoeae

“…It is likely that the coupling mechanism involves the end-dependent dissociation of RecA subunits from the 5′-proximal end of the RecA filament [60]. In addition to the increased ATPAse activity of RecA Ng relative to RecA Ec , the conversion to the NC form in early time points by RecA Ng was also significantly higher than that of RecA Ec (Figure 2).…”

RecA K72R Filament Formation Defects Reveal an Oligomeric RecA Species Involved in Filament Extension

“…Proteins-The wild type E. coli RecA, RecA K72R, RecA E38K/K72R, and SSB proteins were purified as previously described (45,48,(51)(52)(53), and their concentrations were determined using native extinction coefficients: ⑀ 280 ϭ 2.23 ϫ 10 (55), and apparent K d values were determined using the Prism software.…”

“…It was used to demonstrate that very little disassembly of RecA filaments occurs from the interior of the filament (46), that RecA filaments on dsDNA are more dynamic than those bound to ssDNA (46), that RecA filaments disassemble over the course of DNA strand exchange (47), and that RecA filament disassembly is necessary to complete exchange of long homologous DNA substrates (48). If differing ratios of the wild type and the mutant RecA proteins are premixed before incubating them with circular single-stranded DNA (cssDNA), the decrease in ATP hydrolysis rate is greater than that expected if the hydrolysis-deficient RecA K72R were simply occluding binding sites on the DNA (46).…”

Directed Evolution of RecA Variants with Enhanced Capacity for Conjugational Recombination

“…The E . coli RecX, Neisseria gonorrhoeae RecX [ 90 ], SSB [ 90 ] and the wild type RecA protein [ 155 , 156 ] were purified as previously described. The RecA V79L, RecA I102L, RecA E86G/C90G mutant proteins were purified by the same means as the wild type RecA protein with the following modifications.…”