RecA Protein Filaments Disassemble in the 5′ to 3′ Direction on Single-stranded DNA

Abstract

RecA protein forms filaments on both single-and double-stranded DNA. Several studies confirm that filament extension occurs in the 5 to 3 direction on singlestranded DNA. These filaments also disassemble in an end-dependent fashion, and several indirect observations suggest that the disassembly occurs on the end opposite to that at which assembly occurs. By labeling the 5 end of single-stranded DNA with a segment of duplex DNA, we demonstrate unambiguously that RecA filaments disassemble uniquely in the 5 to 3 direction.The active form of the bacterial RecA protein is a filament formed on DNA. RecA has no known activities when it is not part of such a filamentous complex. Further, the action of several other bacterial proteins appears to be directed, at least in part, to the modulation of RecA filament assembly and disassembly on DNA (1-5). An understanding of the formation and disassembly of these filaments is thus critical to a broader understanding of RecA function in recombination processes and to an understanding of the activities of many other recombination proteins.Filament formation generally includes a distinct and ratelimiting nucleation step followed by rapid filament extension. RecA filaments are formed most readily on ssDNA, 1 where nucleation is followed by extension uniquely in the 5Ј to 3Ј direction (1, 6). Filament formation on dsDNA is very slow above pH 7 due to slow nucleation (7,8). On a gapped DNA substrate, RecA will nucleate within the single-stranded gapped region, and the subsequent extension will encompass any adjacent duplex DNA on the 3Ј-proximal end of the gap (1, 6, 9 -11). The RecA protein forms a right-handed helical filament on DNA with visible striations seen using EM (12, 13). Within the filament, bound DNA is highly extended and underwound (12, 14 -18).The bacterial SSB protein modulates many aspects of RecA filament dynamics. Prebound SSB inhibits the nucleation step in RecA filament assembly on ssDNA (19). However, once RecA filament formation is initiated, SSB facilitates the subsequent filament extension by removing secondary structure in the DNA (20). Even though filament nucleation is inhibited by SSB, the same SSB is readily displaced during the extension phase of filament formation. When SSB is overproduced within the Escherichia coli cell, recombination of UV-irradiated bacteriophage DNA is specifically inhibited (21).A net end-dependent disassembly of RecA protein filaments has been observed on dsDNA (11, 22) and ssDNA (1). Net filament disassembly from dsDNA and ssDNA is observed at neutral and higher pH values. On ssDNA, observation of a net disassembly is dependent upon the presence of SSB and ATP (1) and proceeds at a rate of about 60 -70 monomers min Ϫ1 at 37°C. In the EM, filament disassembly on linear singlestranded DNA appears to occur only on one end (1). Because filament extension occurs unambiguously in the 5Ј to 3Ј direction, and because extension followed by recession at the same end seemed unlikely, we previously argued that filament disassembly must b...

The C Terminus of the Escherichia coli RecA Protein Modulates the DNA Binding Competition with Single-stranded DNA-binding Protein

“…As previously observed, a small net disassembly occurs in the absence of SSB, as indicated by the slight decline in the rate of ATP hydrolysis with time ( Fig. 2, dashed lines), and disassembly and reassembly of filaments is doubtless occurring at a steady state (5,56,58). The rate of hydrolysis by RecA protein in the absence of SSB is somewhat higher at 10 mM Mg 2ϩ than at 3 mM.…”

“…Previously, it was shown that much more prebound RecA is displaced from poly(dT) by SSB than from M13mp8 ssDNA (7). This occurs to a large extent because of end-dependent disassembly of RecA filaments (5,56,58), which is readily observed only on linear DNAs, where filament extension at one end does not compensate for disassembly at the other. Here we show that at 10 mM Mg 2ϩ , wild-type RecA protein (3 nt/monomer) is almost completely displaced from poly(dT) by SSB in a process that is complete in less than 10 min (Fig.…”

“…Alternatively, they could reflect a greater steady-state level of binding to the DNA. On the linear poly(dT) DNA substrates used here, net DNA binding reflects the balance between filament assembly and end-dependent disassembly (5,56,58). We evaluated the intrinsic rate of ATP hydrolysis by comparing mutants side by side with excess protein on M13mp8 circular ssDNA and with SSB added after the RecA protein (data not shown).…”

The Requirement for ATP Hydrolysis by Saccharomyces cerevisiae Rad51 Is Bypassed by Mating-Type Heterozygosity or RAD54 in High Copy

“…It has been suggested that ATP hydrolysis is required for disassembly of the RecA filament to allow more-efficient recycling of the protein (6,24,27). This model attractively explains the phenotype conferred by the rad51-K191R allele.…”

SSB Antagonizes RecX-RecA Interaction

“…There are discrete nucleation and extension phases in the assembly process, with extension proceeding primarily 5Ј to 3Ј relative to the ssDNA (17, 18). Disassembly also proceeds primarily 5Ј to 3Ј, with monomeric subunits being added on one end and subtracted from the other (19,20). Dissociation at the disassembly end is coupled to ATP hydrolysis (21,22).…”