Quantitative analysis of the kinetics of end-dependent disassembly of RecAfilaments from ssDNA

On linear single-stranded DNA, RecA filaments assemble and disassemble in t he 5' to 3' direction. Monomers (or other units) associate at one end and d issociate from the other. ATP hydrolysis occurs throughout the filament. Di ssociation can result when ATP is hydrolyzed by the monomer at the disassem bly end. We have developed a comprehensive model for the end-dependent fila ment disassembly process. The model accounts not only for disassembly, but also for the limited reassembly that occurs as DNA is vacated by disassembl ing filaments. The overall process can be monitored quantitatively by follo wing the resulting decline in DNA-dependent Am hydrolysis. The rate of disa ssembly is highly pH dependent, being negligible at pH 6 and reaching a max imum at pH values above 7.5. The rate of disassembly is not significantly a ffected by the concentration of free RecA protein within the experimental u ncertainty. For filaments on single-stranded DNA, the monomer k(cat) for AT P hydrolysis is 30 min(-1), and disassembly proceeds at a maximum rate of 6 0-70 monomers per minute per filament end. The latter rate is that predicte d if the ATP hydrolytic cycles of adjacent monomers are not coupled in any way. (C) 1999 Academic Press.