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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 single-stranded 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.
Highlights
Filament formation generally includes a distinct and ratelimiting nucleation step followed by rapid filament extension
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 be occurring at the 5Ј-proximal end [1]
Experimental Design—To demonstrate by EM that RecA protein filaments disassembled in the 5Ј to 3Ј direction, we needed a method to label one end of the ssDNA
Summary
Enzymes and Biochemicals—The E. coli RecA protein was purified by a procedure developed for the RecA K72R mutant protein [23]. The linear dsDNA fragment (M13mp ϫ PstI ϫ BsrGI) substrate for the RecA reaction was prepared as follows. Reactions contained 7 M RecA, 2 M SSB, 3 mM ATP, 20 M M13mp8.1037 circular ssDNA, and 20 M M13mp ϫ PstI ϫ BsrGI linear dsDNA. The entire reaction mixture was digested with PvuII for 60 min at 37 °C to generate a linear gapped DNA molecule with 1894 bp of dsDNA at the 5Ј end of the long linear ssDNA region. The reactions included 2 M RecA protein, 3 M linear gapped DNA (2.4 M nucleotides ssDNA), and 0.24 M SSB. The RecA protein was preincubated with the linear gapped DNA for 10 min at 37 °C in the presence of all reaction components except ATP and SSB. The judgments were again checked for accuracy and bias by comparison with direct measurements from electron micrographs (on average 5 filaments/experiment)
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