Abstract
The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA (ssDNA), align it with a homologous duplex, and promote an extensive strand exchange between them. Both reactions have properties, including a tolerance of base analog substitutions that tend to eliminate major groove hydrogen bonding potential, that suggest a common molecular process underlies the DNA strand exchange promoted by RecA and Rad51. However, optimal conditions for the DNA pairing and DNA strand exchange reactions promoted by the RecA and Rad51 proteins in vitro are substantially different. When conditions are optimized independently for both proteins, RecA promotes DNA pairing reactions with short oligonucleotides at a faster rate than Rad51. For both proteins, conditions that improve DNA pairing can inhibit extensive DNA strand exchange reactions in the absence of ATP hydrolysis. Extensive strand exchange requires a spooling of duplex DNA into a recombinase-ssDNA complex, a process that can be halted by any interaction elsewhere on the same duplex that restricts free rotation of the duplex and/or complex, I.e. the reaction can get stuck. Optimization of an extensive DNA strand exchange without ATP hydrolysis requires conditions that decrease nonproductive interactions of recombinase-ssDNA complexes with the duplex DNA substrate.
Highlights
The bacterial RecA protein and the homologous Rad51 protein in eukaryotes both bind to single-stranded DNA, align it with a homologous duplex, and promote an extensive strand exchange between them
ATP hydrolysis is usually required for extensive DNA strand exchange promoted by RecA protein [10] but not for the reaction promoted by the yeast Rad51 protein [4]
Most of this work focuses on solution requirements for the reactions, some comparative work was done with DNA substrates containing base analogs
Summary
RecA and Rad are frequently cited as functional and structural homologs, based on amino acid sequence similarities [23], the apparent similarity in their filament structures [24], and the DNA pairing and strand exchange reactions they both promote Both proteins require a single strand DNA-binding protein (bacterial SSB or eukaryotic RPA) for an optimal DNA strand exchange reaction. For both RecA and Rad, the requirements for homologous pairing, other than the DNA substrates and recombinase, vary among reports but presumably include ATP (or certain ATP analogs), Mg2ϩ in sufficient concentration to coordinate the nucleotide cofactor, and a multivalent cation. We continue our exploration of the range of DNA substrates tolerated in DNA strand exchange processes [21, 59]
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