RecAf

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Homologous recombination plays a key role in the repair of DNA double-strand breaks, inter-strand cross-linking, and collapsed replication forks, these functions are essential for maintaining genomic integrity. It also plays a key role in generating genetic diversity from bacteria to humans. The core reaction of recombination is the exchange of strands between two homologous DNA molecules and this process is catalyzed by the RecA family of ATPase.

RecA combines with ssDNA in an ATP-dependent manner to form a helical nucleoprotein filament that has ~6.2 RecA proteins per turn and approximately 3 nucleotides per RecA protein. The DNA is underwound to form a form with 18.5 nucleotides per turn, and an average rise of 5.1 Å per nucleotide.

The search for rec genes

Clark and Margulies first identified recA in genetic screening in 1965. They mutagenized F^- bacteria and screened for strains that could not recombine with the Hfr donor strain. Subsequent studies have shown that recA is essential for both the RecBCD- and RecF- pathways of homologous recombination and recA mutants have multiple functions, including influencing DNA repair, SOS mutagenesis, cell division, chromosome separation, and λ prophage induction, etc.

Ten years after the recA gene was discovered, the protein was purified for the first time, and its extensive biochemical activities were reported, including DNA binding, ATP hydrolysis, the ability to form filaments on DNA, and the capacity to exchange homologous DNA strands. Studies have reported that RecA is a 38 kDa protein with approximately 1,000 monomers expressed in each cell and forms right-hand nucleoprotein filaments on ssDNA and dsDNA.

In the absence of DNA damage, the concentration of RecA in vivo is approximately 1 μM; however, during the SOS response, the concentration of RecA will increase approximately 10-fold. ATP-bound nucleoprotein filaments act as surface catalysts for searching and capturing DNA homologous sequences. Once a region of homology is found, the ssDNA strands on the homologous chromosomes are exchanged to produce heteroduplex DNA (that is, each single strand is from a different chromosome).  

The search for structural clues: insight from electron microscopy

In the past thirty years, through high-resolution electron microscopy, people have obtained most of the information of RecA nucleoprotein filament, and thus have a deeper understanding of the morphological changes of the nucleoprotein structure during the recombination process. Under physiological conditions, ssDNA folds into multiple structures, which are stabilized by localized annealing of short regions of complementarity (secondary structure), which inhibit the formation of RecA filaments. DNA secondary structure is largely overcome by ssDNA binding proteins (SSB), which binds ssDNA tightly with SSB, effectively denaturing the secondary structure.

SSB-coated ssDNA is denser than dsDNA and differs in morphology from RecA filaments because each tetramer of SSB is wrapped around 30-70 nucleotides around itself. When RecA filaments are formed together with ATP and hydrolyze to generate a large amount of ADP, the RecA filaments are heterogeneous in pitch along a single filament, which is consistent with the localized and cooperative ATP hydrolysis model. Although the mutant RecA protein has the ability to bind ssDNA and hydrolyze ATP, it cannot extend ssDNA to the canonical filament length, so it cannot catalyze synapsis and DNA strand exchange, indicating that stretching DNA is necessary for DNA strand exchange.

Fig 1. a. Electron microscopy of DNA-free RecA showing heterogeneous oligomerization states of RecA.
b. Electron microscopy of RecA filament-formation on SSB-coated circular ssDNA (Bell, J.C.; Kowalczykowski, S.C. 2016)

Overall structure of the RecA-ssDNA filament

RecA consists of a 30-residue amino-terminal a2b motif, a 240-residue α/β ATPase core and a 64-residue globular domain (CTD), which uses ATPase core to bind to ssDNA. ADP-AlF₄-Mg binds to the interface of the two RecA promoters, and ssDNA is located very close to the filament axis and wraps around it.

The planes of the bases are roughly orthogonal to the filament axis, and their Watson-Crick edge is within 1.0A˚, while the DNA backbone is located at the far end of the axis. The end of ssDNA binds to the N-terminal RecA of the fusion protein.

The RecA-ssDNA filament is different from the previous crystalline filaments, mainly because the direction of the recombinase relative to the filament axis is different, which is different from the RecA-RecA relationship with the position and position for the DNA binding element.

Fig 2. Structure of the presynaptic nucleoprotein filament (Chen, Z.C.; et al. 2008)