Nonfilament-forming RecA dimer catalyzes homologous joint formation

Abstract

Homologous recombination is essential to genome maintenance, and also to genome diversification. In virtually all organisms, homologous recombination depends on the RecA/Rad51-family recombinases, which catalyze ATP-dependent formation of homologous joints—critical intermediates in homologous recombination. RecA/Rad51 binds first to single-stranded (ss) DNA at a damaged site to form a spiral nucleoprotein filament, after which double-stranded (ds) DNA interacts with the filament to search for sequence homology and to form consecutive base pairs with ssDNA (‘pairing’). How sequence homology is recognized and what exact role filament formation plays remain unknown. We addressed the question of whether filament formation is a prerequisite for homologous joint formation. To this end we constructed a nonpolymerizing (np) head-to-tail-fused RecA dimer (npRecA dimer) and an npRecA monomer. The npRecA dimer bound to ssDNA, but did not form continuous filaments upon binding to DNA; it formed beads-on-string structures exclusively. Although its efficiency was lower, the npRecA dimer catalyzed the formation of D-loops (a type of homologous joint), whereas the npRecA monomer was completely defective. Thus, filament formation contributes to efficiency, but is not essential to sequence-homology recognition and pairing, for which a head-to-tail dimer form of RecA protomer is required and sufficient.

Engineered RecA constructs reveal the minimal SOS activation complex

“…Together these results lend further support to the idea that RecA2x is forming transient nuclei on ssDNA rather than stable filaments. 53 RecA WT and each of the constructs from RecA3x to RecA6x showed proficient RecA* filament formation as detected by a change in anisotropy at equilibrium. In comparing the results of RecA WT to the linked oligomeric constructs, the concentration-dependent filament formation curves appeared different.…”

“…Together these results lend further support to the idea that RecA2x is forming transient nuclei on ssDNA rather than stable filaments. 53…”

“…43,46,62,63 As noted earlier, it has been observed that although RecA2x may be capable of binding ssDNA, it does so at a reduced capacity relative to RecA WT. 53 Meanwhile, RecA4x and larger have been shown to be capable of binding to ssDNA stably enough for structural studies via X-ray crystallography. 52 We therefore anticipated a range of ssDNA-binding activity with our RecA series and posited that filament formation could be contributing to the observed pattern of LexA cleavage.…”

Continuum dynamics and statistical correction of compositional heterogeneity in multivalent IDP oligomers resolved by single-particle EM

“…The idea to extract single-particle information directly from individual electron micrograph images is not new (22,(36)(37)(38), but here we exploit geometric characteristics of polymeric systems to create an intuitive and reliable automated approach applicable to the growing, important class of beads-on-string systems (46)(47)(48). We show that the "functional form" of polymeric conformational properties in terms of bead-bead distances and three-bead angles, enabled adequate training based on only a few dozen manually picked oligomers.…”

RecA kinetically selects homologous DNA by testing a five- or six-nucleotide matching sequence and deforming the second DNA

“…The size determined in this manner is slightly smaller than the sizes obtained by other methods, but is clearly larger than three bases. Recent analysis of a non-polymerizing RecA mutant showed that dimer formed by covalent linking can promote DNA pair formation while its monomer form cannot (Shinohara et al ., 2018). Since each RecA monomer covers three bases, this observation suggests that pair formation requires more than a three base match but less than a six base match, which is compatible with our results.…”