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.
Highlights
Homologous recombination is conserved in all organisms
Since the discovery of unique right-handed filamentous structures formed by RecA with dsDNA or ssDNA [12,13,22], it has been generally believed that the filamentous structure that forms around ssDNA consisting of RecA/Rad51 is essential to search and recognition of sequence homology with dsDNA [53,54]
The major finding of this study is that, contrary to this accepted notion, the npRecA dimer, but not the npRecA monomer, catalyzes D-loop formation (Figure 5) without filament formation (Figure 3F and the discussion described below)
Summary
Homologous recombination is conserved in all organisms. It plays an essential role in maintaining genome integrity through the repair of double-strand breaks and in genetic diversification through meiotic recombination, which is required for gametogenesis in sexual reproduction [1,2,3]. Homologous recombination depends on the RecA/Rad51-family recombinases. The RecA/Rad51-family recombinases catalyze ATP-dependent homologous joint formation. In this reaction, a single-stranded (ss) tail generated by resection at a double-strand break forms base pairs with the complementary strand of the homologous sequence within the intact double-stranded (ds) DNA. Many details of RecA/Rad51-catalyzed homologous joint formation have long been studied, this question remains unanswered
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