Cellular & Molecular Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan.
Nucleic Acids Res. 2018 Nov 16;46(20):10855-10869. doi: 10.1093/nar/gky877.
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.
同源重组对于基因组的维持和多样化至关重要。在几乎所有的生物中，同源重组都依赖于 RecA/Rad51 家族重组酶，它可以催化 ATP 依赖性的同源连接的形成，这是同源重组的关键中间产物。RecA/Rad51 首先结合到受损部位的单链 DNA 上形成螺旋核蛋白丝，然后双链 DNA 与丝相互作用，寻找序列同源性，并与 ssDNA 形成连续碱基对（“配对”）。序列同源性如何被识别以及丝的形成的确切作用仍然未知。我们探讨了丝的形成是否是同源连接形成的前提条件。为此，我们构建了一个非聚合（np）头尾融合的 RecA 二聚体（npRecA 二聚体）和一个 npRecA 单体。npRecA 二聚体结合到 ssDNA 上，但在与 DNA 结合时不会形成连续的丝；它只形成串珠样结构。尽管效率较低，但 npRecA 二聚体仍能催化 D 环的形成（一种同源连接），而 npRecA 单体则完全失效。因此，丝的形成有助于提高效率，但对于序列同源性的识别和配对并非必需，RecA 单体的头尾二聚体形式是必需和充分的。
