Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae. Issue 6 (21st February 2023)

• Record Type: Journal Article
• Authors: Hertzog, Maud; Perry, Thomas Noé; Dupaigne, Pauline; Serres, Sandra; Morales, Violette; Soulet, Anne-Lise; Bell, Jason C; Margeat, Emmanuel; Kowalczykowski, Stephen C; Le Cam, Eric; Fronzes, Rémi; Polard, Patrice
• Is Part Of: Nucleic acids research. Volume 51:Issue 6(2023)
• Page Start: 2800
• Page End: 2817
• Publication Date: 2023-02-21
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gkad080
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae ( Sp RecA), in comparison to the Escherichia coli RecA ( Ec RecA) paradigm. Ec RecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block Ec RecA nucleofilament growth. We report by direct microscopic analysis of Sp RecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of Sp RecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes Sp RecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native Sp RecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the Ec RecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that Sp RecA and Ec RecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.