Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

Hertzog, M.; Perry, T. N.; Dupaigne, P.; Serres, S.; Morales, V.; Soulet, A.-L.; Bell, J.; Margeat, E.; Kowalczykowski, S. C.; Le Cam, E.; Fronzes, R.; Polard, P.

doi:10.1101/2022.07.28.501814

bioRxiv · 10.1101/2022.07.28.501814

Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.

Hertzog, M.·Perry, T. N.·Dupaigne, P.·Serres, S.·Morales, V.·Soulet, A.-L.·Bell, J.·Margeat, E.·Kowalczykowski, S. C.·Le Cam, E.·Fronzes, R.·Polard, P.

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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 (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report that neither of the two paralogous pneumococcal SSBs could assist SpRecA polymerization on ssDNA. Instead, we found that the conserved RadA helicase promotes this SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATP{gamma}S. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.

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Hertzog, M., Perry, T. N., Dupaigne, P., Serres, S., Morales, V., Soulet, A.-L., Bell, J., Margeat, E., Kowalczykowski, S. C., Le Cam, E., Fronzes, R., Polard, P.. 2022-07-28. Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae.. https://doi.org/10.1101/2022.07.28.501814

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