Real-time tracking reveals catalytic roles for the two DNA binding sites of Rad51.

Nature Communications
Kentaro ItoHiroshi Iwasaki

Abstract

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.

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Citations

Dec 22, 2020·Biochimica Et Biophysica Acta. Molecular Cell Research·Lukas GrätzAgo Rinken
Jun 11, 2021·DNA Repair·Bilge ArgunhanHideo Tsubouchi
Jul 3, 2021·Genes·Irina S ShkundinaAlexander V Mazin
Jul 11, 2021·Current Opinion in Genetics & Development·Hideo TsubouchiHiroshi Iwasaki

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Methods Mentioned

BETA
fluorescence
circular dichroism
coimmunoprecipitation
FRET
electrophoresis
gel filtration
Assay
co-IP

Software Mentioned

Visual Data Tools
DynaFit
Excel
DataGraph
MODELLER

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