Invited Speaker Australian Society for Microbiology Annual Scientific Meeting 2022

Half a century after their discovery, cryo-EM structures of Redβ and Erf annealases provide insights into their evolution and molecular mechanisms (82588)

Timothy Newing 1 2 , MariaKatarina Lambourne 1 2 3 , Jodi L. Brewster 1 2 3 , Haibo Yu 1 , Nikolas P. Johnston 4 , Lucy J. Fitschen 1 2 , Gökhan Tolun 1 2 3
  1. School of Chemistry and Molecular Bioscience, Molecular Horizons, University of Wollongong, Wollongong, NSW, Australia
  2. Illawarra Health and Medical Research Institute, Wollongong, NSW, Australia
  3. ARC Industrial Transformation Training Centre for Cryo-electron Microscopy of Membrane Proteins, UOW Node, Wollongong, NSW, Australia
  4. School of Chemistry and Molecular Bioscience, University of Wollongong, Wollongong, NSW, Australia

Single-strand annealing homologous DNA recombination (SSA) is a process conserved throughout evolution from bacteriophages to humans1, emphasising its importance for life. It is particularly vital in viruses, as it catalyses circularisation and concatamerisation of the viral DNA, and is involved in viral genome repair2.

SSA is catalysed by EATR (Exonuclease Annealase Two-component Recombinase) complexes. The exonuclease component binds to dsDNA ends, digesting one strand 5ʹ to 3ʹ, generating a 3′ ssDNA overhang. The annealase component then binds to this nascent ssDNA and catalyses a homology search and subsequent annealing to a homologous DNA sequence. Despite half a century of extensive research into SSA and EATRs, the molecular mechanistic details are poorly understood3.

Erf (essential recombination function) is the annealase of the bacteriophage P22 that infects Salmonella enterica s. Typhimurium, and is the defining member of the Erf protein family. Redβ is the annealase of phage λ that infects Escherichia coli and is one of the defining members of the Redβ/RecT protein family. Structures Redβ and Erf were still not available, despite their discovery in 19664 and 19705, respectively. We report the first atomic structures of the N-terminal domains of Redβ and Erf annealases, more than 50 years after their discovery.

Our 3.3 Å cryo-EM structure of Redβ177 bound to two complementary 27mer oligonucleotides shows both how it forms homo-oligomeric helical filaments, and how the DNA-annealing intermediate is formed during recombination. Our 2.47 Å cryo-EM structure of Erf shows how it forms nonadecameric (19 subunits) rings. These structures reveal a striking similarity to the human Rad52 annealase, containing a β-β-β-α fold forming the DNA-binding site.

We discuss the implications of our findings from both evolutionary and molecular mechanistic perspectives.

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