Single strand annealing homologous DNA recombination (SSA) and DNA replication are processes found in virtually all life.
SSA is particularly important in the double-strand DNA viruses. It is catalysed by two proteins: An Exonuclease and an Annealase that form a Two-component Recombinase complex (EATR). The exonuclease digests a broken dsDNA from its end to generate a single-strand DNA (ssDNA) overhang, to which the annealase binds and anneals it to a homologous ssDNA strand, catalysing recombination. This re-pairing of ssDNAs ultimately results in repairing of dsDNA breaks.
DNA replication machinery duplicates DNA before cell division. Several proteins and protein complexes are involved in this highly dynamic process. In E. coli, the DnaB helicase unwinds the double-stranded parental DNA, and the DnaG primase synthesises short RNA primers; these two proteins form a primosome complex.
Despite more than half a century of research, our knowledge of the molecular mechanistic details of SSA and DNA replication have various gaps. We use a multi-disciplinary approach to better understand how these bio-nano-machines work, with a focus on cryo-EM. Our work-in-progress results are presented.
We show that the phage P22 annealase Erf forms 19-mer oligomeric rings with a central density and hook-like protrusions outside the ring. We also show that a truncated form of Redβ annealase of phage λ forms solenoids (i.e., helical filaments).
The preliminary cryo-EM structure of a disulphide-crosslinked engineered DnaB helicase in complex with the helicase-binding domain of DnaG shows a structure similar to that of a similar complex from another bacterium.
Further work in determining the high resolution cryo-EM structures of these protein complexes to reveal their mechanistic details, is underway.
Acknowledgments: Redβtr was provided by Nina Constantino, Dr. Gary Shaw, Dr. Donald L. Court, and Dr. Xinhua Ji. Erf was provided by Dr. Kenan C. Murphy.