The single-strand annealing homologous recombination (SSA) pathway is one of many used for repairing dsDNA breaks. SSA is mediated by an Exonuclease-Annealase Two-component Recombinase (EATR) complex. Two viral examples we study are the bacteriophage λ EATR consisting of the exonuclease λExo and annealase Redβ, and the Herpes Simplex Virus 1 (HSV-1) EATR composed of exonuclease UL12 and annealase ICP8.
While there are structures available for both phage λ Redβ and HSV-1 ICP8, these structures are of truncated versions, and therefore, incomplete. The structures have greatly aided our understanding of how the annealase proteins in EATRs function, however the molecular mechanistic details of how these proteins process DNA and form an EATR complex with exonucleases remain unknown. To our knowledge, this work demonstrates the first structural investigations into the phage λ EATR complex and the continued structural investigations into the ICP8 complex with DNA.
The ICP8 crystal structure is without DNA bound and in a conformation that is possibly physiologically irrelevant. Therefore, our goal is to obtain a cryo-EM structure of ICP8 bound to DNA that reveals the detailed mechanism of annealing. Negative staining electron microscopy (NS-EM) was also used to examine the multimeric ring and filament complexes formed by ICP8 with complementary ssDNAs.
How Redβ interacts with its partner λExo to form an EATR and function as a complex is still unknown. We used NS-EM to examine the λ EATR proteins. Our results show that the treatment of a dsDNA with λExo and Redβ results in the formation of helical filaments.
Functional assays were also performed after successful protein purification of ICP8, λExo and Redβ. The activities of the annealase proteins were tested via a FRET (Fluorescence Resonance Energy Transfer) based EMSA (electromobility shift assay). The activity of λExo was examined using a single-molecule characterization assay determining the rate of digestion of dsDNA by λExo via TIRF microscopy.
The results we present demonstrate the continued functional characterization of viral EATR proteins and show progress towards more complete structural data for the phage λ EATR and the ICP8+DNA complex via cryo-EM.