The Structural Determination of Nucleic Acid Binding Proteins by Cryogenic Electron Microscopy

<p dir="ltr">Nucleic acids carry the genetic information essential for life and are among the most important biomolecules. The proteins that interact with nucleic acids constitute an incredibly complex and diverse network and are thus an intriguing subject for study. A strong relationship exists between the structure of such proteins and the functions they carry out. Structural determination of macromolecules therefore represents a key avenue for investigating and characterising these complexes, to shed light on their mechanisms. To this end, cryogenic electron microscopy has become an invaluable tool for investigating these structures, particularly since it is applicable to a variety of biological specimens which can be imaged in their near-native state.</p><p dir="ltr">This thesis describes the characterisation of several proteins that bind and interact with nucleic acids. BALF2 is an essential single-stranded binding protein and annealase from Epstein-Barr virus. It recruits the viral replisome to replication compartments and through binding activity stabilises the replication fork. As an annealase, it is involved in single strand annealing homologous recombination, for genomic maintenance and to increase genetic diversity. ORF6, a homologue from Kaposi’s Sarcoma-associated Herpesvirus, and Erf, an analogue from bacteriophage p22, are also investigated. PhoH2 is a toxin protein found in several bacteria and archaea, including Mycobacterium tuberculosis. The protein enables these organisms to enter a state of hibernation through the sequence-specific degradation of RNA, during which they show enhanced antibiotic resistance.</p><p dir="ltr">The work presented details the structural determination of BALF2, captured as a filamentous annealing intermediate, with its DNA-binding properties characterised by negative-stain EM and gel-shift assays. The formation of analogous annealing intermediates by both ORF6 and Erf was also assessed. In addition, the structure of PhoH2 was determined as an ADP-bound toroid, and its oligomeric behaviour investigated, revealing a nucleotide-dependent transition to an active heptamer. Together, these findings provide structural and mechanistic insight into diverse nucleic acid binding proteins, their modes of assembly, and their roles in nucleic acid processing and regulation.</p>