Specific Binding Affinity of the Non-Catalytic Domain of Eukaryotic Like Type IB Topoisomerase of vaccinia Virus

Topoisomerases are ubiquitous proteins that alter supercoiling in double stranded DNA (dsDNA) during transcription and replication and. vaccinia and the closely related poxvirus variola virus, at 314 amino acids in length, encode the smallest of the type I topoisomerases(TopIB). TopIB is a two domain protein that recognizes the sequence 5’-T/CCCTT, cleaves at the 3’-end and relaxes supercoiling through rotation. The C-terminal domain (CTD) alone contains the catalytic activity and specificity. Deletion of the N-terminal domain results in a greatly reduced rate of relaxation and rapid dissociation. Biochemical data suggests that the N-terminal domain (NTD) is important for pre-cleavage binding and affinity for the target site. A combination of NMR-based interaction studies, the measurement of backbone dynamics using 15N relaxation measurements, and isothermal calorimetry (ITC) is used in this work to show that the NTD is capable of independently binding to DNA. Additionally, it is shown that the nature of the engagement of dsDNA by the NTD, in terms of affinity and characteristics of the binding modes, differs between sequences containing the 5’-CCCTT segment from those that do not. An attempt is made to extend these observations to the full length protein