Functional analysis of the bovine papillomavirus E1 protein: Bending, phasing and interactions with the E2 protein

Cooperative binding of the viral E1 and E2 proteins to the origin of replication is an essential step in viral DNA replication. Two protein-protein interactions between the E1 and E2 proteins of BPV are required for cooperative binding. One interaction occurs between the DNA Binding Domain (DBD) of E2 and the DBD of E1. We show that an important consequence of this interaction is the induction of a sharp bend in the origin of replication. This bend serves an architectural role and promotes a second interaction between the activation domain (AD) of E2 and the E1 helicase domain. Mutations in the E2 DBD that affect the interaction with the E1 DBD disrupts bending and simultaneously disrupts the interaction between the E2 AD and the E1 helicase domain, resulting in loss of replication in vivo. Furthermore, mutations in the DNA sequence between the E1 and E2 binding sites demonstrate no specific sequence requirement; however, mutations that alter the bendability of the DNA result in loss of bending and loss of interaction between the two DBDs.;Analysis presented in this dissertation further indicates that the DBD of the E2 protein interacts exclusively with the DBD of the E1 protein. The second, productive interaction occurs solely between the Activation Domain of the E2 protein and the C terminal helicase domain of the E1 protein.