A conserved regulatory module at the C terminus of the papillomavirus E1 helicase domain controls E1 helicase assembly.

Unlabelled: Viruses frequently combine multiple activities into one polypeptide to conserve coding capacity. This strategy creates regulatory challenges to ascertain that the combined activities are compatible and do not interfere with each other. The papillomavirus E1 protein, as many other helicases, has the intrinsic ability to form hexamers and double hexamers (DH) that serve as the replicative DNA helicase.

Dynamic look at DNA unwinding by a replicative helicase.

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models.

CK2 phosphorylation inactivates DNA binding by the papillomavirus E1 and E2 proteins.

Papillomaviruses have complex life cycles that are understood only superficially. Although it is well established that the viral E1 and E2 proteins play key roles in controlling viral transcription and DNA replication, how these factors are regulated is not well understood. Here, we demonstrate that phosphorylation by the protein kinase CK2 controls the biochemical activities of the bovine papillomavirus E1 and E2 proteins by modifying their DNA binding activity.

Mechanistic analysis of local ori melting and helicase assembly by the papillomavirus E1 protein.

Preparation of DNA templates for replication requires opening of the duplex to expose single-stranded (ss) DNA. The locally melted DNA is required for replicative DNA helicases to initiate unwinding. How local melting is generated in eukaryotic replicons is unknown, but initiator proteins from a handful of eukaryotic viruses can perform this function.

Structure-based mutational analysis of the bovine papillomavirus E1 helicase domain identifies residues involved in the nonspecific DNA binding activity required for double trimer formation.

The papillomavirus E1 protein is a multifunctional initiator protein responsible for preparing the viral DNA template for initiation of DNA replication. The E1 protein encodes two DNA binding activities that are required for initiation of DNA replication. A well-characterized sequence-specific DNA binding activity resides in the E1 DBD and is used to tether E1 to the papillomavirus ori.

Adjacent residues in the E1 initiator beta-hairpin define different roles of the beta-hairpin in Ori melting, helicase loading, and helicase activity.

We have analyzed two residues in the helicase domain of the E1 initiator protein. These residues are part of a highly conserved structural motif, the beta-hairpin, which is present in the helicase domain of all papovavirus initiator proteins. These proteins are unique in their ability to transition from local template melting activity to unwinding.

ATP-dependent minor groove recognition of TA base pairs is required for template melting by the E1 initiator protein.

Template melting is an essential step in the initiation of DNA replication, but the mechanism of template melting is unknown for any replicon. Here we demonstrate that melting of the bovine papillomavirus type 1 ori is a sequence-dependent process which relies on specific recognition of TA base pairs in the minor groove by the E1 initiator. We show that correct template melting is a prerequisite for the formation of a stable double hexamer with helicase activity and that ori mutants that fail to melt correctly are defective for ori unwinding and DNA replication in vivo.

Surface mutagenesis of the bovine papillomavirus E1 DNA binding domain reveals residues required for multiple functions related to DNA replication.

The E1 protein from papillomaviruses is a multifunctional protein with complex functions required for the initiation of viral DNA replication. We have performed a surface mutagenesis of the well-characterized E1 DNA binding domain (DBD). We demonstrate that substitutions of multiple residues on the surface of the E1 DBD are defective for DNA replication without affecting the DNA binding activity of the protein.

Assembly of a double hexameric helicase.

Viral initiators perform multiple functions in initiation of DNA replication including ori binding, melting, and unwinding, culminating in the formation of a double hexameric (DH) helicase. We have recapitulated the assembly of the papillomavirus E1 initiator DH helicase, providing the first description of how such a complex is formed. We have identified an intermediate, a double trimer (DT), which relies on two cooperating DNA binding activities to melt double-stranded DNA and generate a substrate for formation of the DH helicase.

Role of papillomavirus E1 initiator dimerization in DNA replication.

Viral initiator proteins are polypeptides that form oligomeric complexes on the origin of DNA replication (ori). These complexes carry out a multitude of functions related to initiation of DNA replication, and although many of these functions have been characterized biochemically, little is understood about how the complexes are assembled. Here we demonstrate that loss of one particular interaction, the dimerization between E1 DNA binding domains, has a severe effect on DNA replication in vivo but has surprisingly modest effects on most individual biochemical activities in vitro.