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. The formation of the DT is dependent on nucleotide binding, while formation of the DH also requires hydrolysable ATP. The DNA binding properties of the DT explain how E1, which binds to DNA as a dimer, can effect a transition to ring structures, such as the double hexamer. These results provide new insight into the intricate machinery that initiates DNA replication.

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. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.ATPase | molecular motors D NA replication is the most fundamental of all of life's processes. One of the key requisites in the initiation of replication is the separation of the two strands of the double helix, which is carried out by a hexameric helicase. Despite their prominent roles in biology, some of the basic aspects of these helicases, whether they use a strand-exclusion mechanism or whether they translocate along double-stranded DNA, for example, have been subjects of considerable debate (1, 2). Viral replicative helicases, such as SV40 Large-T antigen (LTag) and papillomavirus E1, have provided the opportunity to study some of these basic features largely owing to their homohexameric architecture. These viral helicases recognize their respective origin of DNA replication (ori) through their dsDNA-binding domains (DBDs) and assemble in a stepwise fashion, ultimately forming double-hexameric (DH) structures on their ori and unwind the DNA bidirectionally.E1 consists of an N-terminal domain, a DBD, an oligomerization domain (OD), a helicase/ATPase domain (HD), and a C-terminal acidic tail (Fig. 1A). Biochemical and structural data have demonstrated that the DBDs bound to the pseudopalindromic E1 binding site are at the center of the double hexamer and that the helicase domains that bind to the flanks of the ori are on either end in a head-to-head arrangement (3, 4). This arrangement is supported by EM studies of LTag that show a dumbbell-shaped structure for the DH (5, 6), with each half of the dumbbell containing two lobes: a larger HD outer lobe and a smaller DBD inner lobe. The assembled DH appears to place the HD of E1 proximal to the dsDNA to be unwound.However, in the structure of the E1 helicase with ssDNA and Mg 2+ -ADP, the ssDNA is oriented such that the N-terminal part of the polypeptide (the OD) is closest to the 5′ end of the DNA (7). The 3′→5′ polarity of E1 helicase indicates that the translocating helicase moves with the N-terminal OD leading and the C-terminal helicase domain trailing, or alternatively, that the DNA is pumped through the helicase from the OD side...

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. We demonstrate that the beta-hairpin has two functions. First, it is the tool used by the E1 double trimer (DT) to pry open and melt double-stranded DNA. Second, it is required for the unwinding activity of the hexameric E1 helicase. The fact that the same structural element, but not the same residues, contacts both dsDNA in the DT for melting and ssDNA in the double hexamer (DH) for helicase activity provides a link between local origin melting and DNA helicase activity and suggests how the transition between these two states comes about.

Summary 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. Here we dissect the local melting process carried out by the papillomavirus E1 protein. We characterize the melting process kinetically and identify mutations in the E1 helicase and in the ori that arrest the local melting process. We show that a subset of these mutants have specific defects for melting of the center of the ori containing the binding sites for E1 and demonstrate that these mutants fail to untwist the ori DNA. This newfound understanding of how E1 generates local melting suggests possible mechanisms for local melting in other replicons.

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. Phosphorylation at multiple sites in the Nterminal domain in E1 results in the loss of sequence-specific DNA binding activity, a feature that is also conserved in human papillomavirus (HPV) E1 proteins. The bovine papillomavirus (BPV) E2 protein, when phosphorylated by CK2 on two specific sites in the hinge, also loses its site-specific DNA binding activity. Mutation of these sites in E2 results in greatly increased levels of latent viral DNA replication, indicating that CK2 phosphorylation of E2 is a negative regulator of viral DNA replication during latent viral replication. In contrast, mutation of the N-terminal phosphorylation sites in E1 has no effect on latent viral DNA replication. We propose that the phosphorylation of the N terminus of E1 plays a role only in vegetative viral DNA replication, and consistent with such a role, caspase 3 cleavage of E1, which has been shown to be necessary for vegetative viral DNA replication, restores the DNA binding activity to phosphorylated E1.T he study of papillomaviruses has resulted in a fair understanding of the overall strategy that these viruses employ to infect their hosts and to generate new virus particles. Papillomaviruses infect the basal layers of the epithelium, where the early viral genes are expressed and the viral DNA is replicated at a low level (1). As the infected cells migrate toward the skin surface and differentiate into keratinocytes, the viral DNA is replicated at high levels, viral capsid proteins are produced, and new virus particles are assembled (1). In contrast to other well-studied viruses, reproduction of the viral life cycle in vitro is difficult but can be achieved with low efficiency (2-4). Consequently, although the general functions of the virus-encoded polypeptides are known, many subtleties, including the consequences of modifications of the viral polypeptides, ranging from alternative splicing to posttranslational modifications, have been difficult to analyze and are poorly understood.The viral E1 and E2 proteins have been studied biochemically, genetically, and structurally and are among the best-studied polypeptides encoded by the papillomaviruses (5, 6). The E1 protein is a site-specific DNA binding protein that binds to the viral origin of DNA replication (ori) and opens the DNA duplex in preparation for initiation of DNA replication and also serves as the replicative DNA helicase (7-15). The E2 protein is a DNA binding transcription factor that can regulate viral transcription by binding to specific sites in the viral genome (16-21). The E2 protein is also required...