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 first step in initiation of DNA replication is the binding of an initiator protein to the replication origin (ori). Local melting of the DNA sequences in the ori and subsequent loading of the replicative DNA helicase follow this recognition step. Although it is well established that these processes must occur in all organisms with double-stranded DNA (dsDNA) genomes, the mechanistic understanding of these events is limited. For example, it is known that in Escherichia coli the initiator protein DnaA recognizes ori C and generates local melting, but the mechanism by which melting occurs is not known, although various models have been proposed (6, 13). Viral initiator proteins such as T-ag from simian virus 40 (SV40) and E1 protein from papillomaviruses provide good systems for the study of the early events in initiation of DNA replication since these multifunctional proteins can carry out all of the activities required to prepare the template for initiation of DNA replication in vitro. The papillomavirus initiator E1 is an ∼70-kDa AAA+ (ATPases associated with various cellular activities) protein and belongs to the SF3 helicase family (18). E1 proteins provide at least four activities required for replication initiation: specific and nonspecific DNA binding, local DNA melting of ori, and unwinding activity carried out by the E1 DNA helicase in front of the replication fork (11, 12, 14, 16, 20, 24, 26, 28, 30, 32). In recent years the study of viral initiator proteins such as E1 and SV40 T-ag has received a significant boost from a combination of biochemistry and structural biology (2, 8, 10, 15, 17, 19, 21, 27). The E1 DNA binding domain (DBD) has a high specificity for E1 binding sites (E1 BS) in the papillomavirus ori (28, 29). However, this sequence-specific DNA binding activity is masked in the full-length protein by a nonspecific DNA binding activity present in the E1 helicase domain (21, 28). The combination of these two DNA binding activities results in a modest specificity of E1 for binding to the ori, since the nonspecific DNA binding activity is dominant (28). Due to the low sequence specificity of E1, binding of E1 to the ori in vivo requires the virus-encoded transcription factor E2. E1 and E2 bind cooperatively to the ori in the form of an E12E22 ori complex that has high specificity for ori because the activation domain of E2 interacts physically with the helicase domain of E1 (1, 28). This interaction prevents nonspecific DNA binding by the E1 helicase domain, and the E12E22 complex therefore binds to the ori with much higher sequence specificity than E1 alone (28). After the E12E22 complex has bound to ori, the E2 dimer is displaced and further E1 molecules are added to the complex, eventually resulting in the formation of a complex containing six E1 molecules bound to the ori as a head-to-head E1 double trimer (DT). In vitro, in the absence of competitor DNA, the low specificity of E1 is sufficient to form the DT in the absence of E2 (21). The E1 DT serves as a required precursor for the functional helicase, which is an E1 double hexamer (DH). In the transition from a DT to a DH the template is melted locally, allowing the formation of the DH helicase on single-stranded DNA (ssDNA) (17, 21). The formation of the DT requires the cooperation of the sequence-specific DNA binding activity present in the E1 DBD and the nonspecific DNA binding activity present in the E1 helicase domain (21). E1 DBD and its binding to the E1 BS have been studied extensively by biochemical, genetic, and structural biology approaches and is well understood (3-5, 7, 9, 29). Much less is known about the nonspecific DNA binding activity and how the E1 helicase domain contacts dsDNA in a sequence-nonspecific manner. Structural information for the E1 helicase domain is available from several structures: a complex between the E1 helicase domain and the transactivation domain of E2, as well as a fragment containing the oligomerization and helicase domains of E1 as a hexamer assembled around ssDNA (1, 8). In the hexamer structure, the six helicase domains form a channel enclosing the ssDNA. Each helicase domain projects a specific motif, the β-hairpin, into the channel in a right-handed staircase pattern. The oligomerization domains contact each other to form a rigid collar that holds the six subunits together. This structural information does not provide direct information about the interaction between the E1 helicase domain and dsDNA since the structure contains ssDNA. However, based on previous studies, two conserved residues at the tip of the β-hairpin (K506 and H507) have been implicated in non-sequence-specific binding to dsDNA (2, 17, 21). Examination of the E1 structure provides additional candidates for residues that could be involved in dsDNA binding. We performed a mutagenesis screen of these surface residues in the E1 helicase domain together with some residues in the oligomerization domain to determine whether these were involved in non-sequence-specific DNA binding. In this screen we identified five residues in the E1 helicase domain that are required for sequence nonspecific DNA binding. Three of these residues—F464, R505, and K506—have dual function and affect both the helicase activity and the DT formation of E1, while the other two show severe defects only for DT formation.