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. 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.     

Chaperone proteins abrogate inhibition of the human papillomavirus (HPV) E1 replicative helicase by the HPV E2 protein

Human papillomavirus (HPV) DNA replication requires the viral origin recognition protein E2 and the presumptive viral replicative helicase E1. We now report for the first time efficient DNA unwinding by a purified HPV E1 protein. Unwinding depends on a supercoiled DNA substrate, topoisomerase I, single-stranded-DNA-binding protein, and ATP, but not an origin. Electron microscopy revealed completely unwound molecules. Intermediates contained two single-stranded loops emanating from a single protein complex, suggesting a bidirectional E1 helicase which translocated the flanking DNA in an inward direction. We showed that E2 protein partially inhibited DNA unwinding and that Hsp70 or Hsp40, which we reported previously to stimulate HPV-11 E1 binding to the origin and promote dihexameric E1 formation, apparently displaced E2 and abolished inhibition. Neither E2 nor chaperone proteins were detected in unwinding complexes. These results suggest that chaperones play important roles in the assembly and activation of a replicative helicase in higher eukaryotes. An E1 mutation in the ATP binding site caused deficient binding and unwinding of origin DNA, indicating the importance of ATP binding in efficient helicase assembly on the origin.     

The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization

The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.     

Characterization of simian virus 40 T-antigen double hexamers bound to a replication fork. The active form of the helicase

Large T-antigen (T-ag) is a viral helicase required for the initiation and elongation of simian virus 40 DNA replication. The unwinding activity of the helicase is powered by ATP hydrolysis and is critically dependent on the oligomeric state of the protein. We confirmed that the double hexamer is the active form of the helicase on synthetic replication forks. In contrast, the single hexamer cannot unwind synthetic forks and remains bound to the DNA as ATP is hydrolyzed. This inability of the T-ag single hexamer to release the DNA fork is the likely explanation for its poor helicase activity. We characterized the interactions of T-ag single and double hexamers with synthetic forks and single-stranded (ss) DNA. We demonstrated that DNA forks promote the formation of T-ag double hexamer. The lengths of the duplex region and the 3' tail of the synthetic forks are the critical factors in assembly of the double hexamer, which is bound to a single fork. We found that the cooperativity of T-ag binding to ss oligonucleotides increased with DNA length, suggesting that multiple consecutive subunits in the hexamer engage the ssDNA.     

Crystal structure of the DNA binding domain of the replication initiation protein E1 from papillomavirus

Papillomaviral infection causes both benign and malignant lesions and is a necessary cause of cervical carcinoma. Replication of this virus requires the replication initiation proteins E1 and E2, which bind cooperatively at the origin of replication (ori) as an (E1)2-(E2)2-DNA complex. This is a precursor to larger E1 complexes that distort and unwind the ori. We present the crystal structure of the E1 DNA binding domain refined to 1.9 A resolution. Residues critical for DNA binding are located on an extended loop and an alpha helix. We identify the E1 dimerization surface by selective mutations at an E1/E1 interface observed in the crystal and propose a model for the (E1)2-DNA complex. These and other observations suggest how the E1 DNA binding domain orchestrates assembly of the hexameric helicase on the ori.     

Crystal structures of two intermediates in the assembly of the papillomavirus replication initiation complex

Initiation of DNA replication of the papillomavirus genome is a multi-step process involving the sequential loading of viral E1 protein subunits onto the origin of replication. Here we have captured structural snapshots of two sequential steps in the assembly process. Initially, an E1 dimer binds to adjacent major grooves on one face of the double helix; a second dimer then binds to another face of the helix. Each E1 monomer has two DNA-binding modules: a DNA-binding loop, which binds to one DNA strand and a DNA-binding helix, which binds to the opposite strand. The nature of DNA binding suggests a mechanism for the transition between double- and single-stranded DNA binding that is implicit in the progression to a functional helicase.     

Characterization of the minimal DNA binding domain of the human papillomavirus e1 helicase: fluorescence anisotropy studies and characterization of a dimerization-defective mutant protein

The E1 helicase of papillomaviruses is required for replication of the viral double-stranded DNA genome, in conjunction with cellular factors. DNA replication is initiated at the viral origin by the assembly of E1 monomers into oligomeric complexes that have unwinding activity. In vivo, this process is catalyzed by the viral E2 protein, which recruits E1 specifically at the origin. For bovine papillomavirus (BPV) E1 a minimal DNA-binding domain (DBD) has been identified N-terminal to the enzymatic domain. In this study, we characterized the DBD of human papillomavirus 11 (HPV11), HPV18, and BPV E1 using a quantitative DNA binding assay based on fluorescence anisotropy. We found that the HPV11 DBD binds DNA with an affinity and sequence requirement comparable to those of the analogous domain of BPV but that the HPV18 DBD has a higher affinity for nonspecific DNA. By comparing the DNA-binding properties of a dimerization-defective protein to those of the wild type, we provide evidence that dimerization of the HPV11 DBD occurs only on two appropriately positioned E1 binding-sites and contributes approximately a 10-fold increase in binding affinity. In contrast, the HPV11 E1 helicase purified as preformed hexamers binds DNA with little sequence specificity, similarly to a dimerization-defective DBD. Finally, we show that the amino acid substitution that prevents dimerization reduces the ability of a longer E1 protein to bind to the origin in vitro and to support transient HPV DNA replication in vivo, but has little effect on its ATPase activity or ability to oligomerize into hexamers. These results are discussed in light of a model of the assembly of replication-competent double hexameric E1 complexes at the origin.     

Two patches of amino acids on the E2 DNA binding domain define the surface for interaction with E1

The E1 and E2 proteins from bovine papillomavirus bind cooperatively to the viral origin of DNA replication (ori), forming a complex which is essential for initiation of DNA replication. Cooperative binding has two components, in which (i) the DNA binding domains (DBDs) of the two proteins interact with each other and (ii) the E2 transactivation domain interacts with the helicase domain of E1. By generating specific point mutations in the DBD of E2, we have defined two patches of amino acids that are involved in the interaction with the E1 DBD. These same mutations, when introduced into the viral genome, result in severely reduced replication of the viral genome, as well as failure to transform mouse cells in tissue culture. Thus, the interaction between the E1 and E2 DBDs is important for the establishment of the viral genome as an episome and most likely contributes to the formation of a preinitiation complex on the viral ori.     

cis-Acting components of human papillomavirus (HPV) DNA replication: linker substitution analysis of the HPV type 11 origin.

Papillomavirus DNA replication requires the viral trans-acting factors E1 and E2 in addition to the host cell's general replication machinery. The origins of DNA replication in bovine and human papillomavirus genomes have been localized to a specific part of the upstream regulatory region (URR) which includes recognition sites for E1 and E2 proteins. To fine map cis-acting elements influencing human papillomavirus type 11 (HPV-11) DNA replication and to determine the relative contributions of such sites, we engineered consecutive linker substitution mutations across a region of 158 bp in the HPV-11 origin and tested mutant origins for replication function in a cell-based transient replication assay. Our results both confirm and extend the findings of others. E2 binding sites are the major cis components of HPV-11 DNA replication, and there is evidence for synergy between these sites. Differential capacity of the three E2 binding sites within the origin to affect replication may be attributed, at least in part, to context. At least one E2 binding site is essential for replication. The imperfect AT-rich palindrome of the E1 helicase binding site is not essential since replication occurs even in the absence of this sequence. However, replication is enhanced by the presence of the palindromic sequence in the HPV-11 origin. Sequence components adjacent to the E1 and E2 binding sites, comprising AT-rich and purine-rich elements and the consensus TATA box sequence, probably contribute to the overall efficiency of replication, though they are nonessential. None of the other cis elements of the HPV-11 origin region analyzed seems to influence replication significantly in the system described. The HPV-11 origin of DNA replication therefore differs from those of the other papovaviruses, simian virus 40 and polyomavirus, inasmuch as an intact helicase binding site and adjacent AT-rich components, while influential, are not absolutely essential.     

SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.     

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. Our results also indicate that melting of the DNA is achieved by destabilization of the double helix along its length through multiple interactions with E1, each of which is responsible for melting of a few base pairs, resulting in the extensive melting that is required for initiation of DNA replication.     

Functional Mapping of the DNA Binding Domain of Bovine Papillomavirus E1 Protein

Bovine papillomavirus type 1 (BPV-1) requires viral proteins E1 and E2 for efficient DNA replication in host cells. E1 functions at the BPV origin as an ATP-dependent helicase during replication initiation. Previously, we used alanine mutagenesis to identify two hydrophilic regions of the E1 DNA binding domain (E1DBD), HR1 (E1(179-191)) and HR3 (E1(241-252)), which are critical for sequence-specific recognition of the papillomavirus origin. Based on sequence and structure, these regions are similar in spacing and location to DNA binding regions A and B2 of T antigen, the DNA replication initiator of simian virus 40 (SV40). HR1 and A are both part of extended loops which are supported by residues from the HR3 and B2 alpha-helices. Both elements contain basic residues which may contact DNA, although lack of cocrystal structures for both E1 and T antigen make this uncertain. To better understand how E1 interacts with origin DNA, we used random mutagenesis and a yeast one-hybrid screen to select mutations of the E1DBD which disrupt sequence-specific DNA interactions. From the screen we selected seven single point mutants and one double point mutant (F175S, N184Y/K288R, D185G, V193M, F237L, K241E, R243K, and V246D) for in vitro analysis. All mutants tested in electrophoretic mobility shift assays displayed reduced sequence-specific DNA binding compared to the wild-type E1DBD. Mutants D185G, F237L, and R243K were rescued in vitro for DNA binding by the replication enhancer protein E2. We also tested the eight mutations in full-length E1 for the ability to support DNA replication in Chinese hamster ovary cells. Only mutants D185G, F237L, and R243K supported significant DNA replication in vivo which highlights the importance of E1DBD-E2 interactions for papillomavirus DNA replication. Based on the specific point mutations examined, we also assigned putative roles to individual residues in DNA binding. Finally, we discuss sequence and spacing similarities between E1 HR1 and HR3 and short regions of two other DNA tumor virus origin-binding proteins, SV40 T antigen and Epstein-Barr virus nuclear antigen 1 (EBNA1). We propose that all three proteins use a similar DNA recognition mechanism consisting of a loop structure which makes base-specific contacts (HR1) and a helix which primarily contacts the DNA backbone (HR3).     

Four different DNA helicases from calf thymus.

Using a strand displacement assay we have followed DNA helicase activities during the simultaneous isolation of several enzymes from calf thymus such as DNA polymerases alpha, delta, and epsilon, proliferating cell nuclear antigen, and replication factor A. Thus we were able to discriminate and isolate four different DNA helicases called A, B, C, and D. DNA helicase A is identical with the enzyme described earlier (Th?mmes, P., and Hübscher, U. (1990) J. Biol. Chem. 265, 14347-14354). The four enzymes can be distinguished by (i) their putative molecular weights after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (ii) glycerol gradient sedimentation under low and high salt conditions, (iii) sensitivity to salt, (iv) binding to DNA, (v) nucleoside- and deoxynucleoside 5'-triphosphate requirements, and (vi) by their direction of movement. DNA helicase A unwinds in the 3'----5' direction on the DNA it was bound to, while DNA helicases B, C, and D do so in the 5'----3' direction. DNA helicase D, and to some extent DNA helicases B and C, are able to unwind long substrates of more than 400 nucleotides. Replication factor A, a single-stranded heterotrimeric DNA binding protein involved in cellular DNA replication and DNA repair stimulates the DNA helicases. The stimulatory effect is most pronounced on DNA helicase A, where replication factor A enables this helicase to unwind longer substrates. DNA helicases B, C, and D are also stimulated by replication factor A. The effect of replication factor A appears to be specific since corresponding single-stranded DNA binding proteins from Escherichia coli and bacteriophage T4 have no or even a negative effect on the four DNA helicases. Heterologous human replication factor A has no stimulatory effect on any of the four DNA helicases suggesting a species specificity of these interactions. Thus it appears that mammalian cells possess, as does E. coli, a variety of different enzymes that can transiently abolish the double helical DNA structure in the cell.     

The inhibitory action of P56 on select functions of E1 mediates interferon's effect on human papillomavirus DNA replication

The interferon (IFN)-induced protein P56 inhibits human papillomavirus (HPV) DNA replication by binding to HPV E1, which has several distinct functions in initiating viral DNA replication. Here, we determined that P56 inhibited HPV type 18 (HPV18) E1's DNA helicase activity, E2 binding, and HPV Ori sequence-specific DNA binding but not nonspecific DNA binding. We observed that deletion of a single amino acid, F399, produced an E1 mutant that could not bind P56. This E1 mutant retained its ability to support Ori DNA replication, but this activity was not inhibited by IFN, demonstrating that P56 is the principal executor of the anti-HPV action of IFN.     

DNA-binding domain of bovine papillomavirus type 1 E1 helicase: structural and functional aspects.

The E1 protein of bovine papillomavirus type 1 is a multifunctional enzyme required for papillomaviral DNA replication. It assists in the initiation of replication both as a site-specific DNA-binding protein and as a DNA helicase. Previous work has indicated that at limiting E1 concentrations, the E2 protein is required for efficient E1 binding to the replication origin. In this study, we have defined the domain of the E1 protein required for site-specific DNA binding. Experiments with a series of truncated proteins have shown that the first amino-terminal 299 amino acids contain the DNA-binding domain; however, the coterminal M protein, which is homologous to E1 for the first 129 amino acids, does not bind origin DNA. A series of small internal deletions and substitution mutations in the DNA-binding domain of E1 show that specific basic residues in this region of the protein, which are conserved in all E1 proteins of the papillomavirus family, likely play a direct role in binding DNA and that a flanking conserved hydrophobic subdomain is also important for DNA binding. A region of E1 that interacts with E2 for cooperative DNA binding is also retained in carboxy-terminal truncated proteins, and we show that the ability of full-length E1 to complex with E2 is sensitive to cold. The E1 substitution mutant proteins were expressed from mammalian expression vectors to ascertain whether site-specific DNA binding by E1 is required for transient DNA replication in the cell. These E1 proteins display a range of mutant phenotypes, consistent with the suggestion that site-specific binding by E1 is important. Interestingly, one E1 mutant which is defective for origin binding but can be rescued for such activity by E2 supports significant replication in the cell.     

Bacteriophage T4 gene 59 helicase assembly protein binds replication fork DNA. The 1.45 A resolution crystal structure reveals a novel alpha-helical two-domain fold

The bacteriophage T4 gene 59 helicase assembly protein is required for recombination-dependent DNA replication, which is the predominant mode of DNA replication in the late stage of T4 infection. T4 gene 59 helicase assembly protein accelerates the loading of the T4 gene 41 helicase during DNA synthesis by the T4 replication system in vitro. T4 gene 59 helicase assembly protein binds to both T4 gene 41 helicase and T4 gene 32 single-stranded DNA binding protein, and to single and double-stranded DNA. We show here that T4 gene 59 helicase assembly protein binds most tightly to fork DNA substrates, with either single or almost entirely double-stranded arms. Our studies suggest that the helicase assembly protein is responsible for loading T4 gene 41 helicase specifically at replication forks, and that its binding sites for each arm must hold more than six, but not more than 12 nucleotides. The 1.45 A resolution crystal structure of the full-length 217-residue monomeric T4 gene 59 helicase assembly protein reveals a novel alpha-helical bundle fold with two domains of similar size. Surface residues are predominantly basic (pI 9.37) with clusters of acidic residues but exposed hydrophobic residues suggest sites for potential contact with DNA and with other protein molecules. The N-terminal domain has structural similarity to the double-stranded DNA binding domain of rat HMG1A. We propose a speculative model of how the T4 gene 59 helicase assembly protein might bind to fork DNA based on the similarity to HMG1, the location of the basic and hydrophobic regions, and the site size of the fork arms needed for tight fork DNA binding. The fork-binding model suggests putative binding sites for the T4 gene 32 single-stranded DNA binding protein and for the hexameric T4 gene 41 helicase assembly.     

The role of template superhelicity in the initiation of bacteriophage lambda DNA replication.

The prepriming steps in the initiation of bacteriophage lambda DNA replication depend on the action of the lambda O and P proteins and on the DnaB helicase, single-stranded DNA binding protein (SSB), and DnaJ and DnaK heat shock proteins of the E. coli host. The binding of multiple copies of the lambda O protein to the phage replication origin (ori lambda) initiates the ordered assembly of a series of nucleoprotein structures that form at ori lambda prior to DNA unwinding, priming and DNA synthesis steps. Since the initiation of lambda DNA replication is known to occur only on supercoiled templates in vivo and in vitro, we examined how the early steps in lambda DNA replication are influenced by superhelical tension. All initiation complexes formed prior to helicase-mediated DNA-unwinding form with high efficiency on relaxed ori lambda DNA. Nonetheless, the DNA templates in these structures must be negatively supertwisted before they can be replicated. Once DNA helicase unwinding is initiated at ori lambda, however, later steps in lambda DNA replication proceed efficiently in the absence of superhelical tension. We conclude that supercoiling is required during the initiation of lambda DNA replication to facilitate entry of a DNA helicase, presumably the DnaB protein, between the DNA strands.     

A ring-opening mechanism for DNA binding in the central channel of the T7 helicase-primase protein

We have investigated the mechanism of binding single-stranded DNA (ssDNA) into the central channel of the ring-shaped T7 gp4A' helicase-primase hexamer. Presteady-state kinetic studies show a facilitated five-step mechanism and provide understanding of how a ring-shaped helicase can be loaded on the DNA during the initiation of replication. The effect of a primase recognition sequence on the observed kinetics suggests that binding to the helicase DNA-binding site is facilitated by transient binding to the primase DNA-binding site, which is proposed to be a loading site. The proposed model involves the fast initial binding of the DNA to the primase site on the outside of the helicase ring, a fast conformational change, a ring-opening step, migration of the DNA into the central channel of the helicase ring, and ring closure. Although an intermediate protein-DNA complex is kinetically stable, only the last species in the five-step mechanism is poised to function as a helicase at the unwinding junction.     

Papillomavirus E1 protein binds to and stimulates human topoisomerase I

The papillomavirus (PV) E1 helicase plays a direct role in recruiting cellular DNA replication factors, such as replication protein A or polymerase alpha-primase, to replicate PV genomes. Here, E1 is shown to bind to human topoisomerase I and stimulate its relaxation activity up to sevenfold. The interaction between E1 and topoisomerase I was mapped to the E1 DNA binding domain and C terminus. These findings imply a mechanism for the recruitment of topoisomerase I to PV DNA replication forks and for stimulating topoisomerase I to allow for efficient relaxation of the torsional stress induced by replication fork progression.     

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. The defects of individual substitutions include failure to form the double trimer that melts the ori and failure to form the double hexamer that unwinds the ori. These results demonstrate that the DBD plays an essential role in multiple DNA replication-related processes apart from DNA binding.