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. We conclude that the dimer interaction is primarily required for initial recognition of ori.     

Sequential and ordered assembly of E1 initiator complexes on the papillomavirus origin of DNA replication generates progressive structural changes related to melting

Multiple binding sites for an initiator protein are a common feature of replicator sequences from various organisms. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA by largely unknown mechanisms. Here we analyze origin of DNA replication (ori) binding by the E1 initiator and show sequential binding to a set of overlapping binding sites. The assembly of these initiator complexes is controlled by a gradual reduction in the dependence of interactions between the initiator and DNA and a gradual increase in the reliance on interactions between initiator molecules, providing a mechanism for sequential and orderly assembly. Importantly, the binding of the initiator causes progressive structural alterations both in the sites and in the sequences flanking the sites, eventually generating severe structural alterations. These results indicate that the process of template melting may be incremental, where binding of each initiator molecule serves as a wedge that upon binding gradually alters the template structure. This mechanism may explain the requirement for multiple initiator binding sites that is observed in many ori's.     

E1 recognition sequences in the bovine papillomavirus type 1 origin of DNA replication: interaction between half sites of the inverted repeats.

The E1 protein encoded by bovine papillomavirus type 1 (BPV-1) is required for viral DNA replication, and it binds site specifically to an A/T-rich palindromic sequence within the viral origin of replication. The protein is targeted to this site through cooperative interactions and binding with the virus-encoded E2 protein. To explore the nature of the E1 binding site, we inserted a series of homologous DNA linkers at the center of dyad symmetry within the E1 recognition palindrome. The effects of these modifications indicated that the E1 recognition palindrome can be separated into functional half sites. The series of insertions manifest a phasing relationship with respect to the wild-type BPV-1 genome in that greater biological activity was measured when full integral turns of the DNA helix separated the palindrome than when the separations were half-turns. This phasing pattern of activity was observed to occur in a variety of biological phenotypes, including transformation efficiency, stable plasmid copy number in cell lines established from pooled foci, and transient replication of full-length viral genomes. For replication reporter constructs where E1 and E2 are supplied in trans by the respective expression vectors, distance between the half sites seems to play a major role, yet the phasing relationships are measurable. DNase I protection studies showed that E1 bound very poorly to the construct containing a 5-bp linker, and binding was close to the wild-type level for the 10-bp insertion, consistent with a requirement for a phasing function between half sites with a modulus of 10 bp. Binding to the 15- and 20-bp insertion mutants was weak, but only for the 20-bp insertions was protection over both halves of the palindrome measurable. As it had been previously reported that the 18-bp palindrome contains sufficient nucleotide sequence information for E1 binding, we speculate that a minimal E1 recognition motif is presented in each half site. A comparison between this sequence and that of an upstream region that also binds E1 (the E2RE1 region) revealed a common pentanucleotide motif of APyAAPy. Mutants with substitutions of the ATAAT elements within E2RE1 failed to bind E1 protein. We present models for how repeats of the pentanucleotide sequence may coordinate E1 binding at the dyad symmetry axis of the origin and compare the DNA sequence organization of BPV-1 with those of the simian virus 40 and polyomaviruses at their origins of DNA replication.     

The E2 binding sites determine the efficiency of replication for the origin of human papillomavirus type 18.

Human papillomaviruses (HPV-s) have been shown to possess transforming and immortalizing activity for many different, mainly keratinocyte cell lines and they have been detected in 90% of anogenital cancer tissues, which suggests a causative role in the induction of anogenital and other tumours. We have exploited a quantitative assay to identify and characterize the origin of replication of the human papillomavirus type 18 (HPV-18), one of the most prevalent types in the high-risk HPV group. Replication of HPV origin fragments was studied transiently by cotransfection with a protein expression vector providing replication proteins E1 and E2. We have localized the HPV-18 origin to nucleotides 7767-119. This region contains three E2 binding sites and an essential A/T rich DNA region (nucleotides 9-35) that is partly homologous to the E1 binding site found in bovine papillomavirus type 1 (BPV-1) genome. At least one of the three E2 binding sites was absolutely required for origin function; addition of other E2 sites had cooperative stimulating effect. This is the first quantitative analysis of the E2 binding sites for papillomavirus replication.     

Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin

Minute virus of mice (MVM) replicates via a linearized form of rolling-circle replication in which the viral nickase, NS1, initiates DNA synthesis by introducing a site-specific nick into either of two distinct origin sequences. In vitro nicking and replication assays with substrates that had deletions or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end (5') viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of opposing NS1 binding sites, located near its outboard end, create a 33-bp palindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cruciform, was dispensable for origin function, as was the NS1 binding site occupying the inboard arm of the palindrome. In contrast, the NS1 site in the outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additional NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end origin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each end of the origin to interact, creating a distortion characteristic of a double helical loop.     

Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A

Papovaviruses utilize predominantly cellular DNA replication proteins to replicate their own viral genomes. To appropriate the cellular DNA replication machinery, simian virus 40 (SV40) large T antigen (Tag) binds to three different cellular replication proteins, the DNA polymerase alpha-primase complex, the replication protein A (RPA) complex, and topoisomerase I. The functionally similar papillomavirus E1 protein has also been shown to bind to the DNA polymerase alpha-primase complex. Enzyme-linked immunoassay-based protein interaction assays and protein affinity pull-down assays were used to show that the papillomavirus E1 protein also binds to the cellular RPA complex in vitro. Furthermore, SV40 Tag was able to compete with bovine papillomavirus type 1 E1 for binding to RPA. Each of the three RPA subunits was individually overexpressed in Escherichia coli as a soluble fusion protein. These fusion proteins were used to show that the E1-RPA and Tag-RPA interactions are primarily mediated through the 70-kDa subunit of RPA. These results suggest that different viruses have evolved similar mechanisms for taking control of the cellular DNA replication machinery.     

The nucleotide sequence surrounding the origin of DNA replication of Col E1.

The DNA of Col E1 replicates from a unique origin located at a distance of 17-19% of the genome length from the single Eco RI clevage site. The nucleotide sequence about this site has been determined by a combination of RNA and DNA sequencing techniques. The principal features of the sequence are two palindromes, one of which resembles a palindrome located in the intercistronic region of 0X174. The sequence also contains stretches of purine and pyrimidine clusters of the following compositions: pAT5G, pC2T5G, pGT5G. The origin sequence demonstrates that initiation of DNA replication takes place in an intercistronic region of Col E1DNA, although the possibility that this region makes small polypeptides 30-40 residues long cannot be strictly eliminated at this time.     

Two E2 binding sites alone are sufficient to function as the minimal origin of replication of human papillomavirus type 18 DNA.

Replication of papillomaviruses requires an origin of replication and two virus-encoded proteins, E1 and E2. Using a transient replication assay for human papillomavirus type 18 (HPV-18) DNA, we have found that two adjacent sequences present within the origin of replication can independently support replication. The first, a 77-bp region, contains one E2 binding site (E2BS) and a 16-bp inverted repeat element that probably corresponds to the E1 binding site (E1BS). The other, an 81-bp region, includes two E2BS but lacks the putative E1BS. A synthetic 33-bp oligonucleotide containing two high-affinity E2BS was also found to function as an origin of replication. Replication of all these plasmids was absolutely dependent on the presence of the HPV-18 E1 and E2 proteins. The HPV-1a E1 and E2 proteins were also found to support replication of a plasmid containing the complete HPV-18 origin but failed to replicate a plasmid containing two E2BS alone. Our results suggest that the E2 protein can target E1 to the origin through the formation of an E1-E2 complex which is likely to be involved the initiation of replication.     

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.     

Binding of the E1 and E2 proteins to the origin of replication of bovine papillomavirus.

DNA replication of bovine papillomavirus (BPV) requires two viral proteins encoded from the E1 and E2 open reading frames. E1 and E2 are sequence-specific DNA binding proteins that bind to their cognate binding sites in the BPV origin of replication (ori). The E1 and E2 proteins can interact physically with each other, and this interaction results in cooperative binding when binding sites for both proteins are present. We have analyzed the binding of E1 to the ori in the absence and presence of E2, using DNase I footprint analysis, gel mobility shift assays, and interference analysis. We have also generated a large number of point mutations in the E1 binding site and tested them for binding of E1 as well as for activity in DNA replication. Our results demonstrate that E1 binds to the ori in different forms in the absence and presence of E2 and that E2 has both a quantitative and a qualitative effect on the binding of E1. Our results also suggest that the ori contains multiple overlapping individual E1 recognition sequences which together constitute the E1 binding site and that different subsets of these recognition sequences are used for binding of E1 in the presence and absence of E2.     

Recognition sites for a membrane-derived DNA binding protein preparation in the E. coli replication origin

Summary The DNA binding protein B' preparation, isolated from the membrane of E. coli, recognizes two sites, one of which is locatd in the minimum oriC (35–270 bp) and the other between base pairs 417 and 488. Recognition is only possible when restriction fragments containing these sites are in single-stranded state. At the first site the strand reading 3OH-5P in the direction of the E. coli genetic map is recognized, at the second site the 5P-3OH strand.     

Common determinants in DNA melting and helicase-catalysed DNA unwinding by papillomavirus replication protein E1

E1 and T-antigen of the tumour viruses bovine papillomavirus (BPV-1) and Simian virus 40 (SV40) are the initiator proteins that recognize and melt their respective origins of replication in the initial phase of DNA replication. These proteins then assemble into processive hexameric helicases upon the single-stranded DNA that they create. In T-antigen, a characteristic loop and hairpin structure (the pre-sensor 1β hairpin, PS1βH) project into a central cavity generated by protein hexamerization. This channel undergoes large ATP-dependent conformational changes, and the loop/PS1βH is proposed to form a DNA binding site critical for helicase activity. Here, we show that conserved residues in BPV E1 that probably form a similar loop/hairpin structure are required for helicase activity and also origin (ori) DNA melting. We propose that DNA melting requires the cooperation of the E1 helicase domain (E1HD) and the origin binding domain (OBD) tethered to DNA. One possible mechanism is that with the DNA locked in the loop/PS1βH DNA binding site, ATP-dependent conformational changes draw the DNA inwards in a twisting motion to promote unwinding.     

Ribonucleotide-deoxyribonucleotide linkages at the origin of DNA replication of colicin E1 plasmid

We have shown by nearest-neighbor analysis that ribonucleotide-DNA linkages exist on 6 S L-strand² fragments, the first DNA fragments made on Colicin E1 plasmid, at the origin of replication. The linkages rApdA and rApdC predominate. This is in agreement with the previous finding that located the 5′ end of the 6 S L-fragments on the plasmid DNA sequence (Tomizawa et al., 1977). Most of the molecules that contain ribonucleotides have a single rA residue and a large fraction of this residue is phosphorylated at the 5′ end. A total of 20 to 40% of the 6 S L-fragments contain at least one ribonucleotide.     

Identification of the origin of replication of bovine papillomavirus and characterization of the viral origin recognition factor E1.

Expression of the viral polypeptides E1 and E2 is necessary and sufficient for replication of BPV in mouse C127 cells. By providing these factors from heterologous expression vectors we have identified a minimal origin fragment from BPV that contains all the sequences required in cis for replication of BPV in short term replication assays. This same sequence is also required for stable replication in the context of the entire viral genome. The identified region is highly conserved between different papillomaviruses, and is unrelated to the previously identified plasmid maintenance sequences. The minimal ori sequence contains a binding site for the viral polypeptide E1, which we identify as a sequence specific DNA binding protein, but surprisingly, an intact binding site for the viral transactivator E2 at the ori is not required. The isolated origin shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines.