Human Papillomavirus DNA Replication

Replication of simian virus 40 (SV40) DNA, a model for eukaryotic chromosomal replication, can be reconstituted in vitro using the viral helicase (large tumor antigen, or Tag) and purified human proteins. Tag interacts physically with two cellular proteins, replication protein A and DNA polymerase ␣-primase (pol-prim), constituting the viral primosome. Like the well characterized primosomes of phages T7 and T4, this trio of proteins coordinates parental DNA unwinding with primer synthesis to initiate the leading strand at the viral origin and each Okazaki fragment on the lagging strand template. We recently determined the structure of a previously unrecognized pol-prim domain (p68N) that docks on Tag, identified the p68N surface that contacts Tag, and demonstrated its vital role in primosome function. Here, we identify the p68N-docking site on Tag by using structure-guided mutagenesis of the Tag helicase surface. A charge reverse substitution in Tag disrupted both p68N-binding and primosome activity but did not affect docking with other pol-prim subunits. Unexpectedly, the substitution also disrupted Tag ATPase and helicase activity, suggesting a potential link between p68N docking and ATPase activity. To assess this possibility, we examined the primosome activity of Tag with a single residue substitution in the Walker B motif. Although this substitution abolished ATPase and helicase activity as expected, it did not reduce pol-prim docking on Tag or primosome activity on single-stranded DNA, indicating that Tag ATPase is dispensable for primosome activity in vitro.De novo DNA replication begins with RNA primer synthesis on single-stranded template DNA, followed by primer extension by a processive DNA polymerase. In prokaryotic replication, the activity of the primase is coordinated with unwinding of duplex DNA by a hexameric replicative helicase and a single-stranded DNA (ssDNA) 2 -binding protein, largely through dynamic physical interactions among the three proteins, which constitute a primosome (1-4). In eukaryotes, the DNA polymerase ␣-primase (pol-prim) complex catalyzes both RNA primer synthesis and extension, yielding RNA-DNA primers of 30 -35 nucleotides (5, 6). Unlike the single subunit prokaryotic primases, pol-prim is a stable heterotetramer composed of the primase heterodimer p48/p58, the catalytic DNA polymerase subunit p180, and a regulatory subunit (B or p68) (7). The eukaryotic replicative helicase complex, Cdc45/ Mcm2-7/GINS, and the ssDNA-binding protein, replication protein A (RPA), appear to coordinate primer synthesis by polprim with parental DNA unwinding, as in prokaryotes (8 -12). However, the nature of the eukaryotic primosome and its operation during chromosome replication, telomere maintenance, and checkpoint signaling at stalled replication forks remain elusive.Because pol-prim is essential for replication of simian virus 40 (SV40) DNA, we utilize this model system here to investigate the functional architecture of a eukaryotic primosome. SV40 DNA replication can be reconstituted in c...

Section: Discussionmentioning

confidence: 99%

A Specific Docking Site for DNA Polymerase α-Primase on the SV40 Helicase Is Required for Viral Primosome Activity, but Helicase Activity Is Dispensable

Huang

Zhao

Arnett

et al. 2010

“…For BPV, it has been demonstrated that ATP hydrolysis is required for displacement of E2 from the origin during assembly of E1 multimeric complexes (17). Either concurrent with or after formation of E1 oligomers, the host polymerase ␣ primase (21)(22)(23)(24)(25) and possibly also replication protein A (22,26) bind to E1, and a replication complex is formed together with additional host factors.…”

Section: From the Department Of Biological Sciences Boehringer Ingelmentioning

confidence: 99%

“…Since E2 affects ATP binding (K m ) rather than hydrolysis (k cat ), we would expect that ATP binding suffices to weaken the interaction of E1 with E2 (75), probably by inducing conformational changes in E1. In virus-infected cells, where E2 is present at low concentrations but ATP is in large excess, this perturbation of E2 binding could promote efficient viral DNA replication, since previous work by us (25) and others (23,24) has suggested that E2 must dissociate from E1 prior to association with the polymerase ␣ primase. Hence, saturation of E1 with ATP subsequent to E1 oligomerization may help release E1 from E2, and allow for its interaction with pol ␣ primase.…”

mentioning

confidence: 99%

Characterization of Recombinant HPV6 and 11 E1 Helicases

White

Pelletier

Brault

et al. 2001

To better characterize the enzymatic activities required for human papillomavirus (HPV) DNA replication, the E1 helicases of HPV types 6 and 11 were produced using a baculovirus expression system. The purified wild type proteins and a version of HPV11 E1 lacking the N-terminal 71 amino acids, which was better expressed, were found to be hexameric over a wide range of concentrations and to have helicase and ATPase activities with relatively low values for K m (ATP) of 12 M for HPV6 E1 and 6 M for HPV11 E1. Interestingly, the value of K m (ATP) was increased 7-fold in the presence of the E2 transactivation domain. In turn, ATP was found to perturb the co-operative binding of E1 and E2 to DNA. Mutant and truncated versions of in vitro translated E1 were used to identify a minimal ATPase domain composed of the C-terminal 297 amino acids. This fragment was expressed, purified, and found to be fully active in ATP hydrolysis, single-stranded DNA binding, and unwinding assays, despite lacking the minimal origin-binding domain.The papillomaviruses (PVs) 1 are small, nonenveloped DNA viruses that infect and replicate in the cutaneous or mucosal epithelia of humans and other mammals. There are over 100 types of human papillomavirus (HPV), which cause conditions ranging from plantar warts (HPV1) and genital warts (HPV6 and -11) to cervical cancer (HPV16, -18, and -31). HPV6 and -11 are also responsible for laryngeal papillomatosis, a rare but very serious infection of the respiratory tract (1). Antiviral agents capable of specifically inhibiting PV replication could play an important role in the treatment of these diseases, but none exist at this time.Despite their host and tissue specificity, all of the PV types share a common genomic organization. The closed, circular genome of ϳ8000 base pairs codes for only 10 proteins: eight early proteins termed E1-E8 and two late proteins, L1 and L2, that make up the viral coat (2). E1 and E2 are the only viral proteins required for HPV DNA replication (3, 4). E2 is a sequence-specific DNA-binding protein that serves to regulate both transcription and DNA replication. E1, a DNA helicase, is the only PV protein that possesses enzymatic activity (5) and is also the most highly conserved of the PV proteins. For these reasons, E1 has been considered as the most attractive molecular target for the development of antiviral agents (6).During the initiation of PV DNA replication, E2 serves as a specificity factor to enhance binding of E1 monomers to the origin, which by themselves bind to double-stranded DNA with little sequence specificity (4, 7-11). E2 dimers bind with high specificity to DNA sequences within the origin and recruit to it E1 monomers, through direct protein-protein interactions (12, 13). Upon binding to DNA, E1 monomers assemble into hexamers (14, 15), but the E1-E2 interaction is not maintained (16,17). Thus, the role of E2 is to catalyze the assembly of hexameric E1 complexes specifically at the origin. ATP hydrolysis is also required for E1 hexamers to distort the orig...

“…E1 protein interacts directly with the p180 and p70 subunits of DNA polymerase ␣/primase (23)(24)(25)(26) and with RPA (27) and is required throughout the initiation and elongation stages, whereas E2 protein is needed only during initiation (28). E2 protein helps recruit E1 protein, preferentially from the homologous HPV genotype, to the origin (21,22,29).…”

mentioning

confidence: 99%

HeLa Cells Are Phenotypically Limiting in Cyclin E/CDK2 for Efficient Human Papillomavirus DNA Replication

Lin

Liu

et al. 2000

Self Cite

Human papillomaviral (HPV) origin-containing plasmids replicate efficiently in human 293 cells or cell extracts in the presence of HPV origin-recognition protein E2 and replication initiation protein E1, whereas cervical carcinoma-derived, HPV-18-positive HeLa cells or cell extracts support HPV DNA replication poorly. We recently showed that HPV-11 E1 interacts with cyclin/ cyclin-dependent kinase (cdk) complexes through an RXL motif and is a substrate for these kinases. E1 mutations in this motif or in candidate cdk phosphorylation sites are impaired in replication, suggesting a role for cdks in HPV replication. We now demonstrate that one limiting activity in HeLa cells is cyclin E/CDK2. Small DNA viruses such as SV40/polyomavirus and papillomaviruses each encode one or two viral origin (ori) recognition and initiator proteins and rely heavily on the cellular DNA replication machinery. Because the interactions among viral and cellular proteins mimic those among the cellular counterparts, these viruses have proven to be particularly useful model systems. The SV40 system has aided in the identification and mechanistic description of several important replication components (reviewed in Ref.2) and more recently has revealed the opposing effects of cyclin E/CDK2 and cyclin A/CDK2 in regulating DNA polymerase ␣/primase via direct phosphorylation (3). The fact that papillomaviruses are highly prevalent and medically important human and animal pathogens also makes them attractive to investigators (reviewed in Ref. 4).The genome of papillomaviruses is an approximately 8-kilobase pair double-stranded circular DNA. Because the productive life cycle takes place only in keratinocytes undergoing terminal squamous differentiation (4), viral DNA replication is studied either in a cell-free system or in transiently transfected cells (reviewed in Refs. 5 and 6). The ori of all papillomaviruses consists of one binding site for the virus-encoded E1 protein and several highly conserved binding sites for the virus-encoded E2 protein. E2 is a critical origin recognition protein (7-15), and E1 is the initiator and a DNA helicase (16 -20). They are functionally equivalent to the cellular origin recognition complex and MCM complexes, respectively, during initiation. Replication is dependent on the cellular DNA replication machinery, including DNA polymerase ␣/primase, DNA polymerase ␦, replication protein A (RPA, the single-stranded DNAbinding protein), the proliferating cell nuclear antigen (the processivity clamp for DNA polymerase ␦), replication factor C (the clamp loader), and topoisomerases I and II (8,14). As a consequence of this high degree of conservation in cis sequence elements and trans-acting factors, E1 and E2 proteins from any one virus can replicate all papillomavirus origins (Refs. 21 and 22 and references therein).E1 protein interacts directly with the p180 and p70 subunits of DNA polymerase ␣/primase (23-26) and with RPA (27) and is required throughout the initiation and elongation stages, whereas E2 protein is nee...