Recruitment and loading of the E1 initiator protein: an ATP-dependent process catalysed by a transcription factor

Sanders, Cyril M.; Stenlund, Arne

doi:10.1093/emboj/17.23.7044

Cited by 103 publications

(144 citation statements)

References 38 publications

(55 reference statements)

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“…Permanganate reactivity assays were performed essentially as described previously (5,25). Binding reaction mixtures (20 mM HEPES [pH 7.9], 100 mM NaCl, 0.1% NP-40, 5% glycerol, 5 mM dithiothreitol [DTT], 5 mM MgCl 2 , and 5 mM ATP) containing ϳ10 fmol of endlabeled probe were assembled and incubated with E1 at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…For eukaryotes, although the origin recognition complex has been identified as the factor that marks the replicator (2), an activity that can melt the DNA in preparation for replication has still not been identified. Viral initiator proteins such as the simian virus 40 (SV40) large T antigen (T-Ag) and the papillomavirus E1 protein have long been known to melt their respective origins of DNA replication, as detected by oxidation with KMnO 4 (4,5,14,22,24,25). However, little information exists about which forms of these proteins execute melting, which parts of the polypeptide are responsible for the melting activity, and whether this process is DNA sequence dependent (5, 27).…”

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confidence: 99%

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ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

Schuck

Stenlund

2007

J Virol

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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.Template melting is an essential step in the initiation of replication of double-stranded DNA. In spite of its fundamental importance for DNA replication, the melting process is almost entirely uncharacterized. For Escherichia coli, the initiator DnaA has long been known to melt OriC, as can be detected by the use of single-stranded DNA (ssDNA)-specific nucleases (6, 32). The mechanism by which the DNA is melted is unknown, but it is established that melting is dependent on nucleotide binding by DnaA (for a review, see reference 16). For eukaryotes, although the origin recognition complex has been identified as the factor that marks the replicator (2), an activity that can melt the DNA in preparation for replication has still not been identified. Viral initiator proteins such as the simian virus 40 (SV40) large T antigen (T-Ag) and the papillomavirus E1 protein have long been known to melt their respective origins of DNA replication, as detected by oxidation with KMnO 4 (4,5,14,22,24,25). However, little information exists about which forms of these proteins execute melting, which parts of the polypeptide are responsible for the melting activity, and whether this process is DNA sequence dependent (5, 27).The E1 proteins from papillomaviruses are ϳ70-kDa polypeptides which, in addition to DNA melting activity, have DNA helicase activity (19,20,30,33,35,37,38,40) and also bind DNA. DNA binding by E1 is the result of two different DNA binding activities. Site-specific DNA binding is provided by the E1 DNA binding domain (DBD), which recognizes and binds to two pairs of E1 binding sites (E1 BS) in the origin of replication (7-9, 11, 15, 17, 31, 36). The E1 helicase domain binds DNA with low sequence specificity, and this activity is required for the ability of the E1 helicase domain to contact the DNA sequences flanking the E1 BS, including a region that has been termed the A/T-rich region (34).Recent advances in the study of E1 and T-Ag have opened up the melting process for more detailed study. Structural studies of E1 and T-Ag have provided important information about the domain structure, how these proteins oligomerize, and how they bind and hydro...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

Schuck

Stenlund

2007

J Virol

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“…Purified E1 protein can initiate in vitro viral replication without the support of the E2 protein (6). However, the interaction of E1 with E2 protein promotes specific binding and assembly of E1 protein on the viral origin to form an active initiation complex (7,8). Additional functions of E1 include recruiting cellular replication factors, such as the DNA polymerase ␣ and replication protein A (9 -11).…”

Section: Bovine Papillomavirus (Bpv)mentioning

confidence: 99%

SUMO-1 Modification of Bovine Papillomavirus E1 Protein Is Required for Intranuclear Accumulation

Rangasamy¹,

Woytek²,

Khan³

et al. 2000

Journal of Biological Chemistry

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The E1 protein is a multifunctional, origin-binding helicase that is essential for replication of papillomaviruses. Recently, bovine papillomavirus E1 was shown to be post-translationally modified by the addition of the SUMO-1 polypeptide. Here we show that the site of sumoylation maps to lysine residue 514. This lysine and the flanking sequences are well conserved in human papillomavirus (HPV) E1 proteins. Both HPV1a and HPV18 E1 proteins are substrates for sumoylation in vitro, which is consistent with this modification being a general property of E1 proteins. Mutations, which impair the sumoylation of bovine papillomavirus E1, prevent normal nuclear accumulation of E1 with a concomitant loss of replication capacity. These results suggest that sumoylation plays a role in nuclear transport and could regulate the E1 replication function by controlling access to the nuclear replication domains. Bovine papillomavirus (BPV)1 E1 protein is the major initiator protein for viral DNA replication and thus plays a critical role in the establishment of episomal viral genomes in the host cell nucleus (1). E1 and E2 proteins are the only two viral proteins required to replicate the viral genome in vivo, whereas the rest of the replication machinery is provided by the host cell (2, 3). E1 is a DNA helicase that binds to specific sequences in the viral origin and unwinds the viral DNA for initiation of replication (4, 5). Purified E1 protein can initiate in vitro viral replication without the support of the E2 protein (6). However, the interaction of E1 with E2 protein promotes specific binding and assembly of E1 protein on the viral origin to form an active initiation complex (7,8). Additional functions of E1 include recruiting cellular replication factors, such as the DNA polymerase ␣ and replication protein A (9 -11). E1 also interacts with histone H1 (12) and SW1/SNF5 (13), suggesting that E1 plays a role in chromatin remodeling at the viral origin.Recently, we demonstrated that BPV E1 protein is posttranslationally modified by a novel process known as sumoylation (14). Sumoylation involves the covalent attachment of the small ubiquitin-related proteins (SUMO-1, -2, or -3) to target substrates (15). Sumoylation is achieved by a multistep process in a manner that is analogous to ubiquitinylation (reviewed in Ref. 16). SUMO is first activated by a heterodimeric enzyme termed Aos1/Uba2, and then the activated SUMO is covalently transferred to the SUMO-specific conjugating enzyme Ubc9 via a thioester linkage (17). Ubc9 physically interacts with various substrates and transfers SUMO to one or more lysine residues in the target protein. Unlike ubiquitinylation in which the primary function is targeting proteins for degradation, the effects of sumoylation are more substrate-specific and can result in alterations in function (18 -20), stability (21), or intracellular location (22-25) of the modified protein. For BPV E1, a mutant unable to interact with Ubc9 showed altered intranuclear distribution (14). As BPV E1 is the first kno...

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“…Formation of this E1-E2-ori ternary complex is dependent on the interaction of both proteins with DNA but also on a critical interaction between the N-terminal transactivation domain (TAD) of E2 and the C-terminal enzymatic domain of E1 (15)(16)(17)(18)(19)(20)(21)(22). Assembly of this initial E1-E2-ori complex serves as a starting point for the recruitment of additional E1 molecules (23,24) and their assembly into hexamers and double hexamers that have ATPase and helicase activity (25,26).…”

mentioning

confidence: 99%

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

White

Titolo

Brault

et al. 2003

Journal of Biological Chemistry

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Human papillomavirus (HPV) DNA replication is initiated by recruitment of the E1 helicase by the E2 protein to the viral origin. Screening of our corporate compound collection with an assay measuring the cooperative binding of E1 and E2 to the origin identified a class of small molecule inhibitors of the protein interaction between E1 and E2. Isothermal titration calorimetry and changes in protein fluorescence showed that the inhibitors bind to the transactivation domain of E2, the region that interacts with E1. These compounds inhibit E2 of the low risk HPV types 6 and 11 but not those of high risk HPV types or of cottontail rabbit papillomavirus. Functional evidence that the transactivation domain is the target of inhibition was obtained by swapping this domain between a sensitive (HPV11) and a resistant (cottontail rabbit papillomavirus) E2 type and by identifying an amino acid substitution, E100A, that increases inhibition by ϳ10-fold. This class of inhibitors was found to antagonize specifically the E1-E2 interaction in vivo and to inhibit HPV DNA replication in transiently transfected cells. These results highlight the potential of the E1-E2 interaction as a small molecule antiviral target.Papillomaviruses are a family of small double-stranded DNA viruses that induce benign and malignant hyperproliferative lesions of the differentiating epithelium (reviewed in Refs. 1-4). Approximately 25 types of human papillomavirus (HPV) 1 infect the anogenital region. These HPV types have been classified as low risk or high risk types depending on whether they cause benign warts or lesions that can progress to invasive cancer, respectively. Current therapies to remove HPV-induced lesions include a variety of ablative or cytodestructive procedures and the use of immunomodulatory molecules such as imiquimod (5, 6) to stimulate a host immune response. Small molecule antivirals for the treatment of HPV infections do not currently exist.The life cycle of HPV is coupled to the cellular differentiation program that occurs in the epithelium (7). Maintenance of the viral genome in infected cells of the basal layer is essential for the viral life cycle and the ensuing pathology. Maintenance of the HPV episome in primary keratinocyte cultures depends on the function of E1 and E2, a 3Ј-5Ј helicase (8) and a sequencespecific DNA-binding protein (9), respectively, which are required for replication of the genome (10). HPV DNA replication is initiated by the co-operative binding of E1 and E2 to specific DNA sequences within the viral origin (11-14). Formation of this E1-E2-ori ternary complex is dependent on the interaction of both proteins with DNA but also on a critical interaction between the N-terminal transactivation domain (TAD) of E2 and the C-terminal enzymatic domain of E1 (15-22). Assembly of this initial E1-E2-ori complex serves as a starting point for the recruitment of additional E1 molecules (23, 24) and their assembly into hexamers and double hexamers that have ATPase and helicase activity (25,26).Any of the protein-pr...

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Recruitment and loading of the E1 initiator protein: an ATP-dependent process catalysed by a transcription factor

Cited by 103 publications

References 38 publications

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

ATP-Dependent Minor Groove Recognition of TA Base Pairs Is Required for Template Melting by the E1 Initiator Protein

SUMO-1 Modification of Bovine Papillomavirus E1 Protein Is Required for Intranuclear Accumulation

Inhibition of Human Papillomavirus DNA Replication by Small Molecule Antagonists of the E1-E2 Protein Interaction

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