Solution structure of the origin DNA-binding domain of SV40 T-antigen

Luo, Xuesong; Sanford, David G.; Bullock, Peter A.; Bachovchin, William W.

doi:10.1038/nsb1296-1034

Cited by 106 publications

(132 citation statements)

References 24 publications

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“…B, domain architecture of SV40 Tag. The DnaJ chaperone domain (72), SV40 OBD (73), and helicase domain (42,43) tution in Tag. Pull-down experiments also showed that K425E, but not K425R, substantially reduced Tag 357-627 binding to p68 1-107 (Fig.…”

Section: Resultsmentioning

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

Journal of Biological Chemistry

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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...

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Section: Resultsmentioning

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

Journal of Biological Chemistry

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“…Intramolecular Contacts between Domains I and II-The available atomic structures and electron microscopic data on native Tag and its oligomeric forms do not provide information on the location of domain I (13)(14)(15)(16)(17)(18)(19)(20)(21)(22). By immunoprecipitating a native tryptic digest of Tag monomers using a monoclonal antibody against an epitope in domain IIb (Pab204), short fragments containing only domain I were detected in the precipitate, implying that the short domain I fragments remained associated with domain II after digestion.…”

Section: Discussionmentioning

confidence: 99%

“…7A. We subdivided domain II into two parts (a and b) based on a Pronase E-resistant fragment generated with origin DNA-bound Tag (30) and the atomic structure of residues 131-259 (21). Monomeric Tag contains an extremely protease-sensitive region in the C-terminal part of domain IIb (darker shading in Fig.…”

Section: Discussionmentioning

confidence: 99%

Partial Proteolysis of Simian Virus 40 T Antigen Reveals Intramolecular Contacts between Domains and Conformation Changes upon Hexamer Assembly

Weißhart¹,

Friedl²,

Taneja

et al. 2004

Journal of Biological Chemistry

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“…Following the melting event, both proteins function as doubly hexameric helicases (Bullock 1997;Simmons 2000). Unexpectedly, structural studies have revealed that the site-specific DNA-binding domains of BPV E1 and T-antigen possess the same fold, despite sharing <10% amino acid sequence identity (Luo et al 1996;Enemark et al 2000). Li et al (2003) have recently determined the crystal structure of the hexameric T-antigen helicase domain.…”

Section: E1 and The Sv40 Large T-antigenmentioning

confidence: 99%

The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2

Abbate¹,

Berger²,

Botchan³

2004

Genes Dev.

147

172

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DNA replication of the papillomaviruses is specified by cooperative binding of two proteins to the ori site: the enhancer E2 and the viral initiator E1, a distant member of the AAA+ family of proteins. Formation of this prereplication complex is an essential step toward the construction of a functional, multimeric E1 helicase and DNA melting. To understand how E2 interacts with E1 to regulate this process, we have solved the X-ray structure of a complex containing the HPV18 E2 activation domain bound to the helicase domain of E1. Modeling the monomers of E1 to a hexameric helicase shows that E2 blocks hexamerization of E1 by shielding a region of the E1 oligomerization surface and stabilizing a conformation of E1 that is incompatible with ATP binding. Further biochemical experiments and structural analysis show that ATP is an allosteric effector of the dissociation of E2 from E1. Our data provide the first molecular insights into how a protein can regulate the assembly of an oligomeric AAA+ complex and explain at a structural level why E2, after playing a matchmaker role by guiding E1 to the DNA, must dissociate for subsequent steps of initiation to occur. Building on previously proposed ideas, we discuss how our data advance current models for the conversion of E1 in the prereplication complex to a hexameric helicase assembly.[Keywords: Crystal structure E1E2 complex; papillomavirus; helicase structure; AAA+ protein; DNA replication] Papillomaviruses (PVs) are small DNA viruses that maintain latency in the stem cells of various epithelial tissues (for review, see Howley 1996). They are the etiological agents of a variety of benign lesions of the epithelium, and certain high-risk variants of the human papillomavirus (HPV), such as HPV-16, 18, and 31, are associated with cervical carcinomas (Bosch et al. 1995). The virus is maintained as an extrachromosomal nuclear plasmid that replicates in S phase with the host genome. Because PVs have few open reading frames (∼8 ORFs), the viral life cycle depends heavily on interaction with the host cell for enzymes and ancillary factors for its own gene expression and replication programs. The PVs have provided important novel insights into the regulation of cell proliferation and cancer etiology; for example, the targeted degradation of p53 directed by the HPV16 E6 protein first revealed how key cellular regulatory proteins could be specifically ubiquitinated by E3 ligases for destruction by the proteosome (Scheffner et al. 1993). The PVs also have provided models to understand how eukaryotic transcription factors coassociate to form loops between segments of DNA (Li et al. 1991) and to explore assembly of specific replication factories on defined origins of replication (Stenlund 2003b).The first two universal steps in the formation of a prereplication complex are origin recognition and subsequent distortion and separation of duplex DNA strands. The simplicity of the PV prereplication complex offers advantages for mechanistic and structural dissection of these steps. Only t...

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Solution structure of the origin DNA-binding domain of SV40 T-antigen

Cited by 106 publications

References 24 publications

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

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

Partial Proteolysis of Simian Virus 40 T Antigen Reveals Intramolecular Contacts between Domains and Conformation Changes upon Hexamer Assembly

The X-ray structure of the papillomavirus helicase in complex with its molecular matchmaker E2

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