scholarly journals The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Dahai Gai ◽  
Damian Wang ◽  
Shu-Xing Li ◽  
Xiaojiang S Chen
Keyword(s):  
Dna Replication ◽  
Biological Process ◽  
Initial Step ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Base Pairs ◽  
Fundamental Biological Process ◽  
Eukaryotic Dna ◽  
Hexameric Helicases

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells.

Structure and function of SV40 large-T antigen

1987 ◽  
Vol 317 (1187) ◽  
pp. 455-469 ◽  
Keyword(s):  
Dna Replication ◽  
Cellular Transformation ◽  
T Antigen ◽  
Viral Gene ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Multifunctional Protein ◽  
Eukaryotic Dna

The small eukaryotic DNA tumour virus, SV40, has long provided a very useful model for the study of eukaryotic DNA replication and cellular transformation. The viral gene product, large-tumour (large-T) antigen, is essential for the initiation of viral DNA replication and the initiation and maintenance of SV40-virus-mediated cellular transformation. The large-T antigen is a complex multifunctional protein, and to delineate its activity more precisely in viral DNA replication and cellular transformation, small functional domains of the protein have been expressed in Escherichia coli and analysed by using a very extensive library of anti-T monoclonal antibodies.

scholarly journals DnaJ/hsp40 chaperone domain of SV40 large T antigen promotes efficient viral DNA replication.

1997 ◽  
Vol 11 (9) ◽  
pp. 1098-1110 ◽  
Author(s):  
K S Campbell ◽  
K P Mullane ◽  
I A Aksoy ◽  
H Stubdal ◽  
J Zalvide ◽  
...  
Keyword(s):  
Dna Replication ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Viral Dna ◽  
Viral Dna Replication

scholarly journals Simian Virus 40 Large T Antigen Can Specifically Unwind the Central Palindrome at the Origin of DNA Replication

2009 ◽  
Vol 83 (7) ◽  
pp. 3312-3322 ◽  
Author(s):  
Weiping Wang ◽  
Daniel T. Simmons
Keyword(s):  
Dna Replication ◽  
Critical Role ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Central Origin ◽  
Sv40 Dna

ABSTRACT The hydrophilic channels between helicase domains of simian virus 40 (SV40) large T antigen play a critical role in DNA replication. Previous mutagenesis of residues in the channels identified one class of mutants (class A: D429A, N449S, and N515S) with normal DNA binding and ATPase and helicase activities but with a severely reduced ability to unwind origin DNA and to support SV40 DNA replication in vitro. Here, we further studied these mutants to gain insights into how T antigen unwinds the origin. We found that the mutants were compromised in melting the imperfect palindrome (EP) but normal in untwisting the AT-rich track. However, the mutants' defect in EP melting was not the major reason they failed to unwind the origin because supplying an EP region as a mismatched bubble, or deleting the EP region altogether, did not rescue their unwinding deficiency. These results suggested that specific separation of the central palindrome of the origin (site II) is an essential step in unwinding origin DNA by T antigen. In support of this, wild-type T antigen was able to specifically unwind a 31-bp DNA containing only site II in an ATPase-dependent reaction, whereas D429A and N515S failed to do so. By performing a systematic mutagenesis of 31-bp site II DNA, we identified discrete regions in each pentanucleotide necessary for normal origin unwinding. These data indicate that T antigen has a mechanism to specifically unwind the central palindrome. Various models are proposed to illustrate how T antigen could separate the central origin.

scholarly journals Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

2000 ◽  
Vol 20 (15) ◽  
pp. 5749-5757 ◽  
Author(s):  
Christopher S. Sullivan ◽  
James D. Tremblay ◽  
Sheara W. Fewell ◽  
John A. Lewis ◽  
Jeffrey L. Brodsky ◽  
...  
Keyword(s):  
Dna Replication ◽  
Jc Virus ◽  
Simian Virus 40 ◽  
Cellular Transformation ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
J Domain ◽  
Species Specific

ABSTRACT The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo.

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