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... which Py T Ag mediates replication of DNA containing the Py replication origin (Py ori-DNA) in murine cell extracts (59, 65). Py T Ag binding to the Py origin palindrome is stimulated by nucleotides =-=(47)-=-, and in the presence of nucleotides it can also form hexamers (85) and melt sites within the origin (4, 43). Additionally, Py T Ag can unwind origin DNA and display DNA helicase activity (71, 84). Li...

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...). Large T antigen bound more strongly to sites A, B, and C than to site 1/2 in the absence of ATP (7, 17, 42), but ATP strongly increased the affinity of large T antigen for DNAs containing site 1/2 =-=(27)-=-, probably by stimulating the formation of hexamers. * Corresponding author. Mailing address: Department of Microbiology and Immunology, McGill University, 3775 University St., Montreal H3A 2B4, Quebe...

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...replication, binding of a Py T antigen double hexamer to the Py origin of replication causes the destabilization of duplex DNA and local unwinding of the origin, followed by binding of mammalian RP-A =-=(17, 34, 71, 72)-=-. The next step in initiation probably involves the association of DNA polymerase �-primase, Downloaded from http://mcb.asm.org/ on February 20, 2013 by PENN STATE UNIVVOL. 15, 1995 SPECIES SPECIFICI...

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... establish the locations of the modified residues. Nitrocellulose filter binding of SV40 T-ag–DNA complexes. The nitrocellulose filter assay for T-ag binding was based on previously published methods =-=(3, 47, 51)-=-. Reaction mixtures (20 �l) contained 7 mM MgCl 2, 0.5 mM DTT, 40 mM creatine phosphate (di-Tris salt [pH 7.6]), 0.48 �g of creatine phosphate kinase (omitted from reaction mixtures containing ADP), 0...

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...o play during many steps from initiation through the process of bidirectional DNA replication. These include sequence-specific DNA-binding activity (9, 10, 18, 26), nucleotide-induced ori DNA binding =-=(4, 16, 31)-=- and hexamer formation (13, 36, 47, 61), DNA-unwinding activity (12, 24, 62, 63), DNA helicase activity (12, 53, 55, 62), and ATPase activity (10, 24). Moreover, both T Ags require and undergo specifi...

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...ces, for T-ag and the T124A mutant. Nitrocellulose filter binding of SV40 T-ag and T124A complexes. The nitrocellulose filter assay for T-ag or T124A binding was based on previously published methods =-=(5, 47, 52, 81)-=-. Reaction mixtures (20 ml) contained 7 mM MgCl2, 0.5 mM DTT, 40 mM creating phosphate (di-Tris salt [pH 7.6]), 0.48 mg of creatine phosphate kinase, 0.2 mg of bovine serum albumin per ml, 0.8 mg of H...

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...s inside indicate genetically defined components, with minimal essential sequences indicated by bold stripes. T-ag binding sites are lightly striped boxes, with the strongest sites marked A, B, and C =-=(52)-=-. DNA binding sites for Py enhancer activation proteins (PEA) 1, 2, and 3 are indicated by solid boxes. Plasmids containing different Py on-core and aux-2 sequences are indicated, and constructions ar...

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...ted by cloning the five GAL4 sites from pG5E4T (32) into the BglII site of pJSCATori (4) and was kindly provided by J. Hassell. p373.A2 carries Py A2 strain DNA inserted into the BamHI site of pAT153 =-=(34)-=-. Proteins.Wild-type and mutant p53 proteins were immunopurified from Sf21 insect cells infected with recombinant baculoviruses as previously reported (52). Py large T antigens were also immunopurifie...

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...38, 41). Like SV40 T antigen, Py T antigen also exhibits ATPase (19) and DNA helicase (42, 53) activities. Furthermore, analogous to SV40 T antigen, Py T antigen displays ATP-dependent origin binding =-=(27)-=- and hexamer formation (54). However, despite their similar functions and structure, SV40 and Py T antigens display distinctly different cell species specificity for mediating viral ori-DNA replicatio...