Model for T-antigen-dependent melting of the simian virus 40 core origin based on studies of the interaction of the beta-hairpin with DNA |
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Authors: | Kumar Anuradha Meinke Gretchen Reese Danielle K Moine Stephanie Phelan Paul J Fradet-Turcotte Amélie Archambault Jacques Bohm Andrew Bullock Peter A |
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Affiliation: | Department of Biochemistry A703, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. |
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Abstract: | The interaction of simian virus 40 (SV40) T antigen (T-ag) with the viral origin has served as a model for studies of site-specific recognition of a eukaryotic replication origin and the mechanism of DNA unwinding. These studies have revealed that a motif termed the "beta-hairpin" is necessary for assembly of T-ag on the SV40 origin. Herein it is demonstrated that residues at the tip of the "beta-hairpin" are needed to melt the origin-flanking regions and that the T-ag helicase domain selectively assembles around one of the newly generated single strands in a manner that accounts for its 3'-to-5' helicase activity. Furthermore, T-ags mutated at the tip of the "beta-hairpin" are defective for oligomerization on duplex DNA; however, they can assemble on hybrid duplex DNA or single-stranded DNA (ssDNA) substrates provided the strand containing the 3' extension is present. Collectively, these experiments indicate that residues at the tip of the beta-hairpin generate ssDNA in the core origin and that the ssDNA is essential for subsequent oligomerization events. |
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