The interaction of SV40 large T antigen with unspecific double-stranded DNA: An electron microscopic study

Wessel, Rainer; Ramsperger, Uwe; Stahl, Hans; Knippers, Rolf

doi:10.1016/0042-6822(92)90705-t

Cited by 17 publications

(16 citation statements)

References 29 publications

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“…These results strongly indicate that a free 3Ј single-stranded DNA tail is needed for the mutant T antigens to unwind replication fork substrates. Previous footprinting data showed that T antigen protects both the singlestranded and duplex regions of replication forks or bubbles (39,50,54,66,67), suggesting a possible role of nonspecific double-stranded DNA binding in the unwinding of duplex DNA (74). In contrast, our data suggest that nonspecific double-stranded DNA binding is not strictly required for helicase activity.…”

Section: Discussioncontrasting

confidence: 55%

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Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin

Jiao

Simmons

2003

J Virol

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Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.The large tumor (T) antigen encoded by simian virus 40 (SV40) is a phosphoprotein that has diverse roles in viral DNA replication and transformation (for reviews, see references 7, 19, 43, and 58). The initiation of DNA replication is a series of tightly regulated events. At first, T antigen binds to the 64-bp core origin of replication in a sequence-specific manner. The two pairs of GAGGC pentanucleotides, termed site II, in the core origin serve as the major binding site for T antigen (16). In the presence of ATP, individual monomers of T antigen multimerize cooperatively into a double hexamer, a bilobed structure encircling the origin (12,28,63,67). Interestingly, preformed hexamers of T antigen were shown to be active in origin DNA binding and unwinding, implying that T antigen is versatile in performing these activities (62).Formation of double hexamers is well supported by either of the two assembly units made up of pentanucleotides 1, 3, and the early palindrome (EP) region or pentanucleotides 2, 4, and the AT track (23,24,52). An intact origin, however, is needed for efficient unwinding (13, 33). After hexamerization, T antigen partially melts the EP region and untwists the AT track (3,5,6,15,32). At the expense of ATP hydrolysis, the distorted origin is subsequently unwound bidirectionally by the helicase activity of T-antigen double hexamers (36,49,54). Once unwinding is initiated, the two hexamers likely remain associated with each other, with the separated single-stranded DNA threading through the hexameric channels (50, 67). At least three other host protei...

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Section: Discussioncontrasting

confidence: 55%

“…As a helicase, T antigen is able to unwind DNA in a 3Ј-to-5Ј fashion (21,54,68). It is capable of unwinding double-stranded DNA (66), artificial replication forks (1,39,49), and replication bubbles (49,50). The region responsible for helicase activity has been mapped to residues 131 to 616 and harbors both DNA binding domains (72).…”

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confidence: 99%

Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin

Jiao

Simmons

2003

J Virol

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“…2, lane 6). T antigen is known to have a weak, nonspecific DNA binding activity (43), and this is shown in Fig. 2, lane 4.…”

Section: Resultsmentioning

confidence: 99%

Simian Virus 40 Large T Antigen Stabilizes the TATA-Binding Protein–TFIIA Complex on the TATA Element

Damania

Lieberman

Alwine

1998

Molecular and Cellular Biology

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Large T antigen (T antigen), the early gene product of simian virus 40 (SV40), is a potent transcriptional activator of both cellular and viral genes. Recently we have shown that T antigen is tightly associated with TFIID and, in this position, performs a TATA-binding protein (TBP)-associated factor (TAF)-like function.Based on this observation, we asked whether T antigen affected steps in preinitiation complex assembly. Using purified components in in vitro complex assembly assays, we found that T antigen specifically enhances the formation of the TBP-TFIIA complex on the TATA element. T antigen accomplishes this by increasing the rate of formation of the TBP-TFIIA complex on the TATA element and by stabilizing the complexes after they are formed on the promoter. In addition, DNA immunoprecipitation experiments indicate that T antigen is associated with the stabilized TBP-TFIIA complexes bound to the DNA. In this regard, it has previously been shown that T antigen interacts with TBP; in the present study, we show that T antigen also interacts with TFIIA in vitro. In testing the ability of T antigen to stabilize the TBP-TFIIA complex, we found that stabilization is highly sensitive to the specific sequence context of the TATA element. Previous studies showed that T antigen could activate simple promoters containing the TATA elements from the hsp70 and c-fos gene promoters but failed to significantly activate similar promoters containing the TATA elements from the promoters of the SV40 early and adenovirus E2a genes. We find that the ability to stabilize the TBP-TFIIA complex on the hsp70 and c-fos TATA elements, and not on the SV40 early and E2A TATA elements, correlates with the ability or inability to activate promoters containing these TATA elements.Large T antigen (T antigen) is one of the early gene products of simian virus 40 (SV40). It is a potent transcriptional activator of both cellular and viral genes and is an oncoprotein capable of interacting with an array of host cellular factors (46). Previous studies from this lab and others have shown that T antigen can activate a simple promoter consisting of a TATA element and one upstream transcription factor binding site (13,15,34). Although T antigen has a specific as well as a nonspecific DNA binding capability, this function is not essential for transcriptional activation (1,12,21). Rather, T antigen appears to mediate its transactivation function through protein-protein interactions. Our lab has previously shown that large T antigen can interact with both the TATA-binding protein (TBP) and several TBP-associated factors (TAFs) (7, 16). In addition, T antigen can interact with a number of transcription factors including TEF-1 and Sp1 (7, 16). We have also established that T antigen is tightly associated with TFIID and, in this position, performs a TAF-like function requiring interactions with upstream bound factors to affect transcriptional activation (7). It is important to note that the additional interaction with the upstream bound factor appears to b...

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“…The SV40 Tag forms a dihexamer that surrounds the DNA at the SV40 origin (33,34). HPV E1 protein binds to DNA mainly as a hexamer (35).…”

Section: Discussionmentioning

confidence: 99%

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to ori _s , an origin of viral DNA replication

Eom

Lehman²

2002

Proc. Natl. Acad. Sci. U.S.A.

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The interaction of SV40 large T antigen with unspecific double-stranded DNA: An electron microscopic study

Cited by 17 publications

References 29 publications

Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin

Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin

Simian Virus 40 Large T Antigen Stabilizes the TATA-Binding Protein–TFIIA Complex on the TATA Element

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to ori _s , an origin of viral DNA replication

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The interaction of SV40 large T antigen with unspecific double-stranded DNA: An electron microscopic study

Cited by 17 publications

References 29 publications

Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin

Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin

Simian Virus 40 Large T Antigen Stabilizes the TATA-Binding Protein–TFIIA Complex on the TATA Element

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to ori s , an origin of viral DNA replication

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The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to ori _s , an origin of viral DNA replication