Download or read book Mapping and Characterization of the Single stranded DNA Binding Domain of Simian Virus 40 Large T Antigen written by Chunxiao Wu and published by . This book was released on 2001 with total page 206 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Mutational Analysis of the Central Channel in the Simian Virus 40 Large T Antigen Helicase written by David Manna and published by . This book was released on 2006 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: I report in this dissertation that residues lining the small tier of the central channel have an important role in DNA binding, especially in single-stranded DNA binding. Defects in helicase activity for the small tier channel mutants can be accounted by their corresponding defects in DNA binding. One of the large tier central channel mutants (R456A) showed a defect in unwinding of origin DNA, but this could not be attributed to a problem in DNA binding. Combined with the results of others, it appears that the large tier is primarily involved in unwinding origin DNA. These findings are incorporated into a model of T antigen helicase activity where single-stranded DNA is looped out of one hexamer through hydrophilic side-channels in the large tier of the hexamer. The looped-out single-stranded DNA runs back into the origin binding domain of the adjacent hexamer. In this model, there is one strand of DNA within each small tier during DNA unwinding from the origin. (Abstract shortened by UMI.).

Download or read book Mutational Analysis of the Origin Binding Domain of Simian Virus 40 Large T antigen Provides Insights Into Its Function in DNA Unwinding written by Erin C. Foster and published by . This book was released on 2010 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: DNA replication is an essential process of cell division required in order to maintain genomic integrity of all dividing cells. Although advancements have been made toward further understanding of this process, much is still unknown about the mechanisms underlying DNA replication, especially in mammalian systems. Eukaryotic DNA replication is a tightly regulated and complex process involving a myriad of proteins and numerous origins. Due to this complexity Simian Virus 40 (SV40) is used as a model system to simplify the investigation of the function of individual protein components. The replication of SV40 DNA employs one multifunctional viral protein, large T-antigen (T-ag). Many structural and genetic studies have provided a wealth of information about T-ag; however much remains unknown about the specific interactions between T-ag, DNA and replication factors and the precise sequence of events during replication. SV40 large T-ag is a multidomain protein that functions in origin recognition and unwinding events during initiation of SV40 DNA replication. The Origin Binding Domain (OBD) of T-ag plays a critical role during initiation to position T-ag on the origin through the use of a DNA binding motif consisting of A1 and B2 loops. After origin recognition, T-ag forms a double hexamer over the origin. Within each hexamer, the OBD adopts a lock washer structure where the origin recognizing A1 and B2 loops face towards the helicase domain and likely become unavailable for DNA binding. New surfaces of the OBD monomers are positioned along the DNA axis during hexamer formation. The role of the central channel of the OBD hexamer in DNA replication was investigated by analyzing the effects of mutations of residues lining the channel. All mutants showed binding defects with origin DNA and ssDNA especially at low protein concentrations, but only half were defective at supporting DNA replication in vitro. All mutants were normal in unwinding linear origin DNA fragments. However, replication defective mutants failed to unwind a small origin containing circular DNA whereas replication competent mutants did so normally. The presence of RPA and/or pol/prim restored circular DNA unwinding activity of compromised mutants probably by interacting with the separated DNA strands on the T-ag surface. The addition of RPA also recovered the ability of replication deficient mutants to support initiation and complete DNA replication in vitro. The addition of pol/prim resulted in the greatest recovery of mutant activity in minicircle unwinding and initiation. Compared to other polymerases tested, pol/prim was the only polymerase able to rescue mutant activity to WT levels indicated that this stimulation is specific to pol/prim. Of the four subunits of the pol/prim complex that could be responsible for stimulating mutant activity, the p180 subunit appeared to be active in recovering circular DNA unwinding. These results indicated a role for the OBD central channel in binding and threading ssDNA during unwinding of circular SV40 DNA. Mixing mutant and WT T-ag in minicircle unwinding assays suggested that only one monomer in an OBD hexamer was necessary for DNA unwinding. Results allowed for the development of a model of DNA threading through the T-ag complex illustrating how single stranded DNA could pass close to a trough generated by key residues in one monomer of the OBD hexamer.

Download or read book Purification and Partial Characterization of a Mutant Within the Simian Virus 40 Large T antigen DNA Binding Domain written by Clyde C. Jones and published by . This book was released on 1996 with total page 168 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Purification and Characterization of Simian Virus 40 Large T Antigen written by Raymond James Paxton and published by . This book was released on 1983 with total page 434 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Initiation of Simian Virus 40 SV40 DNA Replication written by and published by . This book was released on 2005 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: It is well established that during initiation of SV40 DNA synthesis, a double hexamer of T antigen forms over the origin and acts as a bidirectional helicase. The subsequent events are much less clear. In fact, the structure on which DNA synthesis begins is not known, but has been hypothesized to be the double hexamer tightly associated with cellular initiation factors replication protein A (RPA), topoisomerase I (topo I) and more loosely with DNA polymerase & alpha;/primase (pol/prim). I now demonstrate that after a double hexamer forms over small origin fragments, a new structure consisting of a single hexamer associated with DNA is generated in an ATP dependent reaction from double hexamers. This structure, called the preinitiation complex (PIC), requires about 5 minutes to form whereas double hexamers are generated within 20 seconds. Importantly, PICs can associate with RPA and topo I whereas double hexamers, which have not been converted to PICs, can only bind to topo I. PICs are not simply T antigen hexamers associated with single-stranded DNA as the latter structures were shown not to bind RPA or topo I. Topo I can bind to an RPA-associated PIC, but RPA cannot bind to a topo I-associated PIC suggesting that the binding order is RPA then topo I. Importantly, DNA synthesis activity appears to be associated with the PICs but not with double hexamers. In this dissertation, a series of mutants with single-point substitutions in the helicase domain are generated to discover activities required for initiation of DNA replication. Four of these mutants (456 RA, 460ED, 462GA, and 499DA) are normal in their ability to hydrolyze ATP and are capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possess normal ability to bind to single-stranded DNA. However, they fail to melt the early palindrome region of the origin. They also are 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 retain some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity is functional. Intriguingly, these mutants have almost completely lost their ability to bind to double-stranded DNA nonspecifically. Taken together, my data indicate that structural distortion and subsequent unwinding of the origin depend upon T antigen's ability to bind to DNA nonspecifically.

Download or read book Purification and Characterization of Simian Virus 40 Large T Antigen written by Donald Allen Giacherio and published by . This book was released on 1980 with total page 380 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Simian Virus 40 Large Tumor Antigen written by Robin Clark and published by . This book was released on 1986 with total page 222 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterization of Simian Virus 40 Large T antigen Deletion Mutants written by Earl T. Sawai and published by . This book was released on 1990 with total page 294 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterization of Nucleic Acid Binding by SV40 Large T Antigen written by Duncan Lochiel McVey and published by . This book was released on 1990 with total page 368 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterization of Structural Distortion Activity of Simian Virus 40 Large T Antigen written by Lei Chen and published by . This book was released on 1998 with total page 246 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Simian Virus 40 DNA Replication written by Stephen Todd Smale and published by . This book was released on 1986 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Characterization of Simian Virus 40 Late Leader Region Mutants written by Alice Barkan and published by . This book was released on 1983 with total page 516 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Mapping and Characterization of Topoisomerase I Sites in the Simian Virus 40 Origin of Replication written by Shanli Tsui and published by . This book was released on 1990 with total page 278 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book SV40 Large T Antigen Helicase written by and published by . This book was released on 2009 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: To initiate DNA replication, SV40 large T antigen needs to recognize and bind to the origin in SV40 DNA. This is achieved through sequence specific contacts between the T antigen origin binding domain (OBD) and the pentanucleotide regions in the center of the origin, as well as non-sequence specific interactions between the helicase domain and the EP and AT regions of the origin. This leads to the assembly of a double hexamer of T antigen over a melted origin DNA. Subsequently, the DNA becomes unwound bidirectionally, providing SS DNA templates for DNA replication. However, these events are not fully understood. For instance, it is not clear how T antigen separates DS DNA at the origin or how the separated strands become threaded through the double hexamer. In this dissertation, I identified a new unwinding activity of large T antigen. The helicase can unwind the central pentanucleotides in an ATP-hydrolysis dependent way. This activity of T antigen, like its structural distortion activity that acts on the flanking EP and AT regions, is an indispensable component of the unwinding of the entire origin. Specific sequences in the pentanucleotide region are necessary for the activity, and the flanking AT track can stimulate it. The SV40 hexameric helicase consists of a positively charged central channel and six hydrophilic channels in the large tiers. The hydrophilic channels separate adjacent large tier domains within each hexamer. In this dissertation, I generated a series of mutants with single-point substitutions in the hydrophilic channels and studied the function of the hydrophilic channels in SV40 DNA replication. The mutants were characterized in various biochemical assays and grouped into four major classes according to their properties. One class of mutant proteins (class A-D429A, N449S and N515S) displayed normal DNA binding, ATPase, and helicase activities. However, they were defective specifically at unwinding origin DNA. The three mutants were found to be deficient in EP melting activity. Two of them (D429A and N515S) also failed to unwind the central pentanucleotide region of the origin. Taken together, my data indicate that the hydrophilic channels play active roles in unwinding the origin to provide the single-stranded substrates for DNA replication. Based on my results and on published structural and functional information, a model is proposed in this dissertation to illustrate how T antigen could separate the pentanucleotide region at the center of the origin. My data also suggest a mechanism by which the step-wise assembly of the T antigen double hexamer could trigger unwinding of the origin.

Download or read book Dissertation Abstracts International written by and published by . This book was released on 2001 with total page 818 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Genetic and Biochemical Analysis of Simian Virus 40 Large T Antigen Mutants written by Mari Michele Manos and published by . This book was released on 1984 with total page 166 pages. Available in PDF, EPUB and Kindle. Book excerpt: