Mutational analysis of the central channel in the Simian virus 40 large T antigen helicase
| Author(s) | Manna, David | |
| Date Accessioned | 2020-09-08T12:03:06Z | |
| Date Available | 2020-09-08T12:03:06Z | |
| Publication Date | 2006 | |
| Abstract | DNA replication is an essential biochemical process that is needed for all organisms to survive. However, this process is yet to be well understood, especially in mammalian systems. In order to study the extremely complicated process of eukaryotic DNA replication, viral DNA replication systems are often used as a much simpler model. Simian Virus 40 (SV40) Large T antigen is a well-studied protein that is required for many functions of SV40, including acting as a helicase during DNA replication and as an oncogene during cellular transformation. T antigen, like many other helicases, especially the putative human Mcm proteins, function as a hexamer. These molecular motor proteins usually arrange in six-fold symmetry and resemble a propeller shaped 6-point star with a large channel running down the center. Viewed from the side, the T antigen hexameric helicase consists of two tiers, a smaller top tier and a larger bottom tier, and a central channel that runs longitudinally through both tiers. T antigen functions as a double hexamer over the origin DNA, with the origin binding and J domains of each individual hexamer facing each other. The exact mechanism by which single-stranded DNA is unwound and looped out of the helicase is unknown, but models described in the literature show different roles for the central channel in the small and large tiers. Some models have the small tier associating with single-stranded DNA and some have single-stranded DNA completely bypassing it. 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. | en_US |
| Advisor | Simmons, Daniel T. | |
| Degree | M.S. | |
| Department | University of Delaware, Department of Biological Sciences | |
| Unique Identifier | 82134188 | |
| URL | https://udspace.udel.edu/handle/19716/27485 | |
| Publisher | University of Delaware | en_US |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/docview/305324433?accountid=10457 | |
| dc.subject.lcsh | SV40 (Virus) | |
| dc.subject.lcsh | Antigens | |
| dc.subject.lcsh | Mutagens | |
| dc.subject.lcsh | DNA -- Analysis | |
| Title | Mutational analysis of the central channel in the Simian virus 40 large T antigen helicase | en_US |
| Type | Thesis | en_US |