Biochemical characterization of Mgs1 in Saccharomyces cerevisiae
| Author(s) | Khankhoje, Aditi Milind | |
| Date Accessioned | 2022-12-12T12:32:23Z | |
| Date Available | 2022-12-12T12:32:23Z | |
| Publication Date | 2015 | |
| SWORD Update | 2022-08-11T19:08:40Z | |
| Abstract | Mgs1, Maintenance of Genome stability 1, a member of the AAA+ ATPase family is thought to be involved in the fork restart DNA damage response (DDR) pathway activated by replication stress in presence or absence of genotoxic agents. Previous published studies have shown that Mgs1 physically associates with polyubiquitinated PCNA and POL31 subunit of Polymerase δ. Genetic profiling has indicated interactions with the RAD6 epistasis group. It has also been shown that, although deficiency of Mgs1 in not lethal to the cell, it leads to increase in aberrant recombination and mutation. Interactions of Mgs1 with the enzymes involved in both replication and replication repair pathways makes it an interesting protein from a biochemical standpoint, shedding light on its possible role in the maintenance of genomic stability during and after replication. ☐ Throughout the life cycle of a cell, the DNA is damaged as a result of environmental and endogenous factors. Such damage causes aberrant changes in DNA topology which can lead to replication fork stalling and result in erroneous duplication. If stalled replication forks are left unresolved, double strand Breaks (DSB's) would form, causing random recombination events contributing to genome-wide instability. Mutations may accumulate over generations leading to a predisposition to cancer and other genetic diseases. Prokaryotes and eukaryotes have evolved robust DNA damage response mechanisms to overcome genome instability caused by replication fork collapse. Proteins in these biochemical pathways are highly conserved and garner a robust response to replication fork stalling events. It is thus conceivable that deficiencies in these proteins cause genetic disorders, making it imperative that these be studied and characterized in detail. ☐ Plethora of data exists, studying Mgs1 under various genetic backgrounds especially in that of replication stress. Hitherto, detailed biochemical analysis of DNA binding and ATPase activity of Mgs1 is not available. Here, we try to understand the complex nature of ATPase activity and the influence of different DNA structure-substrates on stimulating the activity. We also confirm the ATPase activity by creating a null ATPase mutant; K183A. We study the DNA binding of Mgs1 and its regulation by ATP analogs. ☐ Our data suggests clear difference in stimulation of ATPase activity in the presence of single stranded DNA and double stranded DNA structure. Also we show here that the DNA binding affinities are dictated by the DNA structure and ATP influences this activity. ☐ Our findings help us delineate a mechanistic model for the interaction of Mgs1 with DNA and to place these activities in the perspective of the available genetic data for Mgs1. | |
| Advisor | Zhuang, Zhihao | |
| Degree | M.S. | |
| Department | University of Delaware, Department of Chemistry and Biochemistry | |
| Unique Identifier | 1351386786 | |
| URL | https://udspace.udel.edu/handle/19716/31685 | |
| Language | en | |
| Publisher | University of Delaware | |
| URI | https://login.udel.idm.oclc.org/login?url=https://www.proquest.com/dissertations-theses/biochemical-characterization-mgs1-em/docview/2708570034/se-2?accountid=10457 | |
| Keywords | NADH linked ATPase Assay | |
| Keywords | Mgs1 | |
| Keywords | DNA structure | |
| Keywords | DNA protein interaction | |
| Keywords | DNA Binding | |
| Keywords | AAA+ ATPase | |
| Title | Biochemical characterization of Mgs1 in Saccharomyces cerevisiae | |
| Type | Thesis |