Sanders, Jordan
(2024)
dRnh1-GFP and Drug Discovery: A Toolkit for Better Understanding the Mcm2-7 Replicative Helicase.
Doctoral Dissertation, University of Pittsburgh.
(Unpublished)
Abstract
The faithful replication of the DNA code is essential for the propagation of living cells. At the head of this replication process is the DNA helicase Mcm2-7, which serves to unwind DNA’s double-helix structure and provide access for the DNA replication machinery. Though Mcm2-7 has been extensively characterized by biochemical, single-molecule, and structural studies, questions remain regarding its DNA translocation mechanism, as well as in understanding how Mcm2-7 bypasses “road blocks” caused by RNA polymerase. Here, I discuss the development and preliminary implementation of two tools designed in part to better understand these different aspects of Mcm2-7’s function in Saccharomyces cerevisiae.
The first such tool is a drug discovery pipeline in which the principle of drug synthetic lethality with an mcm2 mutant (mcm2-DENQ) was used to identify novel Mcm2-7 inhibitors. This high-throughput screen, and its secondary assays, identified N-Methyl- β -carboline-3-carboxamide (CMA) as a likely inhibitor of active Mcm2-7. CMA was found to specifically inhibit growth and cell survival of mcm2-DENQ, arrest S-phase DNA replication, and bind to purified Mcm2-7. With this high-throughput assay and secondary assay pipeline established, more novel inhibitors may be discovered and utilized in mechanistic studies of Mcm2-7 as well as for potential chemotherapeutics.
Mcm2-7 mutation can also cause an increase in genomic RNA/DNA hybrids, likely due to an increased rate of unresolved conflicts between transcription and replication machinery. To better understand how Mcm2-7 contributes to/prevents transcription-replication conflicts, I developed a live-cell cytological assay for RNA/DNA hybrid detection in S. cerevisiae. This assay utilizes a catalytically-dead RNaseH1 (dRnh1-GFP) to detect hybrids through formation of easily-detectable nuclear foci. I found that dRnh1-GFP colocalizes with RNA/DNA hybrids as defined by an established immunological assay (S9.6 antibody). Importantly, the percentage of dRnh1-GFP focus-positive cells increased in response to mutations and drug treatments that are known to increase RNA/DNA hybrids, as well as for select drugs known to poison replication forks and promote transcription-replication conflicts. The simplicity and versatility dRnh1-GFP allows for its use outside of Mcm2-7 research and also allows for use in high-throughput screening. With these two tools, we may further our understanding of the Mcm2-7 helicase.
Share
| Citation/Export: |
|
| Social Networking: |
|
Details
| Item Type: |
University of Pittsburgh ETD
|
| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
|
| Date: |
20 December 2024 |
| Date Type: |
Publication |
| Defense Date: |
14 October 2024 |
| Approval Date: |
20 December 2024 |
| Submission Date: |
25 September 2024 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
171 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
Dietrich School of Arts and Sciences > Biological Sciences |
| Degree: |
PhD - Doctor of Philosophy |
| Thesis Type: |
Doctoral Dissertation |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
dRnh1, R-loop, Mcm2-7, Transcription-replication conflicts |
| Date Deposited: |
20 Dec 2024 14:10 |
| Last Modified: |
20 Dec 2024 14:10 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/46984 |
Metrics
Monthly Views for the past 3 years
Plum Analytics
Actions (login required)
 |
View Item |
![[feed]](/images/feed-icon-32x32.png){kind=icon}