Page 162 |
Save page Remove page | Previous | 162 of 171 | Next |
|
small (250x250 max)
medium (500x500 max)
Large (1000x1000 max)
Extra Large
large ( > 500x500)
Full Resolution
All (PDF)
|
This page
All
|
147
Appendix D: Deactivation of Cdc7 delays completion of DNA replication
Figure 37. Strains containing the temperature sensitive cdc7-4 allele are delayed in completion of bulk DNA synthesis in unperturbed conditions and during recovery from MMS. (A) WT (SSy187) and cdc7-4 (SSy224) cells were arrested with-factor and released into YEPD at 23oC. After allowing time for early origin firing, half of each culture was shifted to 34oC (restrictive temperature, red time points). Samples were collected at the indicated times for DNA content analysis. (B) Strains from (A) were arrested in late G1 with -factor and released into YEPD containing 0.033% MMS at 23oC for 15min. Cultures were shifted to 34oC for 45min while still in MMS (total time in MMS = 60min). After which, MMS was quenched, cells were pelleted and resuspended in 23oC YEPD, which was then allowed to “warm up” to 34oC. Samples were collected at the indicated times for DNA content analysis. Cdc7 is a key component of DDK and is required for origin initiation. The replication defect of cdc7-4 cells after temperature shift (A) likely reflects faulty origin initiation. Completion of replication is delayed >2.5hrs during MMS recovery compared to WT cells (B). Potentially, Cdc7 could be required for fork stabilization or fork restart during recovery from MMS insult. However, I should note that Cdc7 was inactivated 15 minutes after -factor release. Possibly, early inactivation of Cdc7 could have allowed only the earliest of early origins to fire. Therefore, the delay in cdc7-4 could be due to only a subset of early origins firing or a defect in fork stabilization/restart. BrdU incorporation and 2D gel experiments analyzing initiation and fork progression in these mutants should be performed to discern between these possibilities.
Object Description
| Title | The intra-S phase checkpoint and its effect on replication fork dynamics in saccharomyces cerevisiae |
| Author | Szyjka, Shawn Joseph |
| Author email | sszyjka@gmail.com; hammonds77@gmail.com |
| Degree | Doctor of Philosophy |
| Document type | Dissertation |
| Degree program | Molecular Biology |
| School | College of Letters, Arts and Sciences |
| Date defended/completed | 2008-07-25 |
| Date submitted | 2008 |
| Restricted until | Unrestricted |
| Date published | 2008-10-21 |
| Advisor (committee chair) | Aparicio, Oscar |
| Advisor (committee member) |
Forsburg, Susan Finkel, Steven E. Qin, Peter Z. Rice, Judd C. |
| Abstract | Duplication of a cell's genetic material is of paramount importance. This task must be completed accurately, efficiently and occur once and only once within any given cell cycle. Origin "firing", which occurs according to a temporal program, results in the establishment of bidirectional replication forks. As replication forks traverse the chromosomal landscape, their stability is threatened by endogenous "obstacles" such as heavily transcribed tRNAs, protein-DNA complexes and "replication slow zones". In addition, fork stability can also be threatened by genotoxic agents. During S phase, in response to genotoxin-induced stress, the cell activates the intra-S phase checkpoint. The intra-S phase checkpoint is comprised of three major groups of proteins: sensors, adaptors and effectors. Sensor proteins detect problems at the replication fork and elicit a phosphorylation cascade that is mediated by adaptor proteins and results in the activation of effector kinases. Together, these proteins act to inhibit late origin firing, slow cell cycle progression, upregulate DNA repair genes, and stabilize replication forks until the stress has been alleviated.; In response to nucleotide depletion, the adaptor protein, Mrc1 (Mediator of the Replication Checkpoint), mediates a checkpoint signal that activates the effector kinase, Rad53. In addition to its checkpoint role, Mrc1 also appears to play a role in unperturbed DNA synthesis. Mrc1 travels with replication forks and mrc1delta cells display slowed bulk DNA synthesis. This phenotype could be due to decreased origin firing, increased fork pausing at endogenous "obstacles", or overall defective fork progression. Using two-dimensional (2D) gel electrophoresis of replication intermediates, we demonstrate that mrc1delta cells are not defective in fork pausing at tRNAs and that Mrc1's replication function is required for efficient progression of replication forks throughout the genome. We hypothesize that Mrc1 maintains association between polymerases and helicases at the replication fork, which results in efficient fork progression.; In response to MMS-induced DNA damage, Rad53 plays a vital role in replication fork stabilization. It is thought that Rad53 maintains the association of replisome components so that forks are poised for efficient restart upon checkpoint deactivation. Despite a wealth of knowledge with respect to checkpoint activation, relatively little is known about checkpoint deactivation and how replication forks restart. Previous work has suggested that replication fork progression along an MMS-damaged template is independent of the checkpoint. However, recent studies lacking Psy2-Pph3, a Rad53 phosphatase responsible for Rad53 deactivation after DNA damage, suggest otherwise, as the completion of S-phase after DNA damage is delayed in the absence of Psy2-Pph3. We have examined the role of Rad53 in the control of replication fork restart by monitoring BrdU incorporation at replication forks in the presence of DNA damage and during recovery from damage. We find that Rad53 deactivation is a key requirement for replication fork restart as cells lacking PPH3 are defective in fork progression in the presence of DNA damage and are delayed in replication restart during recovery. In addition, dominant-negative inactivation of Rad53 in pph3delta cells, enables replication fork restart, arguing that deactivation of Rad53 is sufficient for replication fork restart. Deletion of PTC2, which encodes a second, unrelated Rad53 phosphatase, in addition to PPH3, completely eliminates replication fork progression under DNA damaging conditions and results in lethality. These findings suggest that replication fork stabilization and restart involve a cycle of Rad53 activation and deactivation, and that at least two distinct phosphatases are responsible for regulating this process. |
| Keyword | DNA replication; DNA damage; replication fork; DNA repair; cell cycle checkpoint; phosphatase; Rad53; Mrc1; BrdU; microarray; Claspin; replication fork progression; Rrm3; Pph3; 2-D gel electro |
| Language | English |
| Part of collection | University of Southern California dissertations and theses |
| Publisher (of the original version) | University of Southern California |
| Place of publication (of the original version) | Los Angeles, California |
| Publisher (of the digital version) | University of Southern California. Libraries |
| Provenance | Electronically uploaded by the author |
| Type | texts |
| Legacy record ID | usctheses-m1688 |
| Contributing entity | University of Southern California |
| Rights | Szyjka, Shawn Joseph |
| Repository name | Libraries, University of Southern California |
| Repository address | Los Angeles, California |
| Repository email | cisadmin@lib.usc.edu |
| Filename | etd-Szyjka-2341 |
| Archival file | uscthesesreloadpub_Volume32/etd-Szyjka-2341.pdf |
Description
| Title | Page 162 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@lib.usc.edu |
| Full text | 147 Appendix D: Deactivation of Cdc7 delays completion of DNA replication Figure 37. Strains containing the temperature sensitive cdc7-4 allele are delayed in completion of bulk DNA synthesis in unperturbed conditions and during recovery from MMS. (A) WT (SSy187) and cdc7-4 (SSy224) cells were arrested with-factor and released into YEPD at 23oC. After allowing time for early origin firing, half of each culture was shifted to 34oC (restrictive temperature, red time points). Samples were collected at the indicated times for DNA content analysis. (B) Strains from (A) were arrested in late G1 with -factor and released into YEPD containing 0.033% MMS at 23oC for 15min. Cultures were shifted to 34oC for 45min while still in MMS (total time in MMS = 60min). After which, MMS was quenched, cells were pelleted and resuspended in 23oC YEPD, which was then allowed to “warm up” to 34oC. Samples were collected at the indicated times for DNA content analysis. Cdc7 is a key component of DDK and is required for origin initiation. The replication defect of cdc7-4 cells after temperature shift (A) likely reflects faulty origin initiation. Completion of replication is delayed >2.5hrs during MMS recovery compared to WT cells (B). Potentially, Cdc7 could be required for fork stabilization or fork restart during recovery from MMS insult. However, I should note that Cdc7 was inactivated 15 minutes after -factor release. Possibly, early inactivation of Cdc7 could have allowed only the earliest of early origins to fire. Therefore, the delay in cdc7-4 could be due to only a subset of early origins firing or a defect in fork stabilization/restart. BrdU incorporation and 2D gel experiments analyzing initiation and fork progression in these mutants should be performed to discern between these possibilities. |
