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123
Table 1
Strains used in Chapter 2
Strain Genotype Source
Strains are identical to W303-1a, except as indicated
W303-1a MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 R. Rothstein
can1-100
YJT80 ars608::HIS3 ars609::TRP1 ars305::kanMX (Tercero & Diffley, 2001) ADE2 rad5-535
E1000 GPD-TK(7x)::URA3 (Lengronne et al, 2001)
SSy45 YTJ80 ars305::loxP (Szyjka et al, 2005)
SSy46 SSy45 mrc1::kanMX “
SSy47 SSy46 bar1::LEU2 “
SSy48 SSy45 bar1::LEU2 “
SSy56 SSy46 bar1::URA3 “
SSy62 SSy56 mrc1::kanMX::mrc1-AQ (LEU2) “
SSy69 MAT rrm3::TRP1 RAD5 “
SSy72 SSy185 mrc1::HIS5::mrc1-AQ (LEU2) RAD5 “
SSy81 mrc1::HIS5::mrc1-AQ (LEU2) rrm3::TRP1 (SSy108 segregant) “ SSy108 SSy69 X SSy72 diploid “
SSy109 SSy69 X SSy185 diploid “
SSy116 SSy47 mrc1-AQ “
SSy185 mrc1::HIS5 RAD5 “
CVy29 E1000 mrc1::kanMX ars608::HIS3 bar1::LEU2 “
CVy30 CVy31 mrc1::kanMX “
CVy31 E1000 bar1::LEU2 cdc45::CDC45-HA3 (TRP1) “
rad5-535
CVy39 E1000 ars608::HIS3 bar1::TRP1 “
CVy40 CVy39 mrc1::HIS5::mrc1-AQ (LEU2) “
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 138 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@lib.usc.edu |
| Full text | 123 Table 1 Strains used in Chapter 2 Strain Genotype Source Strains are identical to W303-1a, except as indicated W303-1a MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 R. Rothstein can1-100 YJT80 ars608::HIS3 ars609::TRP1 ars305::kanMX (Tercero & Diffley, 2001) ADE2 rad5-535 E1000 GPD-TK(7x)::URA3 (Lengronne et al, 2001) SSy45 YTJ80 ars305::loxP (Szyjka et al, 2005) SSy46 SSy45 mrc1::kanMX “ SSy47 SSy46 bar1::LEU2 “ SSy48 SSy45 bar1::LEU2 “ SSy56 SSy46 bar1::URA3 “ SSy62 SSy56 mrc1::kanMX::mrc1-AQ (LEU2) “ SSy69 MAT rrm3::TRP1 RAD5 “ SSy72 SSy185 mrc1::HIS5::mrc1-AQ (LEU2) RAD5 “ SSy81 mrc1::HIS5::mrc1-AQ (LEU2) rrm3::TRP1 (SSy108 segregant) “ SSy108 SSy69 X SSy72 diploid “ SSy109 SSy69 X SSy185 diploid “ SSy116 SSy47 mrc1-AQ “ SSy185 mrc1::HIS5 RAD5 “ CVy29 E1000 mrc1::kanMX ars608::HIS3 bar1::LEU2 “ CVy30 CVy31 mrc1::kanMX “ CVy31 E1000 bar1::LEU2 cdc45::CDC45-HA3 (TRP1) “ rad5-535 CVy39 E1000 ars608::HIS3 bar1::TRP1 “ CVy40 CVy39 mrc1::HIS5::mrc1-AQ (LEU2) “ |
