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Nedelcheva MN, Roguev A, Dolapchiev LB, Shevchenko A, Taskov HB, Stewart AF, Stoynov SS (2005) Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex. J Mol Biol 347(3): 509-521
Nguyen VQ, Co C, Li JJ (2001) Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411(6841): 1068-1073
Njagi GD, Kilbey BJ (1982) Mutagenesis in cdc7 strains of yeast. The fate of premutational lesions induced by ultraviolet light. Mutat Res 105(5): 313-318
Noguchi E, Noguchi C, Du LL, Russell P (2003) Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1. Mol Cell Biol 23(21): 7861-7874
Noguchi E, Noguchi C, McDonald WH, Yates JR, 3rd, Russell P (2004) Swi1 and Swi3 are components of a replication fork protection complex in fission yeast. Mol Cell Biol 24(19): 8342-8355
Nougarede R, Della Seta F, Zarzov P, Schwob E (2000) Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation. Mol Cell Biol 20(11): 3795-3806
Nyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36: 617-656
O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006) Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41(5): 577-580
O'Neill BM, Hanway D, Winzeler EA, Romesberg FE (2004) Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response. Nucleic Acids Res 32(22): 6519-6530
O'Neill BM, Szyjka SJ, Lis ET, Bailey AO, Yates JR, 3rd, Aparicio OM, Romesberg FE (2007) Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage. Proc Natl Acad Sci U S A 104(22): 9290-9295
Ooi SL, Shoemaker DD, Boeke JD (2003) DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray. Nat Genet 35(3): 277-286
Osborn AJ, Elledge SJ (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17(14): 1755-1767
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 152 |
| Contributing entity | University of Southern California |
| Repository email | cisadmin@lib.usc.edu |
| Full text | 137 Nedelcheva MN, Roguev A, Dolapchiev LB, Shevchenko A, Taskov HB, Stewart AF, Stoynov SS (2005) Uncoupling of unwinding from DNA synthesis implies regulation of MCM helicase by Tof1/Mrc1/Csm3 checkpoint complex. J Mol Biol 347(3): 509-521 Nguyen VQ, Co C, Li JJ (2001) Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature 411(6841): 1068-1073 Njagi GD, Kilbey BJ (1982) Mutagenesis in cdc7 strains of yeast. The fate of premutational lesions induced by ultraviolet light. Mutat Res 105(5): 313-318 Noguchi E, Noguchi C, Du LL, Russell P (2003) Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1. Mol Cell Biol 23(21): 7861-7874 Noguchi E, Noguchi C, McDonald WH, Yates JR, 3rd, Russell P (2004) Swi1 and Swi3 are components of a replication fork protection complex in fission yeast. Mol Cell Biol 24(19): 8342-8355 Nougarede R, Della Seta F, Zarzov P, Schwob E (2000) Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation. Mol Cell Biol 20(11): 3795-3806 Nyberg KA, Michelson RJ, Putnam CW, Weinert TA (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu Rev Genet 36: 617-656 O'Geen H, Nicolet CM, Blahnik K, Green R, Farnham PJ (2006) Comparison of sample preparation methods for ChIP-chip assays. Biotechniques 41(5): 577-580 O'Neill BM, Hanway D, Winzeler EA, Romesberg FE (2004) Coordinated functions of WSS1, PSY2 and TOF1 in the DNA damage response. Nucleic Acids Res 32(22): 6519-6530 O'Neill BM, Szyjka SJ, Lis ET, Bailey AO, Yates JR, 3rd, Aparicio OM, Romesberg FE (2007) Pph3-Psy2 is a phosphatase complex required for Rad53 dephosphorylation and replication fork restart during recovery from DNA damage. Proc Natl Acad Sci U S A 104(22): 9290-9295 Ooi SL, Shoemaker DD, Boeke JD (2003) DNA helicase gene interaction network defined using synthetic lethality analyzed by microarray. Nat Genet 35(3): 277-286 Osborn AJ, Elledge SJ (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev 17(14): 1755-1767 |
