Gold, Daniel A. (2010) Molecular characterization of the Dbf4/Drf1-dependent kinase (DDK) and the DNA replication checkpoint mediator Claspin in Xenopus egg extracts. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:10232009-160656505
The integrity of DNA replication control and checkpoint mechanisms is essential for preventing tumorigenesis. DNA replication is initiated by the S-CDK and DDK kinases which mediate the unwinding of the replication fork. Genotoxic stress which specifically affects cells in S-phase is detected by the replication checkpoint. Replication blockages activate the ATR kinase which, in turn, activates the downstream effector kinase Chk1 through the mediator protein, Claspin. Chk1 facilitates the arrest of cell cycle progression and the inhibition of replication origin firing.
Claspin has two broadly defined roles, one to mediate Chk1 activation and the other as a component of the replication fork. We have endeavored to study a link between these two facets of Claspin function. Here, we show that Claspin associates with several core replication fork proteins in Xenopus egg extracts. We identified a replication fork-interacting domain on Claspin that associates with the replication fork proteins and is required for Claspin association with chromatin. However, chromatin binding-deficient Claspin proteins can still mediate Chk1 activation in Claspin-depleted extracts, albeit with reduced efficiency. Thus, the localization of Claspin to the replication fork is not required for mediation of Chk1 activation but it does potentiate this process.
Another focus of this study, DDK, is composed of the catalytic subunit Cdc7 and one of two distinct adaptor proteins, Drf1 or Dbf4. Drf1 forms a stable, active complex with Cdc7, even after replication arrest in egg extracts. Accumulation of Drf1 on chromatin in the presence of replication blocks is dependent upon ATR and Claspin but not Chk1. We characterized Xenopus Claspin as a kinase substrate of DDK which forms a stable nuclear complex with Cdc7 and Drf1 under both arrested and unperturbed replication conditions. Moreover, we identified a region of Claspin required for association with DDK that lies within the Chk1-binding domain, which contains a series of repeat sequences. This DDK-associating region is the first, but not the second of these repeat sequences. Furthermore, we have identified two evolutionarily conserved residues within this region required for DDK interaction. Claspin mutant proteins unable to interact with DDK still bind to Chk1 and rescue Chk1 activation in Claspin-depleted extracts. Therefore, we conclude that DDK regulates a largely checkpoint-independent role of Claspin function.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Subject Keywords:||Replication; Checkpoint; S-phase; DNA damage; Cell Cycle; Initiation; Genomic Instability; Tumorigenesis; Cancer; Claspin; DDK; Cdc7; Drf1|
|Degree Grantor:||California Institute of Technology|
|Thesis Availability:||Public (worldwide access)|
|Defense Date:||23 September 2009|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Daniel Gold|
|Deposited On:||30 Aug 2012 18:06|
|Last Modified:||26 Dec 2012 03:18|
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