Roles of replication fork-interacting and Chk1-activating domains from Claspin in a DNA replication checkpoint response.
Claspin is essential for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. Claspin associates with replication forks upon origin unwinding. We show that Claspin contains a replication fork-interacting domain (RFID, residues 265-605) that associates with Cdc45, DNA polymerase epsilon, replication protein A, and two replication factor C complexes on chromatin. The RFID contains two basic patches (BP1 and BP2) at amino acids 265-331 and 470-600, respectively. Deletion of either BP1 or BP2 compromises optimal binding of Claspin to chromatin. Absence of BP1 has no effect on the ability of Claspin to mediate activation of Chk1. By contrast, removal of BP2 causes a large reduction in the Chk1-activating potency of Claspin. We also find that Claspin contains a small Chk1-activating domain (residues 776-905) that does not bind stably to chromatin, but it is fully effective at high concentrations for mediating activation of Chk1. These results indicate that stable retention of Claspin on chromatin is not necessary for activation of Chk1. Instead, our findings suggest that only transient interaction of Claspin with replication forks potentiates its Chk1-activating function. Another implication of this work is that stable binding of Claspin to chromatin may play a role in other functions besides the activation of Chk1.
Lee
Joon
J
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Gold
Daniel A
DA
Shevchenko
Anna
A
Shevchenko
Andrej
A
Dunphy
William G
WG
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D018797:Cell Cycle Proteins / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D004261:DNA Replication; D004527:Egg Proteins / Q000378:metabolism; D004789:Enzyme Activation; D008954:Models, Biological; D011494:Protein Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D051818:Replication Protein C / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; C416368:CLSPN protein, Xenopus; C502211:Cdc45 protein, Xenopus; D018797:Cell Cycle Proteins; D002843:Chromatin; D004527:Egg Proteins; D029867:Xenopus Proteins; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D004257:DNA Polymerase II; D051818:Replication Protein C