Tipin is required for stalled replication forks to resume DNA replication after removal of aphidicolin in Xenopus egg extracts.
Tipin and its interacting partner Tim1 (Timeless) form a complex at replication forks that plays an important role in the DNA damage checkpoint response. Here we identify Xenopus laevis Tipin as a substrate for cyclin E/cyclin-dependent kinases 2 that is phosphorylated in interphase and undergoes further phosphorylation upon entry into mitosis. During unperturbed DNA replication, the Tipin/Tim1 complex is bound to chromatin, and we were able to detect interactions between Tipin and the MCM helicase. Depletion of Tipin from Xenopus extracts did not significantly impair normal replication but substantially blocked the ability of stalled replication forks to recover after removal of a block imposed by aphidicolin. Tipin-depleted extracts also showed defects in the activation of Chk1 in response to aphidicolin, probably because of a failure to load the checkpoint mediator protein Claspin onto chromatin.
Errico
Alessia
A
Clare Hall Laboratories, Cancer Research UK, South Mimms, Hertfordshire EN6 3LD, United Kingdom.
Costanzo
Vincenzo
V
Hunt
Tim
T
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000818:Animals; D016590:Aphidicolin / Q000378:metabolism / Q000494:pharmacology*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D010063:Ovum / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D016196:S Phase; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; C416368:CLSPN protein, Xenopus; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D019927:Cyclin E; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C524403:Tim1 protein, Xenopus; D029867:Xenopus Proteins; C524402:tipin protein, Xenopus; D016590:Aphidicolin; D011494:Protein Kinases; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D051357:Cyclin-Dependent Kinase 2