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Both cdc2 and cdk2 promote S phase initiation in Xenopus egg extracts
S Chevalier1, J P Tassan, R Cox
1Département de Biologie et Génétique du Développement, CNRS, URA 256, Université de Rennes I, France.
Journal of Cell Science
|May 1, 1995
Summary
Cyclin-dependent kinases (cdks) are crucial for initiating DNA replication in Xenopus egg extracts. Both cdk2 and cdc2 can restore S phase initiation, with newly synthesized cdc2 playing a key role.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Xenopus egg extracts replicate DNA in a regulated manner, mimicking in vivo conditions.
- Cyclin-dependent kinases (cdks) and specifically cdk2 are essential for initiating S phase DNA replication.
Purpose of the Study:
- To investigate the roles of cdk2 and cdc2 in initiating S phase DNA replication using Xenopus egg extracts.
- To determine if newly synthesized or pre-existing forms of these kinases are responsible for replication initiation.
Main Methods:
- Depletion of cdk2 and cdc2 from egg extracts using p13suc1 beads.
- Re-addition of synthesized cdk2 or cdc2 proteins (including mutants) via mRNA translation.
- Assessing replication initiation by total DNA synthesis and labeling index.
Main Results:
- Re-addition of either cdk2 or cdc2 mRNA restored S phase initiation in depleted extracts.
- An ATP-binding site mutant of cdk2 (cdk2.R33) failed to stimulate replication and inhibited initiation.
- Both human and Xenopus cdc2 mRNAs rescued replication, indicating a requirement for cdc2 catalytic activity.
Conclusions:
- Newly synthesized p34cdc2, but not the pre-existing form, can induce S phase DNA replication.
- Newly synthesized p34cdc2 may have overlapping functions with p33cdk2 in initiating S phase.