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Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication
Yoshitami Hashimoto1, Haruhiko Takisawa
1Department of Biology, Graduate School of Science, Osaka University, Toyonaka, Japan.
Abstract:
Fission yeast Cut5/Rad4 and its budding yeast homolog Dpb11 are required for both DNA replication and the S-phase checkpoint. Here, we have investigated the role of the Xenopus homolog of Cut5 in the initiation of DNA replication using Xenopus egg extracts. Xenopus Cut5, which shows sequence similarity to DmMus101 and HsTopBP1, is essential for DNA replication in the egg extracts. It is required for the chromatin binding of Cdc45 and DNA polymerases, but not for the formation of pre-replicative complexes or the elongation stage of DNA replication. The chromatin binding of Cut5 consists of two distinct modes. S-phase cyclin-dependent kinase (S-CDK)-independent binding is sufficient for DNA replication while S-CDK-dependent binding is dispensable. Further, S-CDK acts after the chromatin binding of Cut5 and before the binding of Cdc45. These results demonstrate that the chromatin binding of Cut5 is required for the action of S-CDK, which in turn triggers the formation of pre-initiation complexes of DNA replication.
Insights
Xenopus Cut5 is essential for initiating DNA replication by facilitating the binding of Cdc45 and DNA polymerases to chromatin. Its binding is crucial for S-phase cyclin-dependent kinase activity, which then triggers DNA replication initiation.
Area of Science:
- Cell Biology
- Molecular Biology
- Genetics
Background:
- Fission yeast Cut5/Rad4 and budding yeast Dpb11 are vital for DNA replication and the S-phase checkpoint.
- The precise role of Cut5 homologs in DNA replication initiation requires further investigation.
Purpose of the Study:
- To elucidate the function of the Xenopus homolog of Cut5 in initiating DNA replication using Xenopus egg extracts.
- To understand the regulatory mechanisms governing Cut5's role in DNA replication.
Main Methods:
- Utilized Xenopus egg extracts to study DNA replication initiation.
- Investigated the chromatin binding properties of Xenopus Cut5 and its relationship with replication factors.
- Analyzed the impact of S-phase cyclin-dependent kinase (S-CDK) on Cut5 function.
Main Results:
- Xenopus Cut5 is essential for DNA replication in egg extracts, specifically required for the chromatin binding of Cdc45 and DNA polymerases.
- Cut5's chromatin binding occurs independently of S-CDK and is sufficient for replication; S-CDK-dependent binding is dispensable.
- S-CDK acts post-Cut5 chromatin binding and pre-Cdc45 binding, indicating a sequential regulatory role.
Conclusions:
- Chromatin-bound Cut5 is necessary for S-CDK activity, which subsequently triggers the formation of pre-initiation complexes.
- This study reveals a novel regulatory pathway for DNA replication initiation involving Cut5 and S-CDK in Xenopus.
- Findings contribute to understanding the conserved mechanisms of DNA replication control across eukaryotes.