Evidence for Sequential Action of cdc7 and cdk2 Protein Kinases during Initiation of DNA Replication in Xenopus Egg Extracts

Johannes C Walter

doi:10.1074/jbc.m008107200

Abstract

To investigate how the protein kinase cdc7 stimulates DNA replication in metazoans, a soluble cell-free replication system derived from Xenopus eggs was used. DNA was incubated in egg cytosol to form prereplication complexes and then in nucleoplasmic extract to initiate DNA synthesis. We find that cdc7 is greatly enriched in nucleoplasmic extract and that this high concentration is essential for efficient DNA replication, supporting previous models that the nucleus activates replication indirectly by sequestering essential components. cdc7 binds to chromatin at the G(1)/S transition before initiation occurs, and it dissociates from chromatin as S phase progresses. The chromatin association of cdc7 requires chromatin-bound MCM. In turn, cdc7 is required to load the initiation factor cdc45 onto the DNA. Finally, efficient replication is observed when chromatin is exposed first to cdc7 and then to cdk2 but not when it is exposed to cdk2 before cdc7. Therefore, the cdc7- and cdk2-dependent initiation steps can be separated, indicating the existence of a novel, stable initiation intermediate. Moreover, the data suggest that cdk2 can only act after cdc7 has executed its function.

Highlights

Eukaryotic cells regulate the initiation of DNA replication via the ordered assembly and disassembly of replication complexes at origins of replication [1]
The Rate of DNA Replication Is Regulated by the Concentration of cdc7—We previously showed that immunodepletion of egg cytosol and nucleoplasmic extract (NPE) with an antibody raised against Xenopus laevis cdc7 homolog (xCdc7) protein prevented replication initiation [6]
To better understand how xCdc7 contributes to replication initiation, egg cytosol and NPE were immunoblotted with anti-xCdc7 antibody

Summary

Introduction

Eukaryotic cells regulate the initiation of DNA replication via the ordered assembly and disassembly of replication complexes at origins of replication [1]. At the G1/S transition, pre-RCs are activated for replication by an S phase-specific cyclin-dependent kinase (cdk2) and a second cdk-like kinase, cdc. At the G1/S transition, the initiation factor cdc associates with the pre-RC to generate a preinitiation complex In yeast, this event appears to require both the action of cdk and cdc7/ dbf4 [2, 3], and temperature shift experiments suggest that cdk exerts its function before cdc7 [4]. To facilitate the analysis of these kinases, we are using a modification of Xenopus nuclear assembly extracts in which DNA replication occurs in the absence of nuclei [19]. Demembranated sperm chromatin is incubated with membrane-free egg cytosol to form pre-RCs. Second, a nucleoplasmic extract (NPE) is added, which initiates replication, and a complete round of semi-conservative DNA replication takes place [19]. This system has been used to further define the role of xCdc in replication initiation and to understand its functional relationship to cdk

Methods

Results

Conclusion