Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases.. Saccharomyces cerevisiae

Initiation of eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding in vivo studies found that DDK was required for Cdc45 binding at early origins during G1. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of the lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to DNA unwinding and initial RNA primer synthesis. Overall design: We performed chromatin immunoprecipitation (ChIP) against Cdc45 in CDC7 and cdc7-4 cells arrested in G1 phase to assess the requirement of the Dbf4-dependent kinase on the recruitment of Cdc45 to origin DNA during G1.

真核生物DNA复制的起始过程，需要将解旋酶（helicase）加载与解旋酶激活及复制体（replisome）组装在时间上相互分离。我们采用依赖真核生物复制起始位点的体外检测体系，对上述事件的调控机制进行了研究。在完成解旋酶加载后，我们发现依赖Dbf4的Cdc7激酶（DDK）可介导Sld3与解旋酶激活蛋白Cdc45向复制起始位点的初始招募。对应的体内实验研究显示，在G1期，DDK是Cdc45结合早期复制起始位点的必需因子。当S期细胞周期蛋白依赖性激酶（S-CDK）激活后，第二种解旋酶激活蛋白GINS以及复制体的其余组分会被招募至复制起始位点。针对DNA聚合酶招募过程的研究表明，Mcm10蛋白与DNA解旋过程均对滞后链DNA聚合酶的招募至关重要，但对前导链DNA聚合酶的招募无显著影响。本研究明确了DDK与S-CDK在解旋酶激活过程中的不同功能，并支持如下模型：前导链DNA聚合酶的招募发生于DNA解旋与初始RNA引物合成之前。实验整体设计：我们在阻滞于G1期的CDC7与cdc7-4细胞中，针对Cdc45开展了染色质免疫共沉淀（ChIP）实验，以此评估G1期内依赖Dbf4的激酶对Cdc45招募至复制起始位点DNA的必要性。