Molecular mechanism of assembly of replication proteins at replication origins in eukaryotes

在真核细胞的复制起始处，当CDK活性较低时，从M期晚期到G1期形成复制前复合体(preReplicative Complex，Pre-RC)。当CDK在G1期晚期被激活时，许多复制蛋白聚集在前RC上启动DNA复制。然而，它们在起源时是如何组装的还没有被阐明。这是因为在DNA复制的起始步骤中，CDK底物和依赖于CDK的反应是未知的。在这项研究中，我们首先确定SLD2和SLD3是启动DNA复制所必需的CDK底物。CDK磷酸化的SLD2和SLD3都与Dpbl1结合。这些结合对于启动DNA复制是必不可少的，这样当两个结合都被绕过时，DNA复制就会在没有CDK活性的情况下启动。SLD2有11个CDK磷酸化基序。Thr84在其中一个基序上的磷酸化是与Dpbl1结合的单一决定因素，而其他SLD2磷酸化是Thr84磷酸化的先决条件。这一规定似乎有助于微调原点发射。SLD3与一个新的因子SLD7形成一个复合体，具有启动DNA复制的功能。接下来，我们发现预加载复合体(Pre-LC)的形成是一个依赖于CDK的反应。Pre-LC是由ε、GINS、SLD2和Dpbl1组成的一种新的复合体，它的形成依赖于CDK的活性，而与来源或前RC无关。因此，我们提出了这样的模型：当SLD2被CDK磷酸化并与Dpbl1结合时，形成前LC；由于SLD3与G1期的起始点相关联，它在起始点被CDK磷酸化；磷酸化的SLD3通过Dpbl1将前LC招募到起始点；然后，DNA复制启动。

At replication origins in eukaryotic cells, the pre-Replicative Complex (pre-RC) forms from late M phase to G1 phase when CDK activity is low. When CDK is activated at late G1 phase, many replication proteins assemble on the pre-RC to initiate DNA replication. However, how they assemble at origins had not been elucidated. This is because CDK substrates and CDK-dependent reaction at the initiation step of DNA replication was not known. In this study, we first identified Sld2 and Sld3 as CDK substrates essential for initiation of DNA replication. Both CDK-phosphorylated Sld2 and Sld3 bind to Dpbll. These bindings are essential for initiation of DNA replication, so that when both binding are bypassed DNA replication initiates in the absence of CDK activity. Sld2 has 11 CDK phosphorylation motifs. Phosphorylation of Thr84 at one of these motifs is a single determinant for binding to Dpbll while other Sld2 phosphorylations are prerequisites for Thr84 phosphorylation. This regulation seems to contribute to fine-tuning of origin firing. Sld3 forms a complex with a novel factor, Sld7 and functions for initiation of DNA replication. Next, we found formation of the pre-Loading Complex (pre-LC) as a CDK-dependent reaction. The pre-LC is a novel complex containing DNA polymerase ε, GINS, Sld2 and Dpbll, which formation depends on CDK activity but not association with origins or the pre-RC. We thus propose the model; when Sld2 is phosphorylated by CDK and binds to Dpbll the pre-LC forms; since Sld3 associates with origins in G1 phase it is phosphorylated by CDK at origins; the phosphorylated Sld3 recruits the pre-LC to origins through Dpbll; then, DNA replication initiates.