Division of Biology, California Institute of Technology, Pasadena, California 91125, USA.
J Biol Chem. 2010 Apr 23;285(17):12638-46. doi: 10.1074/jbc.M109.077370. Epub 2010 Feb 27.
The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain of Claspin. Furthermore, we have established that two amino acids in this motif, Asp(861) and Gln(866), are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication.
Dbf4/Drf1 依赖性激酶 (DDK) 是真核生物 DNA 复制起始所必需的。另一种蛋白 Claspin 介导细胞复制叉停滞 checkpoint 反应的激活，也是复制的调节剂。在这项研究中，我们发现 DDK 在体外磷酸化 Claspin，并在非洲爪蟾卵提取物中形成包含 Cdc7、Drf1 和 Claspin 的核复合物。此外，纯化的 Claspin 和 DDK 能够进行直接的体外相互作用。我们鉴定了 Claspin 上的一个保守结合位点，该位点对于 DDK 的相互作用是必需的。该位点对应于 Claspin 的 Chk1 结合域中的两个序列重复中的第一个。此外，我们已经确定该基序中的两个氨基酸，天冬氨酸 (Asp861) 和谷氨酰胺 (Gln866)，对于 Claspin 和 DDK 之间的相互作用是必需的。我们发现，不能与 DDK 相互作用的 Claspin 突变体仍然能够与 Chk1 结合并在 DNA 复制受阻时激活它。然而，用这些 Claspin 突变体重建的 Claspin 耗尽的卵提取物进行 DNA 复制的速度更慢。这些发现表明，DDK 与 Claspin 的相互作用介导了与 DNA 复制相关的 Claspin 的无 checkpoint 功能。
