Structural Biology Laboratory, Sincrotrone Trieste, Trieste 34149, Italy.
J Biol Chem. 2012 Feb 3;287(6):4121-8. doi: 10.1074/jbc.M111.285395. Epub 2011 Dec 6.
Cdc45 is an essential protein conserved in all eukaryotes and is involved both in the initiation of DNA replication and the progression of the replication fork. With GINS, Cdc45 is an essential cofactor of the Mcm2-7 replicative helicase complex. Despite its importance, no detailed information is available on either the structure or the biochemistry of the protein. Intriguingly, whereas homologues of both GINS and Mcm proteins have been described in Archaea, no counterpart for Cdc45 is known. Herein we report a bioinformatic analysis that shows a weak but significant relationship among eukaryotic Cdc45 proteins and a large family of phosphoesterases that has been described as the DHH family, including inorganic pyrophosphatases and RecJ ssDNA exonucleases. These enzymes catalyze the hydrolysis of phosphodiester bonds via a mechanism involving two Mn(2+) ions. Only a subset of the amino acids that coordinates Mn(2+) is conserved in Cdc45. We report biochemical and structural data on the recombinant human Cdc45 protein, consistent with the proposed DHH family affiliation. Like the RecJ exonucleases, the human Cdc45 protein is able to bind single-stranded, but not double-stranded DNA. Small angle x-ray scattering data are consistent with a model compatible with the crystallographic structure of the RecJ/DHH family members.
Cdc45 是一种在所有真核生物中都保守的必需蛋白，它既参与 DNA 复制的起始，也参与复制叉的推进。与 GINS 一起，Cdc45 是 Mcm2-7 复制解旋酶复合物的必需辅助因子。尽管它很重要，但关于该蛋白的结构或生物化学性质尚无详细信息。有趣的是，虽然古菌中已经描述了 GINS 和 Mcm 蛋白的同源物，但尚未发现 Cdc45 的对应物。在此，我们报告了一项生物信息学分析，该分析显示真核生物 Cdc45 蛋白与已被描述为 DHH 家族的一大类磷酸酯酶之间存在微弱但显著的关系，该家族包括无机焦磷酸酶和 RecJ ssDNA 外切核酸酶。这些酶通过涉及两个 Mn(2+)离子的机制催化磷酸二酯键的水解。只有协调 Mn(2+)的氨基酸的一个子集在 Cdc45 中保守。我们报告了关于重组人 Cdc45 蛋白的生化和结构数据，这与所提出的 DHH 家族隶属关系一致。与 RecJ 外切核酸酶一样，人 Cdc45 蛋白能够结合单链，但不能结合双链 DNA。小角度 X 射线散射数据与与 RecJ/DHH 家族成员的晶体结构相容的模型一致。
