Grainge Ian, Scaife Sarah, Wigley Dale B
Cancer Research UK, Clare Hall Laboratories, The London Research Institute, Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3LD, UK.
Nucleic Acids Res. 2003 Aug 15;31(16):4888-98. doi: 10.1093/nar/gkg662.
The eukaryotic pre-replication complex is assembled at replication origins in a reaction called licensing. Licensing involves the interactions of a variety of proteins including the origin recognition complex (ORC), Cdc6 and the Mcm2-7 helicase, homologues of which are also found in archaea. The euryarchaeote Archaeoglobus fulgidus encodes two genes with homology to Orc/Cdc6 and a single Mcm homologue. The A.fulgidus Mcm protein and one Orc/Cdc6 homologue have been purified and investigated in vitro. The Mcm protein is an ATP-dependent, hexameric helicase that can unwind between 200 and 400 bp of duplex DNA. Deletion of 112 amino acids from the N-terminus of A.f Mcm produced a protein, which was still capable of forming a hexamer, was competent in DNA binding and was able to unwind at least 1 kb of duplex DNA. The purified Orc/Cdc6 homologue was also able to bind DNA. Both Mcm and Orc/Cdc6 show a preference for specific DNA structures, namely molecules containing a single stranded bubble that mimics early replication intermediates. Nuclease protection showed that the binding sites for Mcm and Orc/Cdc6 overlap. The Orc/Cdc6 protein bound more tightly to these substrates and was able to displace pre-bound Mcm hexamer.
真核生物的前复制复合体在称为许可的反应中于复制起点处组装。许可涉及多种蛋白质的相互作用，包括起源识别复合体（ORC）、Cdc6和Mcm2 - 7解旋酶，古细菌中也发现了它们的同源物。嗜热栖热放线菌编码两个与Orc/Cdc6具有同源性的基因和一个Mcm同源物。嗜热栖热放线菌的Mcm蛋白和一个Orc/Cdc6同源物已被纯化并在体外进行了研究。Mcm蛋白是一种依赖ATP的六聚体解旋酶，能够解开200至400碱基对的双链DNA。从嗜热栖热放线菌Mcm的N端缺失112个氨基酸产生了一种蛋白质，该蛋白质仍能形成六聚体，具有DNA结合能力，并且能够解开至少1千碱基对的双链DNA。纯化的Orc/Cdc6同源物也能够结合DNA。Mcm和Orc/Cdc6都对特定的DNA结构表现出偏好，即含有模拟早期复制中间体的单链泡的分子。核酸酶保护显示Mcm和Orc/Cdc6的结合位点重叠。Orc/Cdc6蛋白与这些底物结合更紧密，并且能够取代预先结合的Mcm六聚体。
