Re-constitution of pre-RC complex

起源识别复合物（originrecognitioncomplex，ORC）是真核生物染色体DNA复制的启动子，通过其亚基Orc 1 p和Orc 5 p与ATP结合。Orc 1 p具有ATP酶活性。DnaA的E.在大肠杆菌启动子中，ATP-DnaA复合物对DNA复制有活性，而ADP-DnaA复合物对DNA复制无活性，因此，DnaA的ATP酶活性使ATP-DnaA复合物失活，从而抑制染色体DNA复制的重新启动。在这项研究中，我们研究了ADP结合ORC的过滤器结合试验。ADP与野生型ORC和ORC-1A（Orc 1 p与步行者A基序缺陷的ORC）结合的Kd值小于10 nM，表明Orc 5 p可以与ADP以类似于ATP的高亲和力结合。ORC-SA（含有带有缺陷的步行者A基序的Orc 5 p的ORC）不与ADP结合，表明ADP-Orc 1 p复合物太不稳定而不能通过过滤结合测定法检测。ADP比ATP更快地从野生型ORC和ORC-1A解离。起源DNA片段不刺激ADP结合任何类型的ORC。在ADP存在下，ORC不能以序列特异性的方式与起始DNA结合。因此，在真核生物中，ADP-ORC复合物可能无法启动染色体DNA复制，并且在这方面它类似于原核生物中的ADP-DnaA复合物。然而，总体控制可能有所不同。在真核生物中，ADP-ORC复合物是不稳定的，表明ADP-ORC复合物可能迅速变成ATP-ORC复合物;而在原核生物中，ADP保持与DnaA结合，使DnaA保持失活，并在一段时间内阻止重新起始。

Origin Recognition Complex (ORC), a possible initiator of chromosomal DNA replication in eukaryotes, binds to ATP through its subunits Orc1p and Orc5p. Orc1p possesses ATPase activity. As for DnaA, the E. coli initiator, the ATP-DnaA complex is active but the ADP-DnaA complex is inactive for DNA replication and therefore, the ATPase activity of DnaA inactivates the ATP-DnaA complex tp suppress the re-initiation of chromosomal DNA replication. In this study, we investigated ADP-binding to ORC by a filter-binding assay. The K_d values for ADP-binding to wild-type ORC and to ORC-1A (ORC containing Orc1p with a defective Walker A motif) were less than 10 nM, showing that Orc5p can bind to ADP with a high affinity, similar to ATP. ORC-SA (ORC containing Orc5p with a defective Walker A motif) did not bind to ADP, suggesting that ADP-Orc1p complex is too unstable to be detected by the filter-binding assay. ADP dissociated more rapidly from wild-type ORC and ORC-1A than ATP did. Origin DNA fragments did not stimulate ADP-binding to any types of ORC. In the presence of ADP, ORC could not bind to origin DNA in a sequence-specific manner. Thus in eukaryotes, the ADP-ORC complex may be unable to initiate chromosomal DNA replication, and in this it resembles the ADP-DnaA complex in prokaryotes. However, overall control may be different. In eukaryotes, the ADP-ORC complex is unstable, suggesting that ADP-ORC complex might rapidly become ATP-ORC complex ; whereas in prokaryotes, ADP remains bound to DnaA, keeping DnaA inactive, and preventing re-initiation for some periods.