Klemm R D, Bell S P
Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8361-7. doi: 10.1073/pnas.131006898.
The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.
复制起点识别复合物（ORC）结合复制起点，并在这些位点指导组装一个高阶蛋白质复合物。ORC在体外能结合并水解ATP。ATP与ORC最大的亚基Orc1p结合，可刺激其与复制起点DNA的特异性结合；然而，ORC水解ATP的功能尚不清楚。为了阐明ATP水解的作用，我们构建了Orc1p内具有显性致死性的突变体。在生理ATP浓度下，这些突变体在无DNA时对ATP水解有缺陷，但对ATP结合无缺陷。这些突变体在过表达时会抑制前复制复合物的形成。同时过表达野生型而非突变型Cdc6p可抑制这些突变型ORC复合物的显性致死表型。我们的研究结果表明，这些水解缺陷型突变体通过使Cdc6p从复制起点脱离而抑制生长。基于这些观察结果，我们提出Cdc6p能特异性识别Orc1p的ATP结合状态，且ATP水解与前复制复合物的解体相关。
