ATP-dependent pre-replicative complex assembly is facilitated by Adk1p in budding yeast.
Pre-replicative complex (pre-RC) assembly is a critical part of the mechanism that controls the initiation of DNA replication, and ATP binding and hydrolysis by multiple pre-RC proteins are essential for pre-RC assembly and activation. Here, we demonstrate that Adk1p (adenylate kinase 1 protein) plays an important role in pre-RC assembly in Saccharomyces cerevisiae. Isolated from a genetic screen, adk1(G20S) cells with a mutation within the nucleotide-binding site were defective in replication initiation. adk1Δ cells were viable at 25 °C but not at 37°C. Flow cytometry indicated that both the adk1-td (temperature-inducible degron) and adk1(G20S) mutants were defective in S phase entry. Furthermore, Adk1p bound to chromatin throughout the cell cycle and physically interacted with Orc3p, whereas the Adk1(G20S) protein had a reduced ability to bind chromatin and Orc3p without affecting the cellular ATP level. In addition, Adk1p associated with replication origins by ChIP assay. Finally, Adk1-td protein depletion prevented pre-RC assembly during the M-to-G(1) transition. We suggest that Adk1p regulates ATP metabolism on pre-RC proteins to promote pre-RC assembly and activation.
Cheng
Xue
X
Section of Biochemistry and Cell Biology, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
Xu
Zhen
Z
Wang
Jiafeng
J
Zhai
Yuanliang
Y
Lu
Yongjun
Y
Liang
Chun
C
D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle / Q000502:physiology*; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000502:physiology*; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D009154:Mutation; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism
D002843:Chromatin; D004271:DNA, Fungal; C497217:ORC3 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases