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ATP-dependent pre-replicative complex assembly is facilitated by Adk1p in budding yeast
Xue Cheng1, Zhen Xu, Jiafeng Wang
1Section of Biochemistry and Cell Biology, Division of Life Science, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China.
The Journal of Biological Chemistry
|July 28, 2010
Summary
Adenylate kinase 1 protein (Adk1p) is crucial for DNA replication initiation in yeast. Adk1p regulates ATP metabolism on pre-replicative complex proteins, ensuring proper assembly and activation for cell cycle progression.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- DNA replication initiation is tightly regulated by pre-replicative complex (pre-RC) assembly.
- ATP binding and hydrolysis are critical for pre-RC function.
- The role of adenylate kinase 1 protein (Adk1p) in this process was previously unknown.
Purpose of the Study:
- To investigate the role of Adk1p in pre-RC assembly and DNA replication initiation in Saccharomyces cerevisiae.
- To elucidate the molecular mechanisms by which Adk1p influences replication initiation.
Main Methods:
- Genetic screening to identify mutations affecting Adk1p function.
- Flow cytometry to assess cell cycle progression.
- Chromatin immunoprecipitation (ChIP) assays to determine protein-DNA interactions.
- Co-immunoprecipitation to study protein-protein interactions.
Main Results:
- Mutations in Adk1p, particularly in the nucleotide-binding site, impaired DNA replication initiation and S phase entry.
- Adk1p binds to chromatin and interacts with Orc3p, a key component of the pre-RC.
- Adk1p depletion or mutation reduced pre-RC assembly during the M-to-G(1) transition.
- Adk1p's interaction with chromatin and Orc3p was diminished in the mutant, independent of cellular ATP levels.
Conclusions:
- Adk1p plays a vital role in the assembly and activation of the pre-replicative complex.
- Adk1p likely regulates ATP metabolism specifically at pre-RC proteins to facilitate DNA replication initiation.
- Adk1p is a novel regulator of DNA replication initiation in yeast.
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