Abstract
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.
Highlights
The initiation of DNA replication governs the genome duplication in eukaryotes, which occurs once and only once per cell cycle
Our study reveals that Adk1p facilitates the ATP-dependent pre-replicative complex (pre-RC) assembly and that Adk1p becomes essential for pre-RC assembly and cell viability at 37 °C
The adk1G20S mutant and wild-type cells have similar in vivo ATP levels and growth rates, indicating that the replication initiation defects of the adk1G20S mutant do not result from an ATP homeostasis imbalance
Summary
Strains, and Antibodies—The original adk1G20S/P138L mutant was isolated by the initiation of DNA replication screen after ethane methyl sulfonate mutagenesis of the YL36 parental strain (ade ade ura his trp leu112 can100), a derivative of W303-1A with the addition of the ade mutation. The adk1td-HA strain was constructed in the GAL-UBR1 background as described [40] using the PCR product generated with forward primer 5Ј-CATTAACGTTTCTCTGGTAAAGTCACCACACAGCATCAAATATAACAGTAAGGGCGAATTGGAGCTCCAC-3Ј and reverse primer 5Ј-GCACCAGGTGGGCCAATTAGGACCATTCTAATGGATTCTGAGCTAGACATCCCTCCTAAAAATGCAGCGT-3Ј. The ADK1-HA strain (3ϫHA-tagged ADK1 at the endogenous ADK1 locus) was constructed in the W303-1A background, and the pJJ244-ADK1-HA and pJJ244-adk1G20S-HA strains were constructed in the integrated pJJ244-ADK1 and pJJ244-adk1G20S background, respectively, using the one-step C-terminal tagging method [41] to transform the respective yeast cells with a PCR fragment amplified by forward primer 5Ј-AACCTCCTGCTACTGTTTGGGCTGACATCTTGAACAAGCTAGGTAAGGATCGGATCCCCGGGTT-3Ј and reverse primer 5Ј-AATTTAAAAAAAAGAAAAGATATTTAGAAGACATTGCGCAAGGTCATTAAGAATTCGAGCTCGT-3Ј. The adk1⌬ strain was constructed in the YL36 background using the one-step gene detection method [41] with a PCR product amplified by forward primer 5Ј-TTTCTCTGGTAAAGTCACCACACAGCATCAAATATAACAGTAATGTACGCTGCAGGTCG-3Ј and reverse primer 5Ј-TAAAAAAAAGAAAAGATATTTAGAAGACATTGCGCAAGGTCATTACGATGAATTCGAGC-3Ј. FACS analysis and chromatin binding assays were performed as described [4, 42, 44]
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