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Molecular basis of the essential s phase function of the rad53 checkpoint kinase
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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2013 (English)In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 33, no 16, 3202-3213 p.Article in journal (Refereed) Published
Abstract [en]

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.

Place, publisher, year, edition, pages
2013. Vol. 33, no 16, 3202-3213 p.
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:umu:diva-79776DOI: 10.1128/MCB.00474-13ISI: 000322224400012PubMedID: 23754745OAI: oai:DiVA.org:umu-79776DiVA: diva2:644767
Available from: 2013-09-02 Created: 2013-09-02 Last updated: 2017-12-06Bibliographically approved
In thesis
1. DNA precursor asymmetries, Mismatch Repair and their effect on mutation specificity
Open this publication in new window or tab >>DNA precursor asymmetries, Mismatch Repair and their effect on mutation specificity
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In order to build any structure, a good supply of materials, accurate workers and quality control are needed. This is even the case when constructing DNA, the so-called “Code of Life.” For a species to continue to exist, this DNA code must be copied with incredibly high accuracy when each and every cell replicates. In fact, just one mistake in the 12 million bases that comprise the genome of budding yeast, Saccharomyces cerevisiae, can be fatal. DNA is composed of a double strand helix made up of just four different bases repeated millions of times. The building blocks of DNA are the deoxyribonucleotides (dNTPs); dCTP, dTTP, dATP and dGTP. Their production and balance are carefully controlled within each cell, largely by the key enzyme Ribonucleotide Reductase (RNR). Here, we studied how the enzymes that copy DNA, the replicative polymerases α, δ and ε, cope with the effects of an altered dNTP pool balance. An introduced mutation in the allosteric specificity site of RNR in a strain of S. cerevisiae, rnr1-Y285A, leads to elevated dCTP and dTTP levels and has been shown to have a 14-fold increase in mutation rate compared to wild type. To ascertain the full effects of the dNTP pool imbalance upon the replicative polymerases, we disabled one of the major quality control systems in a cell that corrects replication errors, the post-replicative Mismatch Repair system. Using both the CAN1 reporter assay and whole genome sequencing, we found that, despite inherent differences between the polymerases, their replication fidelity was affected very similarly by this dNTP pool imbalance. Hence, the high dCTP and dTTP forced Pol ε and Pol α/δ to make the same mistakes. In addition, the mismatch repair machinery was found to correct replication errors driven by this dNTP pool imbalance with highly variable efficiencies. Another mechanism to protect cells from DNA damage during replication is a checkpoint that can be activated to delay the cell cycle and activate repair mechanisms. In yeast, Mec1 and Rad53 (human ATR and Chk1/Chk2) are two key S-phase checkpoint proteins. They are essential as they are also required for normal DNA replication and dNTP pool regulation. However the reason why they are essential is not well understood. We investigated this by mutating RAD53 and analyzing dNTP pools and gene interactions. We show that Rad53 is essential in S-phase due to its role in regulating basal dNTP levels by action in the Dun1 pathway that regulates RNR and Rad53’s compensatory kinase function if dNTP levels are perturbed.

In conclusion we present further evidence of the importance of dNTP pools in the maintenance of genome integrity and shed more light on the complex regulation of dNTP levels.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 36 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1703
Keyword
DNA Replication Fidelity, Mutations, dNTP pools, Mismatch Repair, Checkpoint, Ribonucleotide Reductase, Msh2
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-101817 (URN)978-91-7601-231-4 (ISBN)
Public defence
2015-05-08, BIA201, Biologihuset, Umeå University, Umeå, 09:00 (English)
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Available from: 2015-04-17 Created: 2015-04-13 Last updated: 2015-04-17Bibliographically approved

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