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DNA precursor asymmetries, Mismatch Repair and their effect on mutation specificity
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Andrei Chabes)ORCID iD: 0000-0001-9749-5422
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 [en]
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: urn:nbn:se:umu:diva-101817ISBN: 978-91-7601-231-4 (print)OAI: oai:DiVA.org:umu-101817DiVA: diva2:802924
Public defence
2015-05-08, BIA201, Biologihuset, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2015-04-17 Created: 2015-04-13 Last updated: 2015-04-17Bibliographically approved
List of papers
1. Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity
Open this publication in new window or tab >>Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity
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2014 (English)In: PLOS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 10, no 12, e1004846Article in journal (Refereed) Published
Abstract [en]

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

National Category
Other Basic Medicine
Identifiers
urn:nbn:se:umu:diva-96881 (URN)10.1371/journal.pgen.1004846 (DOI)000346649900047 ()25474551 (PubMedID)
Available from: 2014-12-08 Created: 2014-12-05 Last updated: 2017-12-05Bibliographically approved
2. Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools
Open this publication in new window or tab >>Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools
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2016 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 4, 1669-1680 p.Article in journal (Other academic) Published
Abstract [en]

The absolute and relative concentrations of the four dNTPs are key determinants of DNA replication fidelity, yet the consequences of altered dNTP pools on replication fidelity have not previously been investigated on a genome-wide scale. Here, we use deep sequencing to determine the types, rates and locations of uncorrected replication errors that accumulate in the nuclear genome of a mismatch repair-deficient diploid yeast strain with elevated dCTP and dTTP concentrations. These imbalanced dNTP pools promote replication errors in specific DNA sequence motifs suggesting increased misinsertion and increased mismatch extension at the expense of proofreading. Interestingly, substitution rates are similar for leading and lagging strand replication, but are higher in regions replicated late in S phase. Remarkably, the rate of single base deletions is preferentially increased in coding sequences and in short rather than long mononucleotides runs. Based on DNA sequence motifs, we propose two distinct mechanisms for generating single base deletions in vivo. Collectively, the results indicate that elevated dCTP and dTTP pools increase mismatch formation and decrease error correction across the nuclear genome, and most strongly increases mutation rates in coding and late replicating sequences.

National Category
Cell and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-101931 (URN)10.1093/nar/gkv1298 (DOI)000371519700026 ()26609135 (PubMedID)
Note

Originally published in manuscript form with the title Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by a mutation in ribnucleotide reductase that imbalances dNTP pools.

Available from: 2015-04-16 Created: 2015-04-16 Last updated: 2017-12-04Bibliographically approved
3. Molecular basis of the essential s phase function of the rad53 checkpoint kinase
Open this publication in new window or tab >>Molecular basis of the essential s phase function of the rad53 checkpoint kinase
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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.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:umu:diva-79776 (URN)10.1128/MCB.00474-13 (DOI)000322224400012 ()23754745 (PubMedID)
Available from: 2013-09-02 Created: 2013-09-02 Last updated: 2017-12-06Bibliographically approved

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