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Genome-wide analysis of the specificity and mechanisms of replication infidelity driven by imbalanced dNTP pools
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, NIH, DHHS, Research Triangle Park, NC 27709, USA. (Andrei Chabes)
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). (Andrei Chabes)ORCID iD: 0000-0001-9749-5422
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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.

Place, publisher, year, edition, pages
2016. Vol. 44, no 4, 1669-1680 p.
National Category
Cell and Molecular Biology
Research subject
Molecular Biology
URN: urn:nbn:se:umu:diva-101931DOI: 10.1093/nar/gkv1298ISI: 000371519700026PubMedID: 26609135OAI: diva2:805808

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
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.
Umeå University medical dissertations, ISSN 0346-6612 ; 1703
DNA Replication Fidelity, Mutations, dNTP pools, Mismatch Repair, Checkpoint, Ribonucleotide Reductase, Msh2
National Category
Cell and Molecular Biology
Research subject
Medical Biochemistry; Molecular Biology
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)
Available from: 2015-04-17 Created: 2015-04-13 Last updated: 2015-04-17Bibliographically approved

Open Access in DiVA

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