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Mechanisms controlling DNA damage survival and mutation rates in budding yeast
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Andrei Chabes)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

All living organisms are made of cells, within which genetic information is stored on long strands of deoxyribonucleic acid (DNA). The DNA encodes thousands of different genes and provides the blueprint for all of the structures and activities occurring within the cell. The building blocks of DNA are the four deoxyribonucleotides, dATP, dGTP, dTTP, and dCTP, which are collectively referred to as dNTPs.

The key enzyme in the production of dNTPs is ribonucleotide reductase (RNR). In the budding yeast Saccharomyces cerevisiae, the concentrations of the individual dNTPs are not equal and it is primarily RNR that maintains this balance. Maintenance of the dNTP pool balance is critical for accurate DNA replication and DNA repair since elevated and/or imbalanced dNTP concentrations increase the mutation rate and can ultimately lead to genomic instability and cancer. In response to DNA damage, the overall dNTP concentration in S. cerevisiae increases. Cell survival rates increase as a result of the elevated concentration of dNTPs, but the cells also suffer from a concomitant increase in mutation rates. When the replication machinery encounters DNA damage that it cannot bypass, the replication fork stalls and recruits specialized translesion synthesis (TLS) polymerases that bypass the damage so that replication can continue. We hypothesized that elevated dNTP levels in response to DNA damage may allow the TLS polymerases to more efficiently bypass DNA damage. To explore this possibility, we deleted all known TLS polymerases in a yeast strain in which we could artificially increase the dNTP concentrations. Surprisingly, even though all TLS polymerases had been deleted, elevated dNTP concentrations led to increased cell survival after DNA damage. These results suggest that replicative DNA polymerases may be involved in the bypass of certain DNA lesions under conditions of elevated dNTPs. We confirmed this hypothesis in vitro by demonstrating that high dNTP concentrations result in an increased efficiency in the bypass of certain DNA lesions by DNA polymerase epsilon, a replicative DNA polymerase not normally associated with TLS activity.

We asked ourselves if it would be possible to create yeast strains with imbalanced dNTP concentrations in vivo, and, if so, would these imbalances be recognized by the checkpoint control mechanisms in the cell. To address these questions, we focused on the highly conserved loop2 of the allosteric specificity site of yeast Rnr1p. We introduced several mutations into RNR1-loop2 that resulted in changes in the amino acid sequence of the protein.

Each of the rnr1-loop2 mutation strains obtained had different levels of individual dNTPs relative to the others. Interestingly, all of the imbalanced dNTP concentrations led to increased mutation rates, but these mutagenic imbalances did not activate the S-phase checkpoint unless one or several dNTPs were present at concentrations that were too low to sustain DNA replication. We were able to use these mutant yeast strains to successfully correlate amino acid substitutions within loop2 of Rnr1p to specific ratios of dNTP concentrations in the cells. We also demonstrated that specific imbalances between the individual dNTP levels result in unique mutation spectra. These mutation spectra suggest that the mutagenesis that results from imbalanced dNTP pools is due to a decrease in fidelity of the replicative DNA polymerases at specific DNA sequences where they are more likely to make a mistake. The mutant rnr1-loop2 strains that we have created with defined dNTP pool imbalances will be of great value for in vivo studies of polymerase fidelity, translesion synthesis by specialized DNA polymerases, and lesion recognition by the DNA repair machinery.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2012. , 40 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1493
Keyword [en]
dNTPs, ribonucleotide reductase, translesion synthesis, TLS polymerases
National Category
Other Basic Medicine
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-54203ISBN: 978-91-7459-411-9 (print)OAI: oai:DiVA.org:umu-54203DiVA: diva2:516598
Public defence
2012-05-11, KB3A9, Linnaeus väg 10, 901 87 Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2012-04-20 Created: 2012-04-18 Last updated: 2014-06-12Bibliographically approved
List of papers
1. Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage
Open this publication in new window or tab >>Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage
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2008 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 17, 5660-5667 p.Article in journal (Refereed) Published
Abstract [en]

The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-22414 (URN)10.1093/nar/gkn555 (DOI)18772226 (PubMedID)
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved
2. Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint
Open this publication in new window or tab >>Highly mutagenic and severely imbalanced dNTP pools can escape detection by the S-phase checkpoint
2010 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 38, no 12, 3975-3983 p.Article in journal (Refereed) Published
Abstract [en]

A balanced supply of deoxyribonucleoside triphosphates (dNTPs) is one of the key prerequisites for faithful genome duplication. Both the overall concentration and the balance among the individual dNTPs (dATP, dTTP, dGTP, and dCTP) are tightly regulated, primarily by the enzyme ribonucleotide reductase (RNR). We asked whether dNTP pool imbalances interfere with cell cycle progression and are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. By introducing single amino acid substitutions in loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR, we obtained a collection of strains with various dNTP pool imbalances. Even mild dNTP pool imbalances were mutagenic, but the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity. The S-phase checkpoint was activated by the depletion of one or several dNTPs. In contrast, when none of the dNTPs was limiting for DNA replication, even extreme and mutagenic dNTP pool imbalances did not activate the S-phase checkpoint and did not interfere with the cell cycle progression.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-32433 (URN)10.1093/nar/gkq128 (DOI)000280563400010 ()20215435 (PubMedID)
Note
E-pub ahead of printAvailable from: 2010-03-11 Created: 2010-03-11 Last updated: 2017-12-12Bibliographically approved
3. Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools
Open this publication in new window or tab >>Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools
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2011 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 39, no 4, 1360-1371 p.Article in journal (Refereed) Published
Abstract [en]

The mechanisms by which imbalanced dNTPs induce mutations have been well characterized within a test tube, but not in vivo. We have examined mechanisms by which dNTP imbalances induce genome instability in strains of Saccharomyces cerevisiae with different amino acid substitutions in Rnr1, the large subunit of ribonucleotide reductase. These strains have different dNTP imbalances that correlate with elevated CAN1 mutation rates, with both substitution and insertion-deletion rates increasing by 10- to 300-fold. The locations of the mutations in a strain with elevated dTTP and dCTP are completely different from those in a strain with elevated dATP and dGTP. Thus, imbalanced dNTPs reduce genome stability in a manner that is highly dependent on the nature and degree of the imbalance. Mutagenesis is enhanced despite the availability of proofreading and mismatch repair. The mutations can be explained by imbalanced dNTP-induced increases in misinsertion, strand misalignment and mismatch extension at the expense of proofreading. This implies that the relative dNTP concentrations measured in extracts are truly available to a replication fork in vivo. An interesting mutational strand bias is observed in one rnr1 strain, suggesting that the S-phase checkpoint selectively prevents replication errors during leading strand replication.

Research subject
Medical Biochemistry
Identifiers
urn:nbn:se:umu:diva-41104 (URN)10.1093/nar/gkq829 (DOI)20961955 (PubMedID)
Available from: 2011-03-17 Created: 2011-03-17 Last updated: 2017-12-11Bibliographically approved

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