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dNTPs:  the alphabet of life
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Andrei Chabes)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

From microscopic bacteria to the giant whale, every single living organism on Earth uses the same language of life: DNA. Deoxyribonucleoside triphosphates––dNTPs (dATP, dTTP, dGTP, and dCTP)––are the building blocks of DNA and are therefore the “alphabet of life”. A balanced supply of dNTPs is essential for integral DNA transactions such as faithful genome duplication and repair. The enzyme ribonucleotide reductase (RNR) not only synthesizes all four dNTPs but also primarily maintains the crucial individual concentration of each dNTP in a cell. In this thesis we investigated what happens if the crucial balanced supply of dNTPs is disrupted, addressing whether a cell has a mechanism to detect imbalanced dNTP pools and whether all pool imbalances are equally mutagenic.

To address these questions, we introduced single amino acid substitutions into loop 2 of the allosteric specificity site of Saccharomyces cerevisiae RNR and obtained a collection of yeast strains with different but defined dNTP pool imbalances. These results directly confirmed that the loop 2 is the structural link between the substrate specificity and effector binding sites of RNR. We were surprised to observe that mutagenesis was enhanced even in a strain with mildly imbalanced dNTP pools, despite the availability of the two major replication error correction mechanisms: proofreading and mismatch repair. However, the mutagenic potential of different dNTP pool imbalances did not directly correlate with their severity, and the locations of the mutations in a strain with elevated dTTP and dCTP were completely different from those in a strain with elevated dATP and dGTP. We then investigated, whether dNTP pool imbalances interfere with cell cycle progression and if they are detected by the S-phase checkpoint, a genome surveillance mechanism activated in response to DNA damage or replication blocks. The S-phase checkpoint was activated by the depletion of one or more dNTPs. In contrast, when none of the dNTP pools 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. We also observed an interesting mutational strand bias in one of the mutant rnr1 strains suggesting that the S-phase checkpoint may selectively prevent formation of replication errors during leading strand replication. We further used these strains to study the mechanisms by which dNTP pool imbalances induce genome instability. In addition, we discovered that a high dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions, which are difficult to bypass at normal dNTP concentrations.

Our results broaden the role of dNTPs beyond ‘dNTPs as the building blocks’ and suggest that dNTPs are not only the building blocks of DNA but also that their concentrations in a cell have regulatory implications for maintaining genomic integrity. This is important as all cancers arise as a result of some kind of genomic abnormality.

Place, publisher, year, edition, pages
Umeå: Umeå university , 2010. , 37 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1363
Keyword [en]
ribonucleotide reductase, dNTP pools, s-phase checkpoint, mutagenesis
Research subject
Medical Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-35388ISBN: 978-91-7459-045-6 (print)OAI: oai:DiVA.org:umu-35388DiVA: diva2:343962
Public defence
2010-09-09, KB3A9, KBC, Umeå universitet, Umeå, 21:51 (English)
Opponent
Supervisors
Available from: 2010-08-23 Created: 2010-08-16 Last updated: 2011-03-18Bibliographically 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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