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Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools.

Kumar D, Abdulovic AL, Viberg J, Nilsson AK, Kunkel TA, Chabes A - Nucleic Acids Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

ABSTRACT
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.

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Related in: MedlinePlus

Models for substitutions and deletions resulting from imbalanced dNTP pools. MI: misinsertion; MA: misalignment; PR: primer relocation; RE: rapid extension; IN: incorporation; RA: realignment. Red characters represent the mutational event and green characters represent bases where the dNTP is at an excessively high concentration.
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Figure 1: Models for substitutions and deletions resulting from imbalanced dNTP pools. MI: misinsertion; MA: misalignment; PR: primer relocation; RE: rapid extension; IN: incorporation; RA: realignment. Red characters represent the mutational event and green characters represent bases where the dNTP is at an excessively high concentration.

Mentions: The four deoxyribonucleoside triphosphates, dATP, dTTP, dGTP and dCTP, are essential precursors for the DNA synthesis, which is required for replication, recombination and repair. Because these DNA transactions are needed to maintain genome stability, perturbations in the absolute and relative concentrations of the four dNTPs increase mutation rates by reducing the fidelity of DNA synthesis (1). Changes in dNTPs concentration are mutagenic and may occur due to mutations in enzymes involved in dNTP metabolism or changes in the environment. The mutational mechanisms induced by imbalanced dNTP pools within cells have not been extensively investigated. In vitro studies performed with purified DNA polymerases have revealed several mechanisms by which dNTP perturbations reduce fidelity [reviewed in (2)]. One mechanism predicts that imbalanced dNTP pools increase the probability that a DNA polymerase will misinsert an incorrect dNTP [reviewed in (3)]. For example, an abnormally high ratio of dTTP as compared to dGTP can promote misinsertion (MI) of dTTP opposite a template C (Figure 1, top left). Additional studies in vitro indicate that imbalanced dNTP concentrations can also increase the rate of formation of insertion–deletion (indel) errors during DNA synthesis (4,5). For instance, when the ratio of the correct dNTP to the incorrect dNTP needed for synthesis within a mononucleotide run strongly favors the incorrect dNTP, the probability of DNA strand misalignment (MA) (Figure 1, top right) is increased, thereby increasing indel error rates. Alternatively, a dNTP imbalance may induce an MI that, in the appropriate sequence context, can result in primer relocation [(PR), Figure 1, second pathway from left] to create an indel intermediate with one or more correct terminal base pairs. Another possibility supported by evidence in vitro (5–10) is MA followed by correct incorporation (IN), then realignment (RA), thereby creating a base–base mismatch that was initiated by MA (Figure 1, rightmost pathway). Finally, in vitro studies show that the probability that mismatches will eventually result in substitutions or indels are increased if the nucleotides to be incorporated immediately following the mismatch (colored green in Figure 1) are present at high enough concentrations to promote rapid extension (RE) from the mismatch prior to proofreading and/or RA [reviewed in (3,7)].Figure 1.


Mechanisms of mutagenesis in vivo due to imbalanced dNTP pools.

Kumar D, Abdulovic AL, Viberg J, Nilsson AK, Kunkel TA, Chabes A - Nucleic Acids Res. (2010)

Models for substitutions and deletions resulting from imbalanced dNTP pools. MI: misinsertion; MA: misalignment; PR: primer relocation; RE: rapid extension; IN: incorporation; RA: realignment. Red characters represent the mutational event and green characters represent bases where the dNTP is at an excessively high concentration.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045583&req=5

Figure 1: Models for substitutions and deletions resulting from imbalanced dNTP pools. MI: misinsertion; MA: misalignment; PR: primer relocation; RE: rapid extension; IN: incorporation; RA: realignment. Red characters represent the mutational event and green characters represent bases where the dNTP is at an excessively high concentration.
Mentions: The four deoxyribonucleoside triphosphates, dATP, dTTP, dGTP and dCTP, are essential precursors for the DNA synthesis, which is required for replication, recombination and repair. Because these DNA transactions are needed to maintain genome stability, perturbations in the absolute and relative concentrations of the four dNTPs increase mutation rates by reducing the fidelity of DNA synthesis (1). Changes in dNTPs concentration are mutagenic and may occur due to mutations in enzymes involved in dNTP metabolism or changes in the environment. The mutational mechanisms induced by imbalanced dNTP pools within cells have not been extensively investigated. In vitro studies performed with purified DNA polymerases have revealed several mechanisms by which dNTP perturbations reduce fidelity [reviewed in (2)]. One mechanism predicts that imbalanced dNTP pools increase the probability that a DNA polymerase will misinsert an incorrect dNTP [reviewed in (3)]. For example, an abnormally high ratio of dTTP as compared to dGTP can promote misinsertion (MI) of dTTP opposite a template C (Figure 1, top left). Additional studies in vitro indicate that imbalanced dNTP concentrations can also increase the rate of formation of insertion–deletion (indel) errors during DNA synthesis (4,5). For instance, when the ratio of the correct dNTP to the incorrect dNTP needed for synthesis within a mononucleotide run strongly favors the incorrect dNTP, the probability of DNA strand misalignment (MA) (Figure 1, top right) is increased, thereby increasing indel error rates. Alternatively, a dNTP imbalance may induce an MI that, in the appropriate sequence context, can result in primer relocation [(PR), Figure 1, second pathway from left] to create an indel intermediate with one or more correct terminal base pairs. Another possibility supported by evidence in vitro (5–10) is MA followed by correct incorporation (IN), then realignment (RA), thereby creating a base–base mismatch that was initiated by MA (Figure 1, rightmost pathway). Finally, in vitro studies show that the probability that mismatches will eventually result in substitutions or indels are increased if the nucleotides to be incorporated immediately following the mismatch (colored green in Figure 1) are present at high enough concentrations to promote rapid extension (RE) from the mismatch prior to proofreading and/or RA [reviewed in (3,7)].Figure 1.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus