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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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Cartoon depiction of the hypothesized replication fidelity bias mediated by Pol ε on the leading template when the S-phase checkpoint is activated. Blue and red stars depict hotspot events.
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Figure 5: Cartoon depiction of the hypothesized replication fidelity bias mediated by Pol ε on the leading template when the S-phase checkpoint is activated. Blue and red stars depict hotspot events.

Mentions: This putative asymmetric distribution of mutations on the coding strand in the rnr1-Q288A strain but not in the rnr1-Y285A strain suggests that mutagenesis can be differentially modulated during leading strand and lagging strand replication, depending on the nature of the pool imbalance. How might this occur? Previous analysis of early firing replication origins on the left arm of chromosome five of S. cerevisiae has shown that replication of the CAN1 gene originates from ARS507 and travels through CAN1 towards the telomere (46–48). This predicts that the non-coding strand should be the template for leading strand replication, and that the coding strand should be the template for lagging strand replication. Current evidence suggests that the leading strand template may primarily be replicated by Pol ε (49), whereas the lagging strand template may primarily be replicated by Pol δ (50). These facts are intriguing, given that (i) the rnr1-Y285A strain proliferates normally but the rnr1-Q288A strain proliferates slowly, has a defect in S-phase progression and elicits a checkpoint response (23) and (ii) Pol ε, but not Pol δ, is involved in the S-phase checkpoint response (51,52). Thus, the surprising absence of putative non-coding (i.e. leading) strand hotspots in the rnr1-Q288A strain despite the large dNTP pool imbalance may be related to Pol ε’s combined roles in leading strand replication and in the S-phase checkpoint response. In other words, as Pol ε attempts leading strand replication using too little dCTP and excess dATP and dGTP, certain mismatches that are known to be particularly difficult to extend [e.g. G-dAMP and G-dGMP, (53)] may be prevented from yielding mutations via Pol ε’s checkpoint function. Conceptually, this is similar to the idea that checkpoints initiated by DNA lesions stall replication to provide more time for DNA repair. In the present case, the checkpoint response may provide more time for error correction from proofreading by Pol ε, whose 3′-exonuclease is processive and highly active (54,55). A checkpoint might even allow more time for mismatch repair to correct leading strand replication errors, especially if replication and mismatch repair are coupled (56). Because there are still many coding (i.e. putative lagging) strand mutational hotspots in the CAN1 spectrum in the rnr1-Y285A strain, a checkpoint mechanism to reduce mutagenesis might not apply to lagging strand replication errors made by Pol δ, a polymerase with no reported role in the S-phase checkpoint response (Figure 5). It should also be noted that an activated S-phase checkpoint inhibits replication origin firing (57–59), which could be relevant to the strand biases observed here. Alternatively, it is also possible that differences in the distribution of mutations on the two strands might be related to differences in the efficiency with which pols α/δ versus pol ε extend mismatches driven by high dTTP/dCTP versus those that are driven by high dATP/dGTP. In the future, we plan to further examine this unexpected strand specificity, using rnr1 mutants in combination with mutator alleles of DNA polymerases δ and ε, in a system previously used to study leading and lagging strand replication fidelity in yeast (49,50).Figure 5.


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)

Cartoon depiction of the hypothesized replication fidelity bias mediated by Pol ε on the leading template when the S-phase checkpoint is activated. Blue and red stars depict hotspot events.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Cartoon depiction of the hypothesized replication fidelity bias mediated by Pol ε on the leading template when the S-phase checkpoint is activated. Blue and red stars depict hotspot events.
Mentions: This putative asymmetric distribution of mutations on the coding strand in the rnr1-Q288A strain but not in the rnr1-Y285A strain suggests that mutagenesis can be differentially modulated during leading strand and lagging strand replication, depending on the nature of the pool imbalance. How might this occur? Previous analysis of early firing replication origins on the left arm of chromosome five of S. cerevisiae has shown that replication of the CAN1 gene originates from ARS507 and travels through CAN1 towards the telomere (46–48). This predicts that the non-coding strand should be the template for leading strand replication, and that the coding strand should be the template for lagging strand replication. Current evidence suggests that the leading strand template may primarily be replicated by Pol ε (49), whereas the lagging strand template may primarily be replicated by Pol δ (50). These facts are intriguing, given that (i) the rnr1-Y285A strain proliferates normally but the rnr1-Q288A strain proliferates slowly, has a defect in S-phase progression and elicits a checkpoint response (23) and (ii) Pol ε, but not Pol δ, is involved in the S-phase checkpoint response (51,52). Thus, the surprising absence of putative non-coding (i.e. leading) strand hotspots in the rnr1-Q288A strain despite the large dNTP pool imbalance may be related to Pol ε’s combined roles in leading strand replication and in the S-phase checkpoint response. In other words, as Pol ε attempts leading strand replication using too little dCTP and excess dATP and dGTP, certain mismatches that are known to be particularly difficult to extend [e.g. G-dAMP and G-dGMP, (53)] may be prevented from yielding mutations via Pol ε’s checkpoint function. Conceptually, this is similar to the idea that checkpoints initiated by DNA lesions stall replication to provide more time for DNA repair. In the present case, the checkpoint response may provide more time for error correction from proofreading by Pol ε, whose 3′-exonuclease is processive and highly active (54,55). A checkpoint might even allow more time for mismatch repair to correct leading strand replication errors, especially if replication and mismatch repair are coupled (56). Because there are still many coding (i.e. putative lagging) strand mutational hotspots in the CAN1 spectrum in the rnr1-Y285A strain, a checkpoint mechanism to reduce mutagenesis might not apply to lagging strand replication errors made by Pol δ, a polymerase with no reported role in the S-phase checkpoint response (Figure 5). It should also be noted that an activated S-phase checkpoint inhibits replication origin firing (57–59), which could be relevant to the strand biases observed here. Alternatively, it is also possible that differences in the distribution of mutations on the two strands might be related to differences in the efficiency with which pols α/δ versus pol ε extend mismatches driven by high dTTP/dCTP versus those that are driven by high dATP/dGTP. In the future, we plan to further examine this unexpected strand specificity, using rnr1 mutants in combination with mutator alleles of DNA polymerases δ and ε, in a system previously used to study leading and lagging strand replication fidelity in yeast (49,50).Figure 5.

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