Still the most interesting enzyme in the world

Mathews, Christopher K.

doi:10.1096/fj.201800790r

Cited by 13 publications

(10 citation statements)

References 17 publications

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“…To characterize and compare mutation profiles between different isogenetic backgrounds, we genetically altered dNTP pools using previously characterized mutations in RNR1, the large subunit of RNR. We chose RNR because alterations in dNTP pool levels generate distinct mutation profiles and RNR is well-associated with cancer (XU et al 2008;KUMAR et al 2010;KUMAR et al 2011;BUCKLAND et al 2014;AYE et al 2015;MATHEWS 2015;WATT et al 2016;MATHEWS 2018). The rnr1D57N mutation, in the activity site, leads to a balanced two-fold increase in each of the four dNTPs (CHABES et al 2003).…”

Section: Rates Of Canavanine Resistance In Different Genetic Backgroundsmentioning

confidence: 99%

“…RNR1 encodes the large subunit of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step in dNTP synthesis. RNR expression and activity is tightly regulated to promote replication fidelity and is misregulated in many different types of tumors and cancer cells (AYE et al 2015;MATHEWS 2015;MATHEWS 2018). Altered RNR activity is associated with cancer and nucleobase analogs are frequently used as chemotherapeutics, commonly targeting RNR (JORDHEIM et al 2013;KOHNKEN et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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A targeted next generation sequencing approach to develop robust, genotype-specific mutation profiles and uncover novel variants inSaccharomyces cerevisiae

Lamb

Bard

Buck

et al. 2020

Preprint

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Determining mutation signatures is becoming the standard for understanding the etiology of human tumors and informing cancer treatment. Multiple levels of replication fidelity prevent mutagenesis that leads to carcinogenesis, including regulation of free deoxyribonucleoside triphosphate (dNTP) pools and the mismatch repair (MMR) pathway. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to determine mutational signatures associated with both skewed and balanced increases in dNTP levels and/or defects in MMR substrate recognition. Notably, even small changes in dNTP pools led to distinct mutation profiles, as did deletions of each MMR recognition subunit MSH2, MSH3 or MSH6. Altered dNTP levels increased and altered the mutational load, therefore combining rnr1 and msh mutations allowed us to observe previously unreported specificities of Msh2-Msh3 and Msh2-Msh6. Msh2-Msh3 is uniquely able to direct repair of G/C single base deletions in GC runs, while Msh2-Msh6 specifically directs repair of substitutions at G/C dinucleotides. We also identified broader sequence contexts that influence variant profiles in different genetic backgrounds and found that there was not necessarily a simple additive relationship of mutation profiles in double mutants. Our results have implications for interpreting mutation signatures from human tumors.

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Section: Rates Of Canavanine Resistance In Different Genetic Backgroundsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A targeted next generation sequencing approach to develop robust, genotype-specific mutation profiles and uncover novel variants inSaccharomyces cerevisiae

Lamb

Bard

Buck

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…Altered dNTP pools compromise replication fidelity by: (1) increasing the frequency of misinsertion and misalignment by replicative DNA polymerases and (2) biasing those polymerases toward synthesis at the expense of proofreading, promoting extension beyond a mispair ( Phear et al 1987 ; Kunkel and Soni 1988 ; Kumar et al 2010 ). Notably, altered RNR activity is associated with cancer and nucleobase analogs are frequently used as chemotherapeutics, commonly targeting RNR ( Jordheim et al 2013 ; Aye et al 2015 ; Kohnken et al 2015 ; Mathews 2015 , 2018 ). Overexpression of Rrm2 , encoding the small subunit of RNR, induced lung neoplasms in a mouse model, consistent with an elevated mutator phenotype ( Xu et al 2008 ).…”

Section: Introductionmentioning

confidence: 99%

A selection-based next generation sequencing approach to develop robust, genotype-specific mutation profiles in Saccharomyces cerevisiae

Lamb

Bard

Buck

et al. 2021

G3 Genes|Genomes|Genetics

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Distinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature can therefore predict the underlying mechanism of mutagenesis. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds. Mutations in the activity and selectivity sites of rnr1 lead to elevated and/or unbalanced dNTP levels, which compromises replication fidelity and increases mutation rates. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed previously unidentified, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity.

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“…RNR has been described as a textbook example of allosteric regulation in enzymes and employs two different allosteric mechanisms to regulate the synthesis of dNTPs ( 21 , 22 ). One common mechanism regulates the balance between the four dNTPs in a sophisticated feedback control at the specificity site (s-site).…”

Section: Introductionmentioning

confidence: 99%

A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant

Grinberg

Lundin

Sahlin

et al. 2018

Journal of Biological Chemistry

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Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor MnIII/MnIV. We show that NrdA from F. ignava can form a catalytically competent RNR with the MnIII/MnIV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis. In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.

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Still the most interesting enzyme in the world

Cited by 13 publications

References 17 publications

A targeted next generation sequencing approach to develop robust, genotype-specific mutation profiles and uncover novel variants inSaccharomyces cerevisiae

A targeted next generation sequencing approach to develop robust, genotype-specific mutation profiles and uncover novel variants inSaccharomyces cerevisiae

A selection-based next generation sequencing approach to develop robust, genotype-specific mutation profiles in Saccharomyces cerevisiae

A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant

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