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Hypermutability and error catastrophe due to defects in ribonucleotide reductase.

ABSTRACT: The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5'-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichia coli RNR that are characterized by altered dNTP pools and increased mutation rates (mutator mutants). Here, we show that one particular set of RNR mutants, carrying alterations at the enzyme's allosteric specificity site, is characterized by relatively modest dNTP pool deviations but exceptionally strong mutator phenotypes, when measured in a mutational forward assay (>1,000-fold increases). We provide evidence indicating that this high mutability is due to a saturation of the DNA mismatch repair system, leading to hypermutability and error catastrophe. The results indicate that, surprisingly, even modest deviations of the cellular dNTP pools, particularly when the pool deviations promote particular types of replication errors, can have dramatic consequences for mutation rates.

SUBMITTER: Ahluwalia D

PROVIDER: S-EPMC3832021 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Hypermutability and error catastrophe due to defects in ribonucleotide reductase.

Ahluwalia Deepti D Schaaper Roel M RM

Proceedings of the National Academy of Sciences of the United States of America 20131028 46

The enzyme ribonucleotide reductase (RNR) plays a critical role in the production of deoxynucleoside-5'-triphosphates (dNTPs), the building blocks for DNA synthesis and replication. The levels of the cellular dNTPs are tightly controlled, in large part through allosteric control of RNR. One important reason for controlling the dNTPs relates to their ability to affect the fidelity of DNA replication and, hence, the cellular mutation rate. We have previously isolated a set of mutants of Escherichi ...[more]

PMID: 24167285

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Project description:Template-dependent RNA replication mechanisms render picornaviruses susceptible to error catastrophe, an overwhelming accumulation of mutations incompatible with viability. Viral RNA recombination, in theory, provides a mechanism for viruses to counteract error catastrophe. We tested this theory by exploiting well-defined mutations in the poliovirus RNA-dependent RNA polymerase (RDRP), namely, a G64S mutation and an L420A mutation. Our data reveal two distinct mechanisms by which picornaviral RDRPs influence error catastrophe: fidelity of RNA synthesis and RNA recombination. A G64S mutation increased the fidelity of the viral polymerase and rendered the virus resistant to ribavirin-induced error catastrophe, but only when RNA recombination was at wild-type levels. An L420A mutation in the viral polymerase inhibited RNA recombination and exacerbated ribavirin-induced error catastrophe. Furthermore, when RNA recombination was substantially reduced by an L420A mutation, a high-fidelity G64S polymerase failed to make the virus resistant to ribavirin. These data indicate that viral RNA recombination is required for poliovirus to evade ribavirin-induced error catastrophe. The conserved nature of L420 within RDRPs suggests that RNA recombination is a common mechanism for picornaviruses to counteract and avoid error catastrophe.IMPORTANCE Positive-strand RNA viruses produce vast amounts of progeny in very short periods of time via template-dependent RNA replication mechanisms. Template-dependent RNA replication, while efficient, can be disadvantageous due to error-prone viral polymerases. The accumulation of mutations in viral RNA genomes leads to error catastrophe. In this study, we substantiate long-held theories regarding the advantages and disadvantages of asexual and sexual replication strategies among RNA viruses. In particular, we show that picornavirus RNA recombination counteracts the negative consequences of asexual template-dependent RNA replication mechanisms, namely, error catastrophe.

Project description:A coenzyme B12-dependent ribonucleotide reductase was purified from the archaebacterium Thermoplasma acidophila and partially sequenced. Using probes derived from the sequence, the corresponding gene was cloned, completely sequenced, and expressed in Escherichia coli. The deduced amino acid sequence shows that the catalytic domain of the B12-dependent enzyme from T. acidophila, some 400 amino acids, is related by common ancestry to the diferric tyrosine radical iron(III)-dependent ribonucleotide reductase from E. coli, yeast, mammalian viruses, and man. The critical cysteine residues in the catalytic domain that participate in the thiyl radical-dependent reaction have been conserved even though the cofactor that generates the radical is not. Evolutionary bridges created by the T. acidophila sequence and that of a B12-dependent reductase from Mycobacterium tuberculosis establish homology between the Fe-dependent enzymes and the catalytic domain of the Lactobacillus leichmannii B12-dependent enzyme as well. These bridges are confirmed by a predicted secondary structure for the Lactobacillus enzyme. Sequence similarities show that the N-terminal domain of the T. acidophila ribonucleotide reductase is also homologous to the anaerobic ribonucleotide reductase from E. coli, which uses neither B12 nor Fe cofactors. A predicted secondary structure of the N-terminal domain suggests that it is predominantly helical, as is the domain in the aerobic E. coli enzyme depending on Fe, extending the homologous family of proteins to include anaerobic ribonucleotide reductases, B12 ribonucleotide reductases, and Fe-dependent aerobic ribonucleotide reductases. A model for the evolution of the ribonucleotide reductase family is presented; in this model, the thiyl radical-based reaction mechanism is conserved, but the cofactor is chosen to best adapt the host organism to its environment. This analysis illustrates how secondary structure predictions can assist evolutionary analyses, each important in "post-genomic" biochemistry.

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Hypermutability and error catastrophe due to defects in ribonucleotide reductase.

Publications

Hypermutability and error catastrophe due to defects in ribonucleotide reductase.

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