Increased and Imbalanced dNTP Pools Symmetrically Promote Both Leading and Lagging Strand Replication Infidelity

Abstract

The fidelity of DNA replication requires an appropriate balance of dNTPs, yet the nascent leading and lagging strands of the nuclear genome are primarily synthesized by replicases that differ in subunit composition, protein partnerships and biochemical properties, including fidelity. These facts pose the question of whether imbalanced dNTP pools differentially influence leading and lagging strand replication fidelity. Here we test this possibility by examining strand-specific replication infidelity driven by a mutation in yeast ribonucleotide reductase, rnr1-Y285A, that leads to elevated dTTP and dCTP concentrations. The results for the CAN1 mutational reporter gene present in opposite orientations in the genome reveal that the rates, and surprisingly even the sequence contexts, of replication errors are remarkably similar for leading and lagging strand synthesis. Moreover, while many mismatches driven by the dNTP pool imbalance are efficiently corrected by mismatch repair, others are repaired less efficiently, especially those in sequence contexts suggesting reduced proofreading due to increased mismatch extension driven by the high dTTP and dCTP concentrations. Thus the two DNA strands of the nuclear genome are at similar risk of mutations resulting from this dNTP pool imbalance, and this risk is not completely suppressed even when both major replication error correction mechanisms are genetically intact.

Heterozygous colon cancer-associated mutations of SAMHD1 have functional significance

“…We have previously demonstrated that severely imbalanced dNTP pools strongly decrease DNA replication fidelity in Saccharomyces cerevisiae (8,9) without affecting cell proliferation, as long as none of the dNTPs is limiting for DNA replication (10). An equimolar elevation in dNTP pools also decreases DNA replication fidelity, both in yeast and bacteria, presumably by suppressing the proofreading activity of replicative DNA polymerases and by stimulating lesion bypass by both replicative and translesion DNA polymerases (11)(12)(13)(14)(15)(16).…”

Alterations in cellular metabolism triggered by URA7 or GLN3 inactivation cause imbalanced dNTP pools and increased mutagenesis

“…Similarly, Rrm3 could facilitate the passage through DNA pausing sites resulting from low-fidelity DNA replication conditions, and therefore be preferentially required for polymerases with high processivity (Pole and Polδ) (71). Alternatively, the elevated dNTP pools in rrm3Δ mutant (72, 73) might promote mutagenesis, as it has been described for other mutations causing increased dNTP pools (74,75). Moreover, a recent publication described an alternative helicase-independent Rrm3 function required for restricting DNA replication under replication stress (76), which could potentially enhance DNA replication fidelity.…”

Deoxyribonucleotide metabolism, mutagenesis and cancer

“…Sequence analysis of spontaneous mutant sites in the CAN1 locus, conferring canavanine resistance, confirmed that the mutagenic consequences of specific dNTP imbalances seen in living cells are identical to those described from earlier investigations using in vitro systems. In a more recent analysis 63 , this group has shown, for a yeast mutant containing both dCTP and dTTP in excess, that mutation frequencies and mutant spectra are essentially the same, whether the initial replication error occurs during leading-strand or lagging-strand replication. This finding was unexpected, because leading-strand and lagging-strand replication are carried out by different DNA polymerases (Pol ε and Pol δ, respectively), using some different accessory proteins 64,65 .…”