Dynamic basis for dA•dGTP and dA•d8OGTP misincorporation via Hoogsteen base pairs.

Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. Here, we show that DNA dynamics determine the frequency of misincorporating the A•G mismatch, and altered dynamics explain the high frequency of 8-oxoguanine (8OG) A•8OG misincorporation. NMR measurements revealed that A•G (population (pop.

Structural basis for impaired 5' processing of a mutant tRNA associated with defects in neuronal homeostasis.

SignificanceUnderstanding and treating neurological disorders are global priorities. Some of these diseases are engendered by mutations that cause defects in the cellular synthesis of transfer RNAs (tRNAs), which function as adapter molecules that translate messenger RNAs into proteins. During tRNA biogenesis, ribonuclease P catalyzes removal of the transcribed sequence upstream of the mature tRNA.

Probing conformational transitions towards mutagenic Watson-Crick-like G·T mismatches using off-resonance sugar carbon R relaxation dispersion.

NMR off-resonance R relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson-Crick-like conformations. As Watson-Crick-like G·T mismatches have base pairing geometries similar to Watson-Crick base pairs, we hypothesized that they would mimic Watson-Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R measurements targeting the sugar C3' and C4' nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson-Crick conformation and that transitions toward tautomeric and anionic Watson-Crick-like G·T mismatches restore the canonical Watson-Crick sugar-backbone.

Why are Hoogsteen base pairs energetically disfavored in A-RNA compared to B-DNA?

A(syn)-U/T and G(syn)-C+ Hoogsteen (HG) base pairs (bps) are energetically more disfavored relative to Watson-Crick (WC) bps in A-RNA as compared to B-DNA by >1 kcal/mol for reasons that are not fully understood. Here, we used NMR spectroscopy, optical melting experiments, molecular dynamics simulations and modified nucleotides to identify factors that contribute to this destabilization of HG bps in A-RNA. Removing the 2'-hydroxyl at single purine nucleotides in A-RNA duplexes did not stabilize HG bps relative to WC.

Dynamic basis for dG•dT misincorporation via tautomerization and ionization.

Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (G•T/UG•T/U; population less than 0.4%) and one anionic species (G•T/U; population around 0.

Genomic Analysis of the DNA Replication Timing Program during Mitotic S Phase in Maize () Root Tips.

All plants and animals must replicate their DNA, using a regulated process to ensure that their genomes are completely and accurately replicated. DNA replication timing programs have been extensively studied in yeast and animal systems, but much less is known about the replication programs of plants. We report a novel adaptation of the "Repli-seq" assay for use in intact root tips of maize () that includes several different cell lineages and present whole-genome replication timing profiles from cells in early, mid, and late S phase of the mitotic cell cycle.

Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson-Crick-like Base Pairs.

The replicative and translational machinery utilizes the unique geometry of canonical G·C and A·T/U Watson-Crick base pairs to discriminate against DNA and RNA mismatches in order to ensure high fidelity replication, transcription, and translation. There is growing evidence that spontaneous errors occur when mismatches adopt a Watson-Crick-like geometry through tautomerization and/or ionization of the bases. Studies employing NMR relaxation dispersion recently showed that wobble dG·dT and rG·rU mismatches in DNA and RNA duplexes transiently form tautomeric and anionic species with probabilities (≈0.

A flow cytometric method for estimating S-phase duration in plants.

The duration of the DNA synthesis stage (S phase) of the cell cycle is fundamental in our understanding of cell cycle kinetics, cell proliferation, and DNA replication timing programs. Most S-phase duration estimates that exist for plants are based on indirect measurements. We present a method for directly estimating S-phase duration by pulse-labeling root tips or actively dividing suspension cells with the halogenated thymidine analog 5-ethynl-2'-deoxyuridine (EdU) and analyzing the time course of replication with bivariate flow cytometry.