Structural Insights into DNA Polymerase β Deterrents for Misincorporation Support an Induced-Fit Mechanism for Fidelity

AbstractDNA polymerases generally select the correct nucleotide from a pool of structurally similar molecules to preserve Watson-Crick base-pairing rules. We report the structure of DNA polymerase β with DNA mismatches situated in the polymerase active site. This was achieved by using nicked product DNA that traps the mispair (template-primer, A-C or T-C) in the nascent base pair binding pocket. The structure of each mispair complex indicates that the bases do not form hydrogen bonds with one another, but form a staggered arrangement where the bases of the mispair partially overlap. This prevents closure/opening of the N subdomain that is believed to be required for catalytic cycling. The partially open conformation of the N subdomain results in distinct hydrogen bonding networks that are unique for each mispair. These structures define diverse molecular aspects of misinsertion that are consistent with the induced-fit model for substrate specificity

Stabilization and Enhancement of Sand-modified Root Zones for High Traffic Sports Turfs with Mesh Elements: A Randomly Oriented, Interlocking Mesh Inclusion system.

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Longitudinal changes and correlates of meeting WHO recommended levels of physical activity in the UK during the COVID-19 pandemic: Findings from the HEBECO study

BACKGROUND: The COVID-19 pandemic has seen repeated government enforced restrictions on movement. This study aimed to evaluate longitudinal trends in physical activity (PA) in a self-selected UK-based sample and identify the key correlates of these trends. METHODS: From 23 April 2020 to 30 January 2021, measures of PA engagement were collected in a sample of 1,947 UK-based adults. Generalised estimating equations (GEE) explored trends in PA engagement over time, and how sociodemographic, health and contextual factors impacted participant's attainment of World Health Organization (WHO) recommended levels of PA (constituting muscle strengthening activity (MSA), and moderate or vigorous PA (MVPA)). RESULTS: While one in five achieved the recommended levels of PA in the first UK lockdown in April-June 2020 (19.5%, 95%CI: 17.8-21.3%) and a similar proportion in June-July 2020 (17.7%, 95%CI: 16.1-19.5%), this reduced during the period of eased restrictions in August-September 2020 (15.2%, 95%CI: 13.7-16.9%) and the second UK lockdown in November 2020-January 2021 (14.1%, 95%CI: 12.6-15.9%). Similar trends were observed for MSA and MVPA individually. Better quality of life, higher socioeconomic position and pre-COVID-19 PA levels were associated with meeting the WHO recommended levels of PA, while those living with overweight or obesity, a limiting health condition, or isolating showed the inverse associations. Time-specific associations with MSA or MVPA were observed for gender and age. CONCLUSION: Reductions in PA levels throughout the first strict lockdown continued without reversal during the ensuing period. The association of negative change with socioeconomic and health-related indices points towards deepening health inequities during the pandemic

Revealing the role of the product metal in DNA polymerase β catalysis

DNA polymerases catalyze a metal-dependent nucleotidyl transferase reaction during extension of a DNA strand using the complementary strand as a template. The reaction has long been considered to require two magnesium ions. Recently, a third active site magnesium ion was identified in some DNA polymerase product crystallographic structures, but its role is not known. Using quantum mechanical/ molecular mechanical calculations of polymerase β, we find that a third magnesium ion positioned near the newly identified product metal site does not alter the activation barrier for the chemical reaction indicating that it does not have a role in the forward reaction. This is consistent with time-lapse crystallographic structures following insertion of Sp-dCTPαS. Although sulfur substitution deters product metal binding, this has only a minimal effect on the rate of the forward reaction. Surprisingly, monovalent sodium or ammonium ions, positioned in the product metal site, lowered the activation barrier. These calculations highlight the impact that an active site water network can have on the energetics of the forward reaction and how metals or enzyme side chains may interact with the network to modulate the reaction barrier. These results also are discussed in the context of earlier findings indicating that magnesium at the product metal position blocks the reverse pyrophosphorolysis reaction

Insights into the Conformation of Aminofluorene-Deoxyguanine Adduct in a DNA Polymerase Active Site

The active site conformation of the mutagenic fluoroaminofluorene-deoxyguanine adduct (dG-FAF, N-(2′-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene) has been investigated in the presence of Klenow fragment of Escherichia coli DNA polymerase I (Kfexo−) and DNA polymerase β (pol β) using 19F NMR, insertion assay, and surface plasmon resonance. In a single nucleotide gap, the dG-FAF adduct adopts both a major-groove- oriented and base-displaced stacked conformation, and this heterogeneity is retained upon binding pol β. The addition of a non-hydrolysable 2′-deoxycytosine-5′-[(α,β)-methyleno]triphosphate (dCMPcPP) nucleotide analog to the binary complex results in an increase of the major groove conformation of the adduct at the expense of the stacked conformation. Similar results were obtained with the addition of an incorrect dAMPcPP analog but with formation of the minor groove binding conformer. In contrast, dG-FAF adduct at the replication fork for the Kfexo− complex adopts a mix of the major and minor groove conformers with minimal effect upon the addition of non-hydrolysable nucleotides. For pol β, the insertion of dCTP was preferred opposite the dG-FAF adduct in a single nucleotide gap assay consistent with 19F NMR data. Surface plasmon resonance binding kinetics revealed that pol β binds tightly with DNA in the presence of correct dCTP, but the adduct weakens binding with no nucleotide specificity. These results provide molecular insights into the DNA binding characteristics of FAF in the active site of DNA polymerases and the role of DNA structure and sequence on its coding potential

Persistent starspot signals on M dwarfs: multi-wavelength Doppler observations with the Habitable-zone Planet Finder and Keck/HIRES

Young, rapidly-rotating M dwarfs exhibit prominent starspots, which create quasiperiodic signals in their photometric and Doppler spectroscopic measurements. The periodic Doppler signals can mimic radial velocity (RV) changes expected from orbiting exoplanets. Exoplanets can be distinguished from activity-induced false positives by the chromaticity and long-term incoherence of starspot signals, but these qualities are poorly constrained for fully-convective M stars. Coherent photometric starspot signals on M dwarfs may persist for hundreds of rotations, and the wavelength dependence of starspot RV signals may not be consistent between stars due to differences in their magnetic fields and active regions. We obtained precise multi-wavelength RVs of four rapidly-rotating M dwarfs (AD Leo, G 227-22, GJ 1245B, GJ 3959) using the near-infrared (NIR) Habitable-zone Planet Finder, and the optical Keck/HIRES spectrometer. Our RVs are complemented by photometry from Kepler, TESS, and the Las Cumbres Observatory (LCO) network of telescopes. We found that all four stars exhibit large spot-induced Doppler signals at their rotation periods, and investigated the longevity and optical-to-NIR chromaticity for these signals. The phase curves remain coherent much longer than is typical for Sunlike stars. Their chromaticity varies, and one star (GJ 3959) exhibits optical and NIR RV modulation consistent in both phase and amplitude. In general, though, we find that the NIR amplitudes are lower than their optical counterparts. We conclude that starspot modulation for rapidly-rotating M stars frequently remains coherent for hundreds of stellar rotations, and gives rise to Doppler signals that, due to this coherence, may be mistaken for exoplanets.Comment: Accepted for publication in the Astrophysical Journa

Requirement for transient metal ions revealed through computational analysis for DNA polymerase going in reverse

DNA polymerases use a general two-metal ion mechanism for DNA synthesis. Recent time-lapse crystallographic studies identified additional adjunct metal ions in the polymerase active site. One of these ions correlates with appearance of pyrophosphate and was proposed to be involved in pyrophosphorolysis (reverse reaction of DNA synthesis). Because DNA polymerases can use pyrophosphorolysis to remove chain-terminating nucleotides during chemotherapies, a better understanding of this reaction is warranted. Through site-directed mutagenesis, pyrophosphorolysis measurements, and computational analysis, we examine the role of metal ions in the reverse reaction. The results indicate that the product-associated metal ion facilitates pyrophosphorolysis during the early stages of the reaction but deters the reaction at later stages, suggesting dynamic metal behavior that can modulate the chemical equilibrium