Error-free Fashion Research Articles

Permitted daily exposure limits for noteworthy N-nitrosamines.

A genotoxic carcinogen, N-nitrosodimethylamine (NDMA), was detected as a synthesis impurity in some valsartan drugs in 2018, and other N-nitrosamines, such as N-nitrosodiethylamine (NDEA), were later detected in other sartan products. N-nitrosamines are pro-mutagens that can react with DNA following metabolism to produce DNA adducts, such as O6 -alkyl-guanine. The adducts can result in DNA replication miscoding errors leading to GC>AT mutations and increased risk of genomic instability and carcinogenesis. Both NDMA and NDEA are known rodent carcinogens in male and female rats. The DNA repair enzyme, methylguanine DNA-methyltransferase can restore DNA integrity via the removal of alkyl groups from guanine in an error-free fashion and this can result in nonlinear dose responses and a point of departure or "practical threshold" for mutation at low doses of exposure. Following International recommendations (ICHM7; ICHQ3C and ICHQ3D), we calculated permissible daily exposures (PDE) for NDMA and NDEA using published rodent cancer bioassay and in vivo mutagenicity data to determine benchmark dose values and define points of departure and adjusted with appropriate uncertainty factors (UFs). PDEs for NDMA were 6.2 and 0.6μg/person/day for cancer and mutation, respectively, and for NDEA, 2.2 and 0.04 μg/person/day. Both PDEs are higher than the acceptable daily intake values (96 ng for NDMA and 26.5ng for NDEA) calculated by regulatory authorities using simple linear extrapolation from carcinogenicity data. These PDE calculations using a bench-mark approach provide a more robust assessment of exposure limits compared with simple linear extrapolations and can better inform risk to patients exposed to the contaminated sartans.

Design and Application of a Mutational Sensor for Characterizing AID Activity

Activation‐induced cytidine deaminase (AID) is a mutator enzyme that introduces uracil:guanine mismatches in Immunoglobulin (Igh) genes during the antibody diversification processes of class switch recombination and somatic hypermutation in B lymphocytes. When this happens, one of three pathways is possible. Firstly, the DNA can be directly replicated with the mutation, causing a transition (C → T) mutation. Secondly, during genomic mutation, the repair enzyme Uracil N‐Glycosylase (UNG) can act to base excise the mutation in either an error free fashion, replacing the original cytidine in the mutated spot, or thirdly, an error prone fashion, in which a transversion mutation (C → G/A) will then be introduced. Certain “hotspots” in the DNA sequence increase mutational frequency: cytosine residues are strongly favored when they are part of an RGYW sequence, or its reverse complement, sequence WRCY (where W=A/T, R=A/G, and Y=C/T). However, AID can also produce off target damage to genes, including oncogenes, that result in lymphomas. Therefore, tight regulation of AID mutator activity is necessary for genomic stability. In this study, we designed a cellular mutation reporter assay to determine the role of AID in regulating mutational activity. Mutations introduced by AID were measured with the use of constructs containing a green fluorescent protein (GFP) with premature STOP codons which‐ upon mutation‐ will “turn on” GFP and can be screened by flow cytometry. The use of this construct allows for mutational screening to be done in any cell type in which AID is naturally occurring, such as lymphomas and leukemias, rather than having one cell type with the reporter gene built into the cell line (current method). Once the constructs were successfully created via PCR SOEing reactions, we verified their ability to serve as mutational sensors. To achieve this, cells were co‐transfected with constructs and AID into BOSC cells and analyzed for rates of nucleotide reversion via flow cytometry. AID is shown to reproducibly revert two constructs with premature stop codons in contextual hotspots from GFP− to GFP+, as seen by the increased presence of GFP when analyzed via flow cytometry. The data show that the GFP mutational sensor works and serves as a functional system for determining mutational analysis among BOSC cells via flow cytometry analysis.Support or Funding Information1. Allison Woods is an undergraduate scholar supported in part by the MARC Program at St. Mary's University and the Summer Program in Cancer Research at MD Anderson Cancer Center support by National Cancer Institute (R25CA181004).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Backbone Flexibility Influences Nucleotide Incorporation by Human Translesion DNA Polymerase η opposite Intrastrand Cross-Linked DNA.

Intrastrand cross-links (IaCL) connecting two purine nucleobases in DNA pose a challenge to high-fidelity replication in the cell. Various repair pathways or polymerase bypass can cope with these lesions. The influence of the phosphodiester linkage between two neighboring 2'-deoxyguanosine (dG) residues attached through the O(6) atoms by an alkylene linker on bypass with human DNA polymerase η (hPol η) was explored in vitro. Steady-state kinetics and mass spectrometric analysis of products from nucleotide incorporation revealed that although hPol η is capable of bypassing the 3'-dG in a mostly error-free fashion, significant misinsertion was observed for the 5'-dG of the IaCL containing a butylene or heptylene linker. The lack of the phosphodiester linkage triggered an important increase in frameshift adduct formation across the 5'-dG by hPol η, in comparison to the 5'-dG of IaCL DNA containing the phosphodiester group.

Genetic Control of Replication through N1-methyladenine in Human Cells

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.

Commentary: "Neural signatures of intransitive preferences".

Commentary: "Neural signatures of intransitive preferences".

Mechanisms of Translesion DNA Bypass Across (5′S)‐8,5′‐Cyclo‐2′‐Deoxyguanosine by Sulfolobus Solfataricus DNA Polymerases

The (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine (S‐cdG) DNA adduct is produced from reactions of DNA with hydroxyl radicals generated from ionizing radiation or endogenous oxidative metabolisms. An elevated level of S‐cdG has been detected in Xeroderma pigmentosum, Cockayne syndrome and breast cancer patients, and in aged mice. S‐dG blocks DNA replications in vitro and in human cells, and is repaired by synergetic actions of nucleotide excision repair and translesion DNA synthesis. S‐cdG induces G to A transition in E coli and in human cells, and causes S‐cdG:T mispair in vitro with several translesion DNA polymerases. However, the molecular mechanisms of translesion bypass of S‐cdG remain elusive. We examined kinetic and structural mechanisms of S‐cdG bypass using model DNA polymerases, Sulfolobus Solfataricus DNA Polymerase I (Dpo1) and IV (Dpo4). Both Dpo1 and Dpo4 can moderately bypass S‐cdG with much lower catalytic efficiencies compared to efficiencies with an undamaged template. Dpo1 and Dpo4 bypassed the lesion in a largely error‐free fashion. Pre‐steady‐state kinetics analyses revealed that the lowered catalytic efficiency of Dpo4 in the presence of template S‐cdG derived from a lower DNA binding affinity and an attenuated formation of the productive enzyme‐DNA‐nucleotide complex. To provide structural insights, we solved two X‐ray crystal structures containing Dpo4, S‐cdG‐containing DNA duplex, and the correct dCTP or incorrect dTTP. Together, our study provided mechanistic insights for the translesion bypass across a highly mutagenic cyclopurine lesion.

Kinetics, Structure, and Mechanism of 8-Oxo-7,8-dihydro-2′-deoxyguanosine Bypass by Human DNA Polymerase η

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.

Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases κ and ζ in human cells

Thymine glycol (Tg) is the most common DNA lesion of thymine induced by interaction with reactive oxygen species. Because of the addition of hydroxyl groups at C5 and C6 in a Tg lesion, the damaged base loses its aromatic character and becomes nonplanar; consequently, the C5 methyl group protrudes in an axial direction and that prevents the stacking of the 5' base above the Tg lesion. Because Tg presents a severe block to continued synthesis by replicative DNA polymerases, we determine here how human cells manage to replicate through this lesion. Using a duplex plasmid system where bidirectional replication ensues from an origin of replication, we show that translesion synthesis (TLS) makes a prominent contribution to Tg bypass and that it occurs in a predominantly error-free fashion. Also, we provide evidence that Pol kappa and Pol zeta function together in promoting error-free replication through the lesion, and based on structural and biochemical information, we propose a role for Pol kappa at the insertion step and of Pol zeta at the extension step of Tg bypass. We discuss the implications of these observations and suggest that human cells have adapted the TLS machinery to function in a much more error-free fashion than could have been predicted from the intrinsic catalytic efficiencies and fidelities of TLS polymerases.

Implementation and characterization of active feed-forward for deterministic linear optics quantum computing

In general, quantum computer architectures which are based on the dynamical evolution of quantum states, also require the processing of classical information, obtained by measurements of the actual qubits that make up the computer. This classical processing involves fast, active adaptation of subsequent measurements and real-time error correction (feed-forward), so that quantum gates and algorithms can be executed in a deterministic and hence error-free fashion. This is also true in the linear optical regime, where the quantum information is stored in the polarization state of photons. The adaptation of the photon’s polarization can be achieved in a very fast manner by employing electro-optical modulators, which change the polarization of a trespassing photon upon appliance of a high voltage. In this paper we discuss techniques for implementing fast, active feed-forward at the single photon level and we present their application in the context of photonic quantum computing. This includes the working principles and the characterization of the EOMs as well as a description of the switching logics, both of which allow quantum computation at an unprecedented speed.

Base Pairing and Replicative Processing of the Formamidopyrimidine-dG DNA Lesion

The 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2'-deoxyguanosine (FaPydG) is one of the major DNA lesions found after oxidative stress in cells. To clarify the base pairing and coding potential of this major DNA lesion with the aim to estimate its mutagenic effect, we prepared oligonucleotides containing a cyclopentane based analogue of the DNA lesion (cFaPydG). In addition, oligonucleotides containing the cyclopentane analogue of 2'-deoxyguanosine (cdG), and oligonucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were synthesized. The thermodynamic stability of duplexes containing these building blocks and all canonical counterbases were determined by concentration dependent melting-point measurements (van't Hoff plots). The data reveal that cFaPydG greatly destabilizes a DNA duplex (DeltaDeltaG degrees (298K) approximately 2-4 kcal mol(-1)). The optimal base pairing partner for the cFaPydG lesion is dC. Investigation of duplexes containing dG and cdG shows that the effect of substituting the deoxyribose by a cyclopentane moiety is marginal. The data also provide strong evidence that the FaPydG lesion is unable to form a base pair with dA. Our computational studies indicate that the syn-conformation required for base pairing with dA is energetically unfavorable. This is in contrast to 8-oxodG for which the syn-conformation represents the energetic minimum. Kinetic primer extension studies using S. cerevisiae Pol eta reveal that cFaPydG is replicated in an error-free fashion. dC is inserted 2-3 orders of magnitude more efficiently than dT or dA, showing that FaPydG is a lesion which retains the coding potential of dG. This is also in contrast to 8-oxodG, for which base pairing with dC and dA was established.

UV-induced T-->C transition at a TT photoproduct site is dependent on Saccharomyces cerevisiae polymerase eta in vivo.

UV-induced reversion of the arg4-17 ochre allele in Saccharomyces cerevisiae is largely dependent on translesion polymerase eta (Rad30p), known to bypass cyclobutane-type TT dimers in an error-free fashion. arg4-17 locus reversion was predominantly due to T-->C transition of T127, the 3' T of a TT photoproduct site. This event was at least 20-fold reduced in a rad30 deletion mutant, irrespective of the status of nucleotide excision repair. These data correlate with known properties of 6-4 TT photoproducts and in vitro characteristics of polymerase eta and suggest that polymerase eta plays an important in vivo role in inserting G opposite the 3' T of 6-4 TT photoproducts at this site. Alternatively, an unprecedented error-prone processing of cyclobutane-type photoproducts at this site by polymerase eta must be assumed as the critical mechanism. Whereas photoreactivation results indeed hint at the latter possibility, a possible regulatory influence of reducing the overall UV damage load on the bypass probability of non-cyclobutane-type pyrimidine dimer photoproducts should not be dismissed.

The Translesion DNA Polymerase ζ Plays a Major Role in Ig and bcl-6 Somatic Hypermutation

Ig somatic mutations would be introduced by a polymerase (pol) while repairing DNA outside main DNA replication. We show that human B cells constitutively express the translesion pol ζ, which effectively extends DNA past mismatched bases (mispair extender), and pol η, which bypasses DNA lesions in an error-free fashion. Upon B cell receptor (BCR) engagement and coculture with activated CD4 + T cells, these lymphocytes upregulated pol ζ, downregulated pol η, and mutated the Ig and bcl-6 genes. Inhibition of the pol ζ REV3 catalytic subunit by specific phosphorothioate-modified oligonucleotides impaired Ig and bcl-6 hypermutation and UV damage-induced DNA mutagenesis, without affecting cell cycle or viability. Thus, pol ζ plays a critical role in Ig and bcl-6 hypermutation, perhaps facilitated by the downregulation of pol η.

Random access of nanodevices

Systems containing ultrahigh densities of patterned nanometer scale devices are anticipated to be extremely difficult, if not impossible, to fabricate and access in an error-free fashion using existing technologies. In this work, we show that extremely high densities of on-tube and intertube nonlinear electronic devices are realizable in samples of randomly grown carbon nanotubes. Further we propose and demonstrate a strategy for accessing and exploiting the random collection of nanodevices in such systems.

Theoretical and operational challenges of “high-reliability organizations”: air-traffic control and aircraft carriers

This is an empirical and theoretical examination of high reliability organizations. The empirical referents are the U.S. Navy's nuclear aircraft carriers and the air traffic control system of the U.S. Federal Aviation Administration. Three modes of organizational behavior are observed ranging from routine or bureaucratic to high-tempo to emergency. Each mode has distinctive authority patterns, communications pathways and leadership perspectives. Since 1984, an interdisciplinary group of scholars at Berkeley has been involved in making observations of and theorizing about “high reliability organizations” (HROs), which operate technical systems that are very beneficial, costly, and hazardous. Major operational errors in these organizations are likely to produce catastrophic consequences; therefore, HROs take on the dual goals of sustaining delivery at maximum capacity and operating in nearly error-free fashion. They are so effective that the probability of serious error is very low. Other kinds of organizations systematically use trial-and-error learning. HROs, however, have less confidence in this process for conducting hazardous operations, because their next error may be their last trial. This paper draws on experience with two of the Navy's nuclear aircraft carriers and the Federal Aviation Administration's air-traffic control system. Current theories derive primarily from studies of “failure-tolerant,” trial-and-error operating bureaucracies. Is this literature a sure guide to HROs? Do the phenomena challenge contemporary thinking about complex organizations? “High-reliability” operations reveal a range of theoretical (and operational) surprises. The remainder of this discussion explores a single dimension of this theoretical arena.