Deoxyribophosphate lyase activity of mammalian endonuclease VIII-like proteins

AbstractBase excision repair (BER) protects cells from nucleobase DNA damage. In eukaryotic BER, DNA glycosylases generate abasic sites, which are then converted to deoxyribo-5′-phosphate (dRP) and excised by a dRP lyase (dRPase) activity of DNA polymerase β (Polβ). Here, we demonstrate that NEIL1 and NEIL2, mammalian homologs of bacterial endonuclease VIII, excise dRP by β-elimination with the efficiency similar to Polβ. DNA duplexes imitating BER intermediates after insertion of a single nucleotide were better substrates. NEIL1 and NEIL2 supplied dRPase activity in BER reconstituted with dRPase-null Polβ. Our results suggest a role for NEILs as backup dRPases in mammalian cells

Genome-wide analysis of WD40 protein family and functional characterization of BvWD40-82 in sugar beet

Sugar beet is one of the most important sugar crops in the world. It contributes greatly to the global sugar production, but salt stress negatively affects the crop yield. WD40 proteins play important roles in plant growth and response to abiotic stresses through their involvement in a variety of biological processes, such as signal transduction, histone modification, ubiquitination, and RNA processing. The WD40 protein family has been well-studied in Arabidopsis thaliana, rice and other plants, but the systematic analysis of the sugar beet WD40 proteins has not been reported. In this study, a total of 177 BvWD40 proteins were identified from the sugar beet genome, and their evolutionary characteristics, protein structure, gene structure, protein interaction network and gene ontology were systematically analyzed to understand their evolution and function. Meanwhile, the expression patterns of BvWD40s under salt stress were characterized, and a BvWD40-82 gene was hypothesized as a salt-tolerant candidate gene. Its function was further characterized using molecular and genetic methods. The result showed that BvWD40-82 enhanced salt stress tolerance in transgenic Arabidopsis seedlings by increasing the contents of osmolytes and antioxidant enzyme activities, maintaining intracellular ion homeostasis and increasing the expression of genes related to SOS and ABA pathways. The result has laid a foundation for further mechanistic study of the BvWD40 genes in sugar beet tolerance to salt stress, and it may inform biotechnological applications in improving crop stress resilience

Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the kcat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The kcat values for substrates of different sequences correlate with the DNA twist, while the KM values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the KM and kcat values for different sequences containing oxoG were found

Active voltage damping method with negative DC link current feedback in electric and hybrid electric transmissions

Electric and hybrid electric transmissions in traction drive have a limited capacity power source. Since the traction drive operates in the torque source mode, the DC link voltage becomes unstable and goes into oscillatory mode. This leads to the software protection reaction which prevents the traction inverter overvoltage breakdown. The transition boundary to the oscillatory mode is determined by the power and the value of the capacitance installed in the electric transmission DC link. To increase reliability of the traction inverters, large-capacity electrolytic capacitors are replaced with smallcapacity film capacitors which makes the system more prone to oscillations. To solve this problem, active damping methods are used allowing changing the engine dynamic characteristics by means of the control system. The software methods with power and torque proportional control are most widely used. Proportional power control is the simplest method in which the traction drive simulates an RL load. The torque proportional control method adjusts the torque reference according to the change in the DC link voltage. This paper proposes a new negative DC link feedback method. In this case, the torque is adjusted dynamically depending on the current consumed by the traction inverter from the electric transmission common DC link. Mathematical modeling methods were used to compare the known and proposed methods of DC link voltage active damping. Mathematical models have been developed in the MATLAB Simulink environment which makes it possible to investigate the damping capacity at various values of the power consumed by the traction inverter. It is shown that the proposed method with negative DC link current feedback demonstrated tuning simplicity. In comparison with proportional power and torque control methods, the proposed option is robust when setting parameters, provides a large damping coefficient over the entire range of traction drive power, and has a short duration of the transient process. The proposed method can be used to suppress DC link voltage oscillations on any type of hybrid electric and all-electric vehicles traction inverters and ensures stable and reliable equipment operation

DNA Damage Response and Repair in Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is an approach to the radiotherapy of solid tumors that was first outlined in the 1930s but has attracted considerable attention recently with the advent of a new generation of neutron sources. In BNCT, tumor cells accumulate 10B atoms that react with epithermal neutrons, producing energetic α particles and 7Li atoms that damage the cell’s genome. The damage inflicted by BNCT appears not to be easily repairable and is thus lethal for the cell; however, the molecular events underlying the action of BNCT remain largely unaddressed. In this review, the chemistry of DNA damage during BNCT is outlined, the major mechanisms of DNA break sensing and repair are summarized, and the specifics of the repair of BNCT-induced DNA lesions are discussed

A Low-Activity Polymorphic Variant of Human NEIL2 DNA Glycosylase

Human NEIL2 DNA glycosylase (hNEIL2) is a base excision repair protein that removes oxidative lesions from DNA. A distinctive feature of hNEIL2 is its preference for the lesions in bubbles and other non-canonical DNA structures. Although a number of associations of polymorphisms in the hNEIL2 gene were reported, there is little data on the functionality of the encoded protein variants, as follows: only hNEIL2 R103Q was described as unaffected, and R257L, as less proficient in supporting the repair in a reconstituted system. Here, we report the biochemical characterization of two hNEIL2 variants found as polymorphisms in the general population, R103W and P304T. Arg103 is located in a long disordered segment within the N-terminal domain of hNEIL2, while Pro304 occupies a position in the β-turn of the DNA-binding zinc finger motif. Similar to the wild-type protein, both of the variants could catalyze base excision and nick DNA by β-elimination but demonstrated a lower affinity for DNA. Steady-state kinetics indicates that the P304T variant has its catalytic efficiency (in terms of kcat/KM) reduced ~5-fold compared with the wild-type hNEIL2, whereas the R103W enzyme is much less affected. The P304T variant was also less proficient than the wild-type, or R103W hNEIL2, in the removal of damaged bases from single-stranded and bubble-containing DNA. Overall, hNEIL2 P304T could be worthy of a detailed epidemiological analysis as a possible cancer risk modifier

Distinct Mechanisms of Target Search by Endonuclease VIII-like DNA Glycosylases

Proteins that recognize specific DNA sequences or structural elements often find their cognate DNA lesions in a processive mode, in which an enzyme binds DNA non-specifically and then slides along the DNA contour by one-dimensional diffusion. Opposite to the processive mechanism is distributive search, when an enzyme binds, samples and releases DNA without significant lateral movement. Many DNA glycosylases, the repair enzymes that excise damaged bases from DNA, use processive search to find their cognate lesions. Here, using a method based on correlated cleavage of multiply damaged oligonucleotide substrates we investigate the mechanism of lesion search by three structurally related DNA glycosylases—bacterial endonuclease VIII (Nei) and its mammalian homologs NEIL1 and NEIL2. Similarly to another homologous enzyme, bacterial formamidopyrimidine–DNA glycosylase, NEIL1 seems to use a processive mode to locate its targets. However, the processivity of Nei was notably lower, and NEIL2 exhibited almost fully distributive action on all types of substrates. Although one-dimensional diffusion is often regarded as a universal search mechanism, our results indicate that even proteins sharing a common fold may be quite different in the ways they locate their targets in DNA