The DNA glycosylase MutY, which is a member of the Helix-hairpin-Helix (HhH) DNA glycosylase superfamily, excises adenine from mispairs with 8-oxoguanine and guanine. High-resolution crystal structures of the MutY catalytic core (cMutY), the complex with bound adenine, and designed mutants reveal the basis for adenine specificity and glycosyl bond cleavage chemistry. The two cMutY helical domains form a positively-charged groove with the adenine-specific pocket at their interface. The Watson-Crick hydrogen bond partners of the bound adenine are substituted by protein atoms, confirming a nucleotide flipping mechanism, and supporting a specific DNA binding orientation by MutY and structurally related DNA glycosylases.

8-oxoguanine (8-oxoG), induced by reactive oxygen species and arguably one of the most important mutagenic DNA lesions, is prone to further oxidation. Its one-electron oxidation products include potentially mutagenic guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) because of their mispairing with A or G. All three oxidized base-specific DNA glycosylases of Escherichia coli, namely endonuclease III (Nth), 8-oxoG-DNA glycosylase (MutM) and endonuclease VIII (Nei), excise Gh and Sp, when paired with C or G in DNA, although Nth is less active than the other two. MutM prefers Sp and Gh paired with C (kcat/K(m) of 0.24-0.26 min(-1) x nM(-1)), while Nei prefers G over C as the complementary base (k(cat)/K(m) - 0.15-0.17 min(-1) x nM(-1)). However, only Nei efficiently excises these paired with A. MutY, a 8-oxoG.A(G)-specific A(G)-DNA glycosylase, is inactive with Gh(Sp).A/G-containing duplex oligonucleotide, in spite of specific affinity. It inhibits excision of lesions by MutM from the Gh.G or Sp.G pair, but not from Gh.C and Sp.C pairs. In contrast, MutY does not significantly inhibit Nei for any Gh(Sp) base pair. These results suggest a protective function for MutY in preventing mutation as a result of A (G) incorporation opposite Gh(Sp) during DNA replication.

The linear amino acid sequences of the Escherichia coli DNA repair proteins, MutY and endonuclease III, show significant homology, even though these enzymes recognize entirely different substrates. In this study, proteolysis and molecular modeling of MutY were used to elucidate its domain organization. Proteolysis by trypsin cleaved the enzyme into 26-and 13-kDa fragments. NH 2 -terminal sequencing showed that the p13 domain begins at Gln 226 , indicating that the COOH-terminal portion of MutY, absent in endonuclease III, is organized as a separate domain. The large p26 domain is almost equivalent to the size of endonuclease III. Binding activity of the p26 domain to a DNA substrate containing an A⅐G mismatch was comparable with that of the intact enzyme. In vitro studies show that the p26 domain retains adenine glycosylase and AP lyase activity on DNA containing undamaged adenine opposite guanine or 8-oxo-7,8-dihydro-2-deoxyguanine. Although the activity was somewhat reduced, the above results show that the critical amino acid residues involved in substrate binding and catalysis are present in this domain. The structure predicted by molecular modeling indicates that the region of MutY (Met 1 -Trp 216), which is homologous to endonuclease III exhibits a two domain structure, even though this portion is resistant to proteolysis by trypsin.Proteins are constructed on a modular basis, and frequently these modules or domains are known to have unique functions. Isolation and characterization of these domains provide significant insight into the relationship between particular structural elements of the enzyme and its various activities. The mutY gene of Escherichia coli encodes a 39.1-kDa DNA mismatch repair protein. A significant portion of this protein is homologous to the 26.3-kDa E. coli endonuclease III. These two proteins are 66.3% similar and 23.8% identical over a 181-amino acid region (1). Another enzyme with sequence similarity to MutY is the product of the pdg gene in Micrococcus luteus, which recognizes and incises DNA containing cyclobutane pyrimidine dimers (2). The three enzymes mentioned above contain a [4Fe-4S] 2ϩ cluster, coordinated by four cysteine residues which are perfectly conserved in all three proteins.In contrast to their structural similarities, the functional properties of the above three DNA repair proteins are very different. MutY recognizes and removes the undamaged adenine mispaired with guanine, cytosine, 8-oxo-7,8-dihydro-2Ј-deoxyguanine (8-oxo-dG) 1 and 8-oxo-7,8-dihydro-2Ј-deoxyadenine (3) and is also reported to have AP endonuclease activity (4, 5). In vitro experiments show that MutY also recognizes and removes adenine analogs when they are paired with guanine (5).2 Endonuclease III primarily removes oxidized pyrimidines from DNA (6), and the pdg gene product in M. luteus repairs UV-induced pyrimidine dimer lesions in DNA (2). Thus, despite their structural similarities, these DNA repair enzymes have different substrate specificity. The catalytic mechanism of endonuclease III in...

Mortality is an important endpoint in chronic obstructive pulmonary disease (COPD) trials, although accurately determining cause of death is difficult. In the Understanding the Potential Long-term Impacts on Function with Tiotropium (UPLIFT®) trial, a mortality adjudication committee (MAC) provided systematic, independent and blinded assessment of cause-specific mortality of all 981 reported deaths. Here we describe this process of mortality adjudication and methodological revisions introduced to help standardise the adjudication of two areas recognised to pose particular difficulty; firstly, the classification of fatal COPD exacerbations that occur in the setting of pneumonia and secondly, the categorisation of sudden death. In addition MAC determined cause of death was compared with that reported by site investigators (SIs). MAC-assigned causes of death were: respiratory, 35%; cancer, 25%; cardiovascular, 11%; sudden cardiac death, 4.4%; sudden death, 3.4%; other, 8.8%; unknown, 12.4%. Cancer/cardiac deaths were more common in Global Initiative for Chronic Obstructive Lung Disease stage II, respiratory deaths in stages III and IV. Agreement between MAC and SI regarding cause of death was complete (50.2%), incomplete (18.5%) or none (31.3%). The SI classified deaths as cardiac three-fold more frequently than MAC (incidence rate [IR]/100 patient-years 0.797 vs. 0.257), although IR ratios for cardiac deaths for tiotropium vs. control were similar between SI and MAC. Discrepancies between MAC- and SI-adjudicated causes of death are common, especially increased reporting of cardiac deaths by the SI. Future multicentre COPD trials should plan appropriate infrastructure before study initiation to ensure collection and interpretation of fatal events data.

The Escherichia coli adenine DNA glycosylase, MutY, plays an important role in the maintenance of genomic stability by catalyzing the removal of adenine opposite 8-oxo-7,8-dihydroguanine or guanine in duplex DNA. Although the x-ray crystal structure of the catalytic domain of MutY revealed a mechanism for catalysis of the glycosyl bond, it appeared that several opportunistically positioned lysine side chains could participate in a secondary ␤-elimination reaction. In this investigation, it is established via site-directed mutagenesis and the determination of a 1.35-Å structure of MutY in complex with adenine that the abasic site (apurinic/apyrimidinic) lyase activity is alternatively regulated by two lysines, Lys 142 and Lys 20 . Analyses of the crystallographic structure also suggest a role for Glu 161 in the apurinic/ apyrimidinic lyase chemistry. The ␤-elimination reaction is structurally and chemically uncoupled from the initial glycosyl bond scission, indicating that this reaction occurs as a consequence of active site plasticity and slow dissociation of the product complex. MutY with either the K142A or K20A mutation still catalyzes ␤ and ␤-␦ elimination reactions, and both mutants can be trapped as covalent enzyme-DNA intermediates by chemical reduction. The trapping was observed to occur both pre-and post-phosphodiester bond scission, establishing that both of these intermediates have significant half-lives. Thus, the final spectrum of DNA products generated reflects the outcome of a delicate balance of closely related equilibrium constants.Over the last 15 years, multiple laboratories have investigated the catalytic mechanism of DNA glycosylases that initiate the base excision repair (BER) 1 pathway (reviewed in Refs. 1-3). The elucidation of structure-activity relationships for DNA glycosylases and glycosylase/abasic site (AP) lyases has been facilitated both by solving the crystal structures and cocrystal complexes and analyses of chemical modification and trapping experiments (4 -17). These investigations have been successful in both localizing the active site pocket of these enzymes and identifying the key amino acids that participate in the catalytic events that lead to the excision of the inappropriate base. Determination of the chemical steps in the catalytic mechanism of DNA glycosylases is fundamental for understanding how organisms maintain their genome, despite the inevitable damage caused by exogenous and endogenous agents.DNA glycosylases with an associated AP lyase activity (␤-elimination) can be distinguished from DNA glycosylases without such activity, based on the identity of the nucleophile that attacks C1Ј of the deoxyribose sugar and whether the reaction proceeds through a covalent DNA-enzyme intermediate. DNA glycosylases that also catalyze a ␤-elimination reaction utilize a primary or secondary amine in the active site, whereas monofunctional glycosylases cleave the glycosyl bond via either the activation of a water molecule or a S N 1 attack (reviewed in Refs. 1 and 18). When a pr...

BackgroundMany patients with asthma require frequent rescue medication for acute symptoms despite appropriate controller therapies. Thus, determining the most effective relief regimen is important in the management of more severe asthma. This study’s objective was to evaluate whether ipratropium bromide/albuterol metered-dose inhaler (CVT-MDI) provides more effective acute relief of bronchospasm in moderate-to-severe asthma than albuterol hydrofluoroalkaline (ALB-HFA) alone after 4 weeks.MethodsIn this double-blind, crossover study, patients who had been diagnosed with asthma for ≥1 year were randomized to two sequences of study medication “as needed” for symptom relief (1–7 day washout before second 4-week treatment period): CVT-MDI/ALB-HFA or ALB-HFA/CVT-MDI. On days 1 and 29 of each sequence, 6-hour serial spirometry was performed after administration of the study drug. Co-primary endpoints were FEV1 area under the curve (AUC0–6) and peak (post-dose) forced expiratory volume in 1 s (FEV1) response (change from test day baseline) after 4 weeks. The effects of “as needed” treatment with ALB-HFA/CVT-MDI were analyzed using mixed effect model repeated measures (MMRM).ResultsA total of 226 patients, ≥18 years old, with inadequately controlled, moderate-to-severe asthma were randomized. The study met both co-primary endpoints demonstrating a statistically significant treatment benefit of CVT-MDI versus ALB-HFA. FEV1 AUC0-6h response was 167 ml for ALB-HFA, 252 ml for CVT-MDI (p <0.0001); peak FEV1 response was 357 ml for ALB-HFA, 434 ml for CVT-MDI (p <0.0001). Adverse events were comparable across groups.ConclusionsCVT-MDI significantly improved acute bronchodilation over ALB-HFA alone after 4 weeks of “as-needed” use for symptom relief, with a similar safety profile. This suggests additive bronchodilator effects of β2-agonist and anticholinergic treatment in moderate-to-severe, symptomatic asthma.Trial registrationClinicalTrials.gov No.: NCT00818454; Registered November 16, 2009.Electronic supplementary materialThe online version of this article (doi:10.1186/s12890-016-0223-3) contains supplementary material, which is available to authorized users.

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ClinicalTrials.gov NCT01896297.