Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Natural incorporation of mercury in bone

Mercury (Hg) is a highly toxic element that causes bone defects and malformations. Structure and surface analyses using quantitative x-ray diffraction using the Rietveld method, High-Resolution Transmission Electron Microscopy and nanodiffraction analyses, and Fourier-Transformed Infrared spectroscopy showed that bone enriched naturally with Hg (≤ 2.3 %) contained Hg₃PO₄ [(Hg₂)₃(PO₄)₂] and HgO. Bone [mostly as apatite, verified as carboxyapatite Ca₁₀(PO₄)₄(CO₃)₃(OH)₂(s)] and cinnabar (HgS) dissolved releasing Hg⁺ (existing as dimer Hg₂²⁺) and PO₄³⁻, both of which became immobilized as (Hg₂)₃(PO₄)₂. Besides, released Hg²⁺ became oxidized to form HgO. The outcome of this work is novel, provided that only a handful of stable compounds of Hg₂²⁺ are found in nature.     

Multi-lineage Lung Regeneration by Stem Cell Transplantation across Major Genetic Barriers

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ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

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EXO5-DNA structure and BLM interactions direct DNA resection critical for ATR-dependent replication restart

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A Comparison of Bone Mineral Density and Its Predictors in HIV-Infected and HIV-Uninfected Older Men

ObjectiveBone health in older individuals with HIV infection has not been well studied. This study aimed to compare bone mineral density (BMD), trabecular bone score (TBS), and bone markers between HIV-infected men and age- and body mass index (BMI)-matched HIV-uninfected men aged ≥60 years. We investigated the associations of risk factors related to fracture with BMD, TBS, and bone markers in HIV-infected men.MethodsThis cross-sectional study included 45 HIV-infected men receiving antiretroviral therapy and 42 HIV-uninfected men. Medical history, BMD and TBS measurements, and laboratory tests related to bone health were assessed in all the participants. HIV-related factors known to be associated with bone loss were assessed in the HIV-infected men.ResultsThe mean BMD, TBS, and osteopenia or osteoporosis prevalence were similar among the cases and controls. The HIV-infected men had significantly higher mean N-terminal propeptide of type 1 procollagen and C-terminal cross-linking telopeptide of type I collagen levels. Stepwise multiple linear regression analysis demonstrated that low BMI (lumbar spine, P = .015; femoral neck, P = .018; and total hip, P = .005), high C-terminal cross-linking telopeptide of type I collagen concentration (total hip, P = .042; and TBS, P = .010), and low vitamin D supplementation (TBS, P = .035) were independently associated with low BMD and TBS.ConclusionIn older HIV-infected men with a low fracture risk, the mean BMD and TBS were similar to those of the age- and BMI-matched controls. The mean bone marker levels were higher in the HIV group. Traditional risk factors for fracture, including low BMI, high C-terminal cross-linking telopeptide of type I collagen level, and low vitamin D supplementation, were significant predictors of low BMD and TBS.     

Dietary boron does not affect tooth strength,micro-hardness,and density,but affects tooth mineral composition and alveolar bone mineral density in rabbits fed a high-energy diet

The objective of this study was to determine whether dietary boron (B) affects the strength, density and mineral composition of teeth and mineral density of alveolar bone in rabbits with apparent obesity induced by a high-energy diet. Sixty female, 8-month-old, New Zealand rabbits were randomly assigned for 7 months into five groups as follows: (1) control 1, fed alfalfa hay only (5.91 MJ/kg and 57.5 mg B/kg); (2) control 2, high energy diet (11.76 MJ and 3.88 mg B/kg); (3) B10, high energy diet + 10 mg B gavage/kg body weight/96 h; (4) B30, high energy diet + 30 mg B gavage/kg body weight/96 h; (5) B50, high energy diet + 50 mg B gavage/kg body weight/96 h. Maxillary incisor teeth of the rabbits were evaluated for compression strength, mineral composition, and micro-hardness. Enamel, dentin, cementum and pulp tissue were examined histologically. Mineral densities of the incisor teeth and surrounding alveolar bone were determined by using micro-CT. When compared to controls, the different boron treatments did not significantly affect compression strength, and micro-hardness of the teeth, although the B content of teeth increased in a dose-dependent manner. Compared to control 1, B50 teeth had decreased phosphorus (P) concentrations. Histological examination revealed that teeth structure (shape and thickness of the enamel, dentin, cementum and pulp) was similar in the B-treated and control rabbits. Micro CT evaluation revealed greater alveolar bone mineral density in B10 and B30 groups than in controls. Alveolar bone density of the B50 group was not different than the controls. Although the B treatments did not affect teeth structure, strength, mineral density and micro-hardness, increasing B intake altered the mineral composition of teeth, and, in moderate amounts, had beneficial effects on surrounding alveolar bone.     

DNA ADP-Ribosylation Stalls Replication and Is Reversed by RecF-Mediated Homologous Recombination and Nucleotide Excision Repair

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