Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of DNA polymerase I [KF(exo+)]. For the eight mispairs studied on three DNA sequences, only low levels of discrimination ranging from none to 23-fold were found. The kinetics of dNTP incorporation into the 9/20-mer at low nucleotide concentrations was also determined. A limit of greater than or equal to 250 s-1 was placed on the nucleotide off-rate from the KF(exo+)-9/20-dTTP complex in accord with nucleotide binding being at equilibrium in the overall kinetic sequence. The influence of the relatively short length of the 9/20-mer on the mechanism of DNA replication fidelity was determined by remeasuring important kinetic parameters on a 30/M13-mer with high homology to the 9/20-mer. Pre-steady-state data on the nucleotide turnover rates, the dATP(alpha S) elemental effect, and the burst of dAMP misincorporation into the 30/M13-mer demonstrated that the kinetics were not affected by the length of the DNA primer/template. The effects on fidelity of two site-specific mutations, KF(polA5) and KF(exo-), were also examined. KF(polA5) showed an increased rate of DNA dissociation and a decreased rate of polymerization resulting in less processive DNA synthesis. Nevertheless, with at least one misincorporation event, that of dAMP into the 9/20-mer, KF(polA5) displays an increased replication fidelity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of the Water-Insoluble Procedures for <Emphasis Type="Italic">USP</Emphasis> General Chapter <Emphasis Type="Italic">Residual Solvents</Emphasis> <467>

The water-insoluble procedures in US Pharmacopeia (USP) General Chapter Residual Solvents <467>, which are based on European Pharmacopoeia procedures, were optimized and modified before their inclusion in the chapter to improve their scope, performance, and ruggedness. The optimized procedures use a static headspace introduction system with a gas chromatograph equipped with a flame ionization detector. This article describes some of the key changes made to the USP published procedures, including use of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) as the solvent, addition of 5 mL of water and 1 mL of sample (dissolved in DMSO or DMF) to the headspace vial, use of a 3:1 GC split ratio, and use of new matrix-matched system suitability solutions. These procedures were verified with two different active pharmaceutical ingredients—hydroxyzine pamoate and prednisone. In the investigation, the more polar material (hydroxyzine pamoate) showed greater recoveries for the optimized procedures when prepared in DMSO. The less polar material (prednisone) typically had greater recoveries in DMF for the optimized procedures. During experimentation, insights into sample preparation, additional types of headspace instrumentation, solvent purity, and other parameters were also gained.     

Opposing actions of Notch1 and VEGF in post-natal cardiac valve endothelial cells

The endothelium of the cardiac valves is unique compared the rest of the vasculature in its ability to undergo an endothelial-to-mesenchymal transformation (EMT) in vitro in response to transforming growth factor-β (TGF-β). EMT is a critical event during embryonic valve development, and both VEGF-A and Notch1 have been shown to function in this process. Here we investigate the effects of VEGF-A and Notch1 on EMT in clonal endothelial cell (EC) populations isolated from adult aortic valve leaflets. VEGF-A inhibited TGF-β-induced EMT. Endothelial growth, however, was not affected by VEGF-A or TGF-β. A positive role for Notch1 was revealed in three experiments: (1) TGF-β induced Notch1 mRNA in valve ECs, (2) a γ-secretase inhibitor of Notch1 signaling blocked EMT, and (3) overexpression of a ligand-independent form of Notch1 induced EMT. These results demonstrate, for the first time, that VEGF-A and Notch1 play opposing roles in regulating EMT in post-natal valve endothelium.     

Discovery of the dual polysaccharide composition of emulsan and the isolation of the emulsion stabilizing component

Emulsan has been reported as an emulsion stabilizing amphipathic lipoheteropolysaccharide secreted by the oil-degrading bacterium Acinetobacter venetianus RAG-1. Previously, emulsan was regarded as a single polymer. As a result of developing a new purification process, we have discovered that emulsan is a complex of approximately 80% (w/w) lipopolysaccharide (LPS) and 20% (w/w) high molecular weight exopolysaccharide (EPS). The EPS was purified to 98% (w/w) using tangential flow filtration, Triton X-114 phase extraction, ammonium sulfate precipitation, and hydrophobic interaction chromatography. Several previously reported physical properties of emulsan can be attributed to the LPS fraction, such as charge, fatty acid profile, and solution behavior, while the EPS is responsible for the emulsion stabilization activity. The EPS is believed to be cationic in nature, thus providing an electrostatic binding mechanism for the formation of the emulsan complex.     

Genomic instability and aging-like phenotype in the absence of mammalian SIRT6

The Sir2 histone deacetylase functions as a chromatin silencer to regulate recombination, genomic stability, and aging in budding yeast. Seven mammalian Sir2 homologs have been identified (SIRT1-SIRT7), and it has been speculated that some may have similar functions to Sir2. Here, we demonstrate that SIRT6 is a nuclear, chromatin-associated protein that promotes resistance to DNA damage and suppresses genomic instability in mouse cells, in association with a role in base excision repair (BER). SIRT6-deficient mice are small and at 2-3 weeks of age develop abnormalities that include profound lymphopenia, loss of subcutaneous fat, lordokyphosis, and severe metabolic defects, eventually dying at about 4 weeks. We conclude that one function of SIRT6 is to promote normal DNA repair, and that SIRT6 loss leads to abnormalities in mice that overlap with aging-associated degenerative processes.     

Identification of Small Molecule Inhibitors of Jumonji AT-rich Interactive Domain 1B (JARID1B) Histone Demethylase by a Sensitive High Throughput Screen

JARID1B (also known as KDM5B or PLU1) is a member of the JARID1 family of histone lysine demethylases responsible for the demethylation of trimethylated lysine 27 in histone H3 (H3K4me3), a mark for actively transcribed genes. JARID1B is overexpressed in several cancers, including breast cancer, prostate cancer, and lung cancer. In addition, JARID1B is required for mammary tumor formation in syngeneic or xenograft mouse models. JARID1B-expressing melanoma cells are associated with increased self-renewal character. Therefore, JARID1B represents an attractive target for cancer therapy. Here we characterized JARID1B using a homogeneous luminescence-based demethylase assay. We then conducted a high throughput screen of over 15,000 small molecules to identify inhibitors of JARID1B. From this screen, we identified several known JmjC histone demethylase inhibitors, including 2,4-pyridinedicarboxylic acid and catechols. More importantly, we identified several novel inhibitors, including 2-4(4-methylphenyl)-1,2-benzisothiazol-3(2H)-one (PBIT), which inhibits JARID1B with an IC₅₀ of about 3 μm in vitro. Consistent with this, PBIT treatment inhibited removal of H3K4me3 by JARID1B in cells. Furthermore, this compound inhibited proliferation of cells expressing higher levels of JARID1B. These results suggest that this novel small molecule inhibitor is a lead compound that can be further optimized for cancer therapy.     

Statin consumption as a risk factor for developing colorectal cancer: a retrospective case study

Background

Statins are the backbone of lipid-lowering therapy and are among the most commonly prescribed drugs in the elderly population in Sweden today. Colorectal cancer is the second most common cancer in men and women, after prostate and breast cancer, respectively, with a median age of 72 years at diagnosis. Statins induce mitochondrial damage leading to accumulation of reactive oxygen species in the cell. Reactive oxygen species can cause mutations in mitochondrial as well as nuclear DNA leading to the development of cancer. Our hypothesis was that statins increase the risk for colorectal cancer.

Methods

A case study was performed on consecutive cases of colorectal cancer diagnosed at Norrlands University Hospital (NUS) in Umeå between 2012 and 2015 (n?=?325). Patients diagnosed with diabetes mellitus type II (DM II n?=?65) were excluded in the primary endpoint analysis (occurrence of colorectal cancer). As control, three databases were used to create an age-matched population in order to calculate the proportion of inhabitants using statins in the county of Västerbotten, Sweden. A secondary endpoint was cancer-specific survival among our study group of colorectal cancer patients, including those with DM II, investigating whether there was a difference if the patient was a ‘recent’ statin user or not at the time of diagnosis.

Results

Statin use at the time of colorectal cancer diagnosis in the study group was 23.8%. The corresponding figure in an age-matched population in Västerbotten was 24.6%. Using a one-proportional one-sided z test, there was no significant difference between these (23.8%, 95% CI 18.6–29.0%, p?=?0.601). When comparing groups 20–64 years of age, the difference was greater with recent statin use in 17.8% in the study population and 11.9% in Västerbotten (17.8%, 95% CI 9.0–26.6%, p?=?0.059). When considering cancer-specific survival, no significant difference in survival was seen when comparing ‘former/never’ statin users as reference category with ‘recent’ users diagnosed with colorectal cancer (HR 1.39, 95% CI 0.89–2.16).

Conclusions

No significant increase in risk for developing colorectal cancer among patients (type II diabetics excluded) medicated with statins was found. We found no correlation between ‘recent’ statin use at the time of diagnosis and cancer-specific survival.

     

Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation

Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.