CONFORMATION OF THE C8 SUBSTITUTED GUANINE ADDUCT OF THE CARCINOGEN ACETYLAMINOFLUORENE - MODEL FOR A POSSIBLE Z-DNA MODIFIED STRUCTURE

The general problem of heavy-metal interactions with DNA bases has been investigated in this laboratory for several years. The methylmercury cation, a mutagenic agent, has been used to identify the potential sites of reaction for heavy metals. Results with 1-methylcytosine (mCyt) and unblocked cytosine (Cyt) are described in the present paper

Molecular understanding of mutagenicity using potential energy methods

Our objective, has been to elucidate on a molecular level, at atomic resolution, the structures of DNAs modified by 2-aminofluorene and its N-acetyl derivative, 2-acetylaminofluorene (AAF). The underlying hypothesis is that DNA replicates with reduced fidelity when its normal right-handed B-structure is altered, and one result is a higher mutation rate. This change in structure may occur normally at a low incidence, for example by the formation of hairpin loops in appropriate sequences, but it may be enhanced greatly after covalent modification by a mutagenic substance. We use computational methods and have been able to incorporate the first data from NMR studies in our calculations. Computational approaches are important because x-ray and spectroscopic studies have not succeeded in producing atomic resolution views of mutagen and carcinogen-oligonucleotide adducts. The specific methods that we employ are minimized potential energy calculations using the torsion angle space molecular mechanics program DUPLEX to yield static views. Molecular dynamics simulations, with full solvent and salt, of the important static structures are carried out with the program AMBER; this yields mobile views in a medium that mimics the natural aqueous environment of the cell as well as can be done with current available computing resources

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass

Conformation of Guanosine Cytidine 3',5'-Monophosphate (GpC)

A brief communication of preliminary results of solution of dinucleoside phosphate GpC

Structure of guanosine-3′,5′-cytidine monophosphate. I. Semi-empirical potential energy calculations and model-building

The conformation and packing scheme for guanosine-3’,5’-cytidine monophosphate, GpC, were computed by minimizing the classical potential energy. The lowest energy conformation of the isolated molecule had dihedral angles in the range of helical RNA’s, and the sugar pucker was C3‘ endo. This was used as the starting conformation in a packing search over orientation space, the dihedral angles being flexible in this step also. The packing search was restricted by constraints from our x-ray data, namely, (1) the dimensions of the monoclinic unit cell and its pseudo-C2 symmetry (the real space group is P21), (2) t,he location of the phosphorous atom, and (3) the orientation of the bases. In addition, a geometric function was devised to impose Watson-Crick base pairing. Thus, a trial structure could be sought without explicit inclusion of intermolecular potentials. An interactive computer graphics system was used for visualizing the calculated structures. The packing searches yielded two lowest energy schemes in which the molecules had the same conformation (similar to double-helical RNA) but different orientations within the unit cell. One of these was refined by standard x-ray methods to a discrepancy index of 14.4% in the C2 pseudocell. This served as the starting structure for the subsequent refinement in the real P21 cell

Effect of androgen treatment during foetal and/or neonatal life on ovarian function in prepubertal and adult rats

We investigated the effects of different windows of testosterone propionate (TP) treatment during foetal and neonatal life in female rats to determine whether and when excess androgen exposure would cause disruption of adult reproductive function. Animals were killed prepubertally at d25 and as adults at d90. Plasma samples were taken for hormone analysis and ovaries serial sectioned for morphometric analyses. In prepubertal animals, only foetal+postnatal and late postnatal TP resulted in increased body weights, and an increase in transitory, but reduced antral follicle numbers without affecting total follicle populations. Treatment with TP during both foetal+postnatal life resulted in the development of streak ovaries with activated follicles containing oocytes that only progressed to a small antral (smA) stage and inactive uteri. TP exposure during foetal or late postnatal life had no effect upon adult reproductive function or the total follicle population, although there was a reduction in the primordial follicle pool. In contrast, TP treatment during full postnatal life (d1-25) resulted in anovulation in adults (d90). These animals were heavier, had a greater ovarian stromal compartment, no differences in follicle thecal cell area, but reduced numbers of anti-Mullerian hormone-positive smA follicles when compared with controls. Significantly reduced uterine weights lead reduced follicle oestradiol production. These results support the concept that androgen programming of adult female reproductive function occurs only during specific time windows in foetal and neonatal life with implications for the development of polycystic ovary syndrome in women

Structure and mechanism of human DNA polymerase η

The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase eta (Pol eta), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol eta at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol eta acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol eta orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol eta missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol eta in replicating through D loop and DNA fragile sites

Dclk1 Defines Quiescent Pancreatic Progenitors that Promote Injury-Induced Regeneration and Tumorigenesis

The existence of adult pancreatic progenitor cells has been debated. While some favor the concept of facultative progenitors involved in homeostasis and repair, neither a location nor markers for such cells have been defined. Using genetic lineage tracing, we show that Doublecortin-like kinase-1 (Dclk1) labels a rare population of long-lived, quiescent pancreatic cells. In vitro, Dclk1+ cells proliferate readily and sustain pancreatic organoid growth. In vivo, Dclk1+ cells are necessary for pancreatic regeneration following injury and chronic inflammation. Accordingly, their loss has detrimental effects after cerulein-induced pancreatitis. Expression of mutant Kras in Dclk1+ cells does not affect their quiescence or longevity. However, experimental pancreatitis converts Kras mutant Dclk1+ cells into potent cancer-initiating cells. As a potential effector of Kras, Dclk1 contributes functionally to the pathogenesis of pancreatic cancer. Taken together, these observations indicate that Dclk1 marks quiescent pancreatic progenitors that are candidates for the origin of pancreatic cancer

Need for recovery amongst emergency physicians in the UK and Ireland: A cross-sectional survey

OBJECTIVES: To determine the need for recovery (NFR) among emergency physicians and to identify demographic and occupational characteristics associated with higher NFR scores. DESIGN: Cross-sectional electronic survey. SETTING: Emergency departments (EDs) (n=112) in the UK and Ireland. PARTICIPANTS: Emergency physicians, defined as any registered physician working principally within the ED, responding between June and July 2019. MAIN OUTCOME MEASURE: NFR Scale, an 11-item self-administered questionnaire that assesses how work demands affect intershift recovery. RESULTS: The median NFR Score for all 4247 eligible, consented participants with a valid NFR Score was 70.0 (95% CI: 65.5 to 74.5), with an IQR of 45.5-90.0. A linear regression model indicated statistically significant associations between gender, health conditions, type of ED, clinical grade, access to annual and study leave, and time spent working out-of-hours. Groups including male physicians, consultants, general practitioners (GPs) within the ED, those working in paediatric EDs and those with no long-term health condition or disability had a lower NFR Score. After adjusting for these characteristics, the NFR Score increased by 3.7 (95% CI: 0.3 to 7.1) and 6.43 (95% CI: 2.0 to 10.8) for those with difficulty accessing annual and study leave, respectively. Increased percentage of out-of-hours work increased NFR Score almost linearly: 26%-50% out-of-hours work=5.7 (95% CI: 3.1 to 8.4); 51%-75% out-of-hours work=10.3 (95% CI: 7.6 to 13.0); 76%-100% out-of-hours work=14.5 (95% CI: 11.0 to 17.9). CONCLUSION: Higher NFR scores were observed among emergency physicians than reported in any other profession or population to date. While out-of-hours working is unavoidable, the linear relationship observed suggests that any reduction may result in NFR improvement. Evidence-based strategies to improve well-being such as proportional out-of-hours working and improved access to annual and study leave should be carefully considered and implemented where feasible