Reversal of DNA damage induced Topoisomerase 2 DNA-protein crosslinks by Tdp2.

Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA-protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg(2+)-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. We further identify a Tdp2 active site SNP that ablates Tdp2 Mg(2+) binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons.

The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining.

Nonhomologous end joining (NHEJ) can effectively resolve chromosome breaks despite diverse end structures; however, it is unclear how the steps employed for resolution are determined. We sought to address this question by analysing cellular NHEJ of ends with systematically mispaired and damaged termini. We show NHEJ is uniquely proficient at bypassing subtle terminal mispairs and radiomimetic damage by direct ligation. Nevertheless, bypass ability varies widely, with increases in mispair severity gradually reducing bypass products from 85 to 6%. End-processing by nucleases and polymerases is increased to compensate, although paths with the fewest number of steps to generate a substrate suitable for ligation are favoured. Thus, both the frequency and nature of end processing are tailored to meet the needs of the ligation step. We propose a model where the ligase organizes all steps during NHEJ within the stable paired-end complex to limit end processing and associated errors.

Plasma ghrelin and growth hormone regulation in response to metabolic state in hybrid striped bass: effects of feeding, ghrelin and insulin-like growth factor-I on in vivo and in vitro GH secretion.

The regulation of growth hormone (GH) secretion by ghrelin during variable metabolic states is poorly understood. We examined plasma GH and ghrelin in hybrid striped bass (HSB) undergoing seasonally-based feeding and temperature manipulations. Fasting for 21 days (d) at 24 degrees C resulted in catabolism and up-regulation of plasma GH and ghrelin relative to fed controls. Continued fasting during cold-banking (14 degrees C, 90 d) resulted in a further 43-fold increase in ghrelin while GH remained elevated. A subsequent 19 day refeeding period at 24 degrees C elicited hyperphagic and compensatory growth responses, accompanied by declines in ghrelin and GH. We then tested the role of ghrelin in stimulating GH release in vivo and in vitro. Intraperitoneal injections of ghrelin resulted in dose-dependent increases in plasma GH after 6 hours (h). Ghrelin also increased GH release from HSB pituitaries during 6h incubations. Lastly, we assessed how metabolic state, ghrelin and insulin-like growth factor-I (IGF-I) affect in vitro pituitary GH release. Spontaneous GH release was 5.2-fold higher from pituitaries of fasted compared with fed animals. Ghrelin was equally effective in stimulating GH release from pituitaries of fed and starved animals, while it was ineffective in enhancing GH release from pituitaries of starved (21 d) then refed (4d) HSB. Incubation with IGF-I inhibited GH release regardless of metabolic state. These studies are the first to show that seasonally-based periods of feed deprivation and low temperature yield sustained increases in GH secretion that are likely mediated, at least partially, through elevated ghrelin, reduced IGF-I negative feedback and fasting-induced spontaneous GH release.

Leptin stimulates pituitary prolactin release through an extracellular signal-regulated kinase-dependent pathway.

Leptin was initially identified as a regulator of appetite and weight control centers in the hypothalamus, but appears to be involved in a number of physiological processes. This study was carried out to examine the possible role of leptin in regulating prolactin (PRL) release using the teleost pituitary model system. This advantageous system allows isolation of a nearly pure population of lactotropes in their natural, in situ aggregated state. The rostral pars distalis were dissected from tilapia pituitaries and exposed to varying concentrations of leptin (0, 1, 10, 100 nM) for 1 h. Release of PRL was stimulated by leptin in a potent and concentration-dependent manner. A time-course experiment showed that the strongest response in PRL release with leptin occurs within the first hour (approximately sixfold), and stimulation was sustained after 16 h (approximately twofold). Many of the actions of leptin are mediated by the activation of extracellular signal-regulated kinase (ERK1/2) but nothing is known about the cellular mechanisms by which leptin might regulate PRL secretion in vertebrates. We therefore tested whether ERK1/2 might be involved in the leptin PRL response and found that the ERK inhibitor, PD98059, hindered leptin-induced PRL release. We further analyzed leptin response by quantifying tyrosine and threonine phosphorylation of ERK1/2 using western blots. One hour incubation with leptin induced a concentration-dependent increase in phosphorylated, and thus active, ERK1/2. Our data show that leptin is a powerful stimulator of in vitro PRL release and that its actions occur in part through stimulation of ERK1/2.