Biochemical characterization and DNA repair pathway interactions of Mag1-mediated base excision repair in Schizosaccharomyces pombe

The Schizosaccharomyces pombe mag1 gene encodes a DNA repair enzyme with sequence similarity to the AlkA family of DNA glycosylases, which are essential for the removal of cytotoxic alkylation products, the premutagenic deamination product hypoxanthine and certain cyclic ethenoadducts such as ethenoadenine. In this paper, we have purified the Mag1 protein and characterized its substrate specificity. It appears that the substrate range of Mag1 is limited to the major alkylation products, such as 3-mA, 3-mG and 7-mG, whereas no significant activity was found towards deamination products, ethenoadducts or oxidation products. The efficiency of 3-mA and 3-mG removal was 5–10 times slower for Mag1 than for Escherichia coli AlkA whereas the rate of 7-mG removal was similar to the two enzymes. The relatively low efficiency for the removal of cytotoxic 3-methylpurines is consistent with the moderate sensitivity of the mag1 mutant to methylating agents. Furthermore, we studied the initial steps of Mag1-dependent base excision repair (BER) and genetic interactions with other repair pathways by mutant analysis. The double mutants mag1 nth1, mag1 apn2 and mag1 rad2 displayed increased resistance to methyl methanesulfonate (MMS) compared with the single mutants nth1, apn2 and rad2, respectively, indicating that Mag1 initiates both short-patch (Nth1-dependent) and long-patch (Rad2-dependent) BER of MMS-induced damage. Spontaneous intrachromosomal recombination frequencies increased 3-fold in the mag1 mutant suggesting that Mag1 and recombinational repair (RR) are both involved in repair of alkylated bases. Finally, we show that the deletion of mag1 in the background of rad16, nth1 and rad2 single mutants reduced the total recombination frequencies of all three double mutants, indicating that abasic sites formed as a result of Mag1 removal of spontaneous base lesions are substrates for nucleotide excision repair, long- and short-patch BER and RR.     

K+ channel regulator KCR1 suppresses heart rhythm by modulating the pacemaker current If

Hyperpolarization-activated, cyclic nucleotide sensitive (HCN) channels underlie the pacemaker current I(f), which plays an essential role in spontaneous cardiac activity. HCN channel subunits (HCN1-4) are believed to be modulated by additional regulatory proteins, which still have to be identified. Using biochemistry, molecularbiology and electrophysiology methods we demonstrate a protein-protein interaction between HCN2 and the K(+) channel regulator protein 1, named KCR1. In coimmunoprecipitation experiments we show that KCR1 and HCN2 proteins are able to associate. Heterologously expressed HCN2 whole-cell current density was significantly decreased by KCR1. KCR1 profoundly suppressed I(HCN2) single-channel activity, indicating a functional interaction between KCR1 and the HCN2 channel subunit. Endogenous KCR1 expression could be detected in adult and neonatal rat ventriculocytes. Adenoviral-mediated overexpression of KCR1 in rat cardiomyocytes (i) reduced I(f) whole-cell currents, (ii) suppressed most single-channel gating parameters, (iii) altered the activation kinetics, (iv) suppressed spontaneous action potential activity, and (v) the beating rate. More importantly, siRNA-based knock-down of endogenous KCR1 increased the native I(f) current size and single-channel activity and accelerated spontaneous beating rate, supporting an inhibitory action of endogenous KCR1 on native I(f). Our observations demonstrate for the first time that KCR1 modulates I(HCN2)/I(f) channel gating and indicate that KCR1 serves as a regulator of cardiac automaticity.     

Defining the Far-red Limit of Photosystem I: THE PRIMARY CHARGE SEPARATION IS FUNCTIONAL TO 840 nm*

The far-red limit of photosystem I (PS I) photochemistry was studied by EPR spectroscopy using laser flashes between 730 and 850 nm. In manganese-depleted spinach thylakoid membranes, the primary donor in PS I, P₇₀₀, was oxidized simultaneously with tyrosine Z, the secondary donor in PS II. It was found that at 295 K PS I photochemistry, observed as P₇₀₀⁺ formation, was functional up to 840 nm. This is 30 nm further to the red region than was reported for PS II photochemistry (Thapper, A., Mamedov, F., Mokvist, F., Hammarström, L., and Styring, S. (2009) Plant Cell 21, 2391–2401). The same far-red limit for the P₇₀₀⁺ formation was observed in a PS I reaction center core preparation from Nostoc punctiforme. The reduction of the acceptor side of PS I, observed as reduction of the iron-sulfur centers F_A and F_B by low temperature EPR measurements, was also functional at 15 K with light up to >830 nm. Taken together, these results, obtained from both plants and cyanobacteria, most likely rule out involvement of the red-absorbing antenna chlorophylls in this reaction. Instead we propose the existence of weak charge transfer bands absorbing in the far-red region in the ensemble of excitonically coupled chlorophyll a molecules around P₇₀₀ similar to what has been found in the reaction center of PS II. These charge transfer bands could be responsible for the far-red light absorption leading to PS I photochemistry at wavelengths up to 840 nm.     

Genetic variation in resistance to Phytophthora cinnamomi in seedlings of two Turkish Abies species

Genetic variation in resistance to Phytophthora cinnamomi was investigated for two fir species endemic to the Republic of Turkey. Open-pollinated families of seedlings of Trojan fir (Abies equi-trojani) and Turkish fir (Abies bornmuelleriana) were grown from seed in a greenhouse for approximately 15 months, inoculated with rice grains colonized with P. cinnamomi, and subsequent mortality assessed biweekly for 16 weeks. Final seedling mortality was higher in Trojan fir (56.4 %) compared to Turkish fir (32.9 %). Mortality in both species varied by geographic origin, decreasing from west (59.8 %, ) to east (21.4 %, Karabük). As mortality increased following inoculation, both narrow-sense individual-tree $ \left( {h_i^2} \right) $ and family mean $ ( {h_f^2} ) $ heritabilities increased, plateauing at 0.62?±?0.162 and 0.97?±?0.011 for Trojan fir and 0.50?±?0.102 and 0.96?±?0.01 for Turkish fir, respectively. Terminal and lateral branch bud break assessed under greenhouse conditions were also under strong genetic control. For terminal bud break, individual-tree heritabilities for Trojan and Turkish fir were 0.49?±?0.146 and 0.45?±?0.099, respectively, while family mean heritabilities were 0.88?±?0.035 and 0.88?±?0.027, respectively. The family mean correlation between bud break and final disease mortality was not significant for lateral buds but positive and significant for terminal buds (r?=?0.32) suggesting that selection for resistance would either not alter, or slightly reduce, early bud break. These are encouraging results for ongoing tree improvement efforts in North America and Europe to develop planting stock for the Christmas tree industry.     

Fishery resource use in a subarctic Indian community

Cree Indians of Fort George, James Bay, northern Canada, maintain a large and successful subsistence fishery. Methods used in the fishery, seasons and locations of catch, and yield levels were studied, together with the population biology of two sea-run Coregonus species, cisco and whitefish, that dominate the catch. The fishery was characterized by a high degree of order, social regulation of the fishing effort and the gillnet mesh size, and practices that were identified as adaptations to the subarctic ecosystem. Fishing methods used permit the Cree to control the magnitude of the harvest and the species and size composition of the catch. There is evidence that fishers can alter the scarcity-abundance patterns of the fish stocks, and have a biologically measurable effect on the populations.