An archaeal family-B DNA polymerase variant able to replicate past DNA damage: occurrence of replicative and translesion synthesis polymerases within the B family

A mutant of the high fidelity family-B DNA polymerase from the archaeon Thermococcus gorgonarius (Tgo-Pol), able to replicate past DNA lesions, is described. Gain of function requires replacement of the three amino acid loop region in the fingers domain of Tgo-Pol with a longer version, found naturally in eukaryotic Pol ζ (a family-B translesion synthesis polymerase). Inactivation of the 3′–5′ proof-reading exonuclease activity is also necessary. The resulting Tgo-Pol Z1 variant is proficient at initiating replication from base mismatches and can read through damaged bases, such as abasic sites and thymine photo-dimers. Tgo-Pol Z1 is also proficient at extending from primers that terminate opposite aberrant bases. The fidelity of Tgo-Pol Z1 is reduced, with a marked tendency to make changes at G:C base pairs. Together, these results suggest that the loop region of the fingers domain may play a critical role in determining whether a family-B enzyme falls into the accurate genome-replicating category or is an error-prone translesion synthesis polymerase. Tgo-Pol Z1 may also be useful for amplification of damaged DNA.     

Bulky DNA adducts in human sperm: relationship with fertility,semen quality,smoking, and environmental factors

The integrity of DNA of spermatogenic cells can be affected by endogenous and exogenous genotoxic factors. Resulting DNA damage in spermatozoa may significantly contribute to impaired fertility. Here, the 32P-postlabeling method was used to analyze the levels of bulky DNA adducts in sperm cells in a group of 179 males, either healthy donors or patients with an impaired fertility. When all donors were analyzed, the levels of bulky DNA adducts was 1.2-fold higher in smokers than in non-smokers, but the difference was not statistically significant (P=0.054). However, a statistically significant difference existed between current smokers and never smokers among the healthy individuals (1.7-fold increase, P=0.008). No correlation between alcohol or coffee consumption and sperm DNA adducts was found. The levels of DNA adducts in sperm seemed to be unaffected by environmental and occupational factors. On the other hand, groups of healthy persons and patients with male-factor infertility differed significantly with respect to the level of bulky DNA adducts (P=0.012). A significant negative correlation between DNA adducts and sperm concentration or sperm motility existed among patients with an impaired fertility (n=93; P<0.029, r(S)=-0.225). These results suggest that DNA adducts in sperm cells can be applied as potential biomarkers in studies of human infertility.     

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.     

Stereocontrolled synthesis of diene and enyne sugar-modified nucleosides and their interaction with S-adenosyl-L-homocysteine hydrolase

Conjugated diene 5-7 and enyne 8 analogs derived from adenosine and uridine were synthesized employing Pd-catalyzed cross-coupling reactions.     

Hydrogen cyanide and embryonal dormancy in apple seeds

Embryos of apple ( Malus domestica Borh. cv. Antonówka) were treated with 1 m M gaseous HCN for 6 h and cultured under a 12 h photoperiod. HCN pretreatment stimulated germination, increased the length of hypocotyls, shortened the main root and decreased the percentages of seedlings with asymmetrically grown as well as with asymmetrically greened cotyledons. High activity of β-cyanoalanine synthase (EC 4.4.1.9) and a sharp increase in cyanogen content during embryo culture suggested very low levels of endogenous HCN. despite the activity of HCN releasing enzymes. The obtained data allow us to postulate an important role for cyanide in the regulatory complex controlling dormancy in apple seeds. Experiments with respiratory inhibitors indicated, however, that HCN pretreatment affected neither the alternative electron transport pathway nor residual respiration.     

Cyanide controls enzymes involved in lipid and sugar catabolism in dormant apple embryos during culture

The activities of alkaline lipase (EC 3.1.1.3, AlkL), isocitrate lyase (EC 4.1.3.1. ICL), pyruvate kinase (EC 2.7.1.40. PK) and glucose–6–phosphate dehydrogenase (EC 1.1.1.49, G6PDH) were determined in cultured, dormant embryos of apple (Malus domestica Borb. cv. Antonówka), pretreated with gaseous HCN. The C₆/C₁ , ratio was estimated in the same material. The activities of AlkL and ICL were not stimulated by HCN pretreatment until the period of maximum stimulation of germination. The activity of G6PDH was inhibited by cyanide only late during the culture of embryos. Therefore, the changes in these activities are considered to be the result and not the cause of enhanced germination. On the other hand, also PK, active very early during the culture of embryos, was modified as a result of the treatment. The cyanide-induced changes in activity of this enzyme in cotyledons (inhibition followed by stimulation) were similar to those in the whole embryo, whereas its changes in the embryonal axis (stimulation followed by inhibition) resembled CN-induced changes in PK in axes of apple seeds submitted to cold stratification (Bogatek and Lewak 1988). The estimation of C₆/C₁ ratios partly confirmed these observations. A role of HCN-induced modifications of PK activity in embryonal dormancy is proposed.     

Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae

Summary In the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H⁺-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to man the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu⁺ and Pma⁺ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5 end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.Publication n^o 2456 from the Biology Directorate of the Commission of European Communities