Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers: evidence for the occurrence of long-range interactions between the enzyme and substrate

Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, excises uracil from DNA. Crystal structures of several UDGs have identified residues important for their exquisite specificity in detection and removal of uracil. Of these, Y66 and N123 in Escherichia coli UDG have been proposed to restrict the entry of non-uracil residues into the active site pocket. In this study, we show that the uracil excision activity of the Y66F mutant was similar to that of the wild-type protein, whereas the activities of the other mutants (Y66C, Y66S, N123D, N123E and N123Q) were compromised approximately 1000-fold. The latter class of mutants showed an increased dependence on the substrate chain length and suggested the existence of long-range interactions of the substrate with UDG. Investigation of the phosphate interactions by the ethylation interference assay reaffirmed the key importance of the -1, +1 and +2 phosphates (with respect to the scissile uracil) to the enzyme activity. Interestingly, this assay also revealed an additional interference at the -5 position phosphate, whose presence in the substrate had a positive effect on substrate utilisation by the mutants that do not possess a full complement of interactions in the active site pocket. Such long-range interactions may be crucial even for the wild-type enzyme under in vivo conditions. Further, our results suggest that the role of Y66 and N123 in UDG is not restricted merely to preventing the entry of non-uracil residues. We discuss their additional roles in conferring stability to the transition state enzyme-substrate complex and/or enhancing the leaving group quality of the uracilate anion during catalysis.     

Pollination systems of trees in Kakachi, a mid-elevation wet evergreen forest in Western Ghats, India

A long-term survey (1990-2000) of pollination modes of 86 tree species was carried out at Kakachi, a mid-elevation wet forest site in southern Western Ghats, India. Observations were made on 86 tree species. This comprises 80% of the total arborescent species present in the site. Approximately 75% of these species were specialized to a single pollinator group such as bee, beetle, or moth. Pollinators from diverse groups pollinate the remaining 25% of the tree species. Global comparison with other wet forest sites showed that diversity and specialized pollination modes observed in Kakachi bore closer resemblance to other lowland than montane forest sites described so far. However, the number of pollinators involved in pollination was comparable with montane sites. We examine the consequences that might have led to selection of the observed pollination modes in Kakachi. We discuss the conservation implications of these results.