The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed

Ion pairs of indobenzimidazolo cyanines: a structural study based on conductivity, absorption, fluorescence and H-1-NMR

Asymmetric benzimidazolo carbo, di- and tricarbocyanines form ion pairs of the solvent-separated and contact types with different counterions in tetrahydrofuran, toluene and toluene-nitrile mixtures. The dissociation constants of the ion pairs in tetrahydrofuran, evaluated from conductivity data, do not depend on the length of the polymethine chain and show only a small decrease with decreasing counterion size. The absorption and fluorescence excitation spectra of the contact ion pairs exhibit a pronounced hypsochromic shift with respect to the solvated ions and the solvent-separated ion pairs. H-1-NMR experiments have provided information about the electronic structures of the ions of both the asymmetric dyes and the corresponding symmetric carbocyanines. They have also revealed different preferred anion locations in the contact ion pairs of the symmetric indocarbocyanine on one hand, and of the benzimidazolo carbocyanine and the asymmetric dyes on the other. This structural difference is suggested to be a cause of the observed opposite effects of ion pairing on the isomerization kinetics of the two groups of dyes. (C) 1998 Elsevier Science B.V. All rights reserved

PHOTOISOMERIZATION OF ASYMMETRIC INDOBENZIMIDAZOLO CYANINE DYES

The isomerization kinetics of three indobenzimidazolo polymethine cyanines have been investigated and compared with the corresponding properties of the parent symmetric dyes. A significant alternation of the pi bond orders in the polymethine chain was found in the ground states (S-0) of the asymmetric dyes. A similarly alternant behaviour was exhibited by the calculated S-0 potential energy barriers for twisting around the polymethine chain bonds of the asymmetric carbocyanine, whereas uniform barriers were found for the symmetric parent compounds. The experimentally observed sequence of back-isomerization activation energies was interpreted on the basis of these theoretical results. It was suggested by some spectral and kinetic fluorescence properties, and it was confirmed by the calculated polymethine-chain pi bond orders of the lowest singlet excited slates (S-1) that the electronic asymmetry induced by the different terminal heterocycles was strongly reduced upon excitation of these dyes to their S-1 states. In spite of this, the trans-cis photoisomerization of asymmetric cyanines occurred invariably around those bonds having the highest pi bond orders in the ground state. A tentative theoretical explanation of this systematic behaviour is reported