[Reagents for modification of protein-nucleic complexes. III. Site-specific photomodification of elongation complex of DNA polymerase beta with arylazide derivatives of primers sensitized with fluorescent ATP gamma-amide]. / Reagenty dlia modofikatsii belkovo-nukleinovykh kompleksov. III. Sait-spetsificheskaia fotomodifikatsiia élongiruiushchego kompleksa DNK-polimerazy beta arilazidnymi proizvodnymi praimerov, sensibilizirovannaia fluorestsentnymi gamma-amidami ATP.

ATP gamma-amides containing in gamma-N-position 1-methylpyrene, 9-methylanthracene, 10-chloro-9-methylanthracene, and 3-methylperylene residues were synthesized and characterized. These compounds were used as sensitizers of site-specific photomodification of the reconstituted elongating complex of the mammalian DNA polymerase beta. The photomodification was carried out with the use of photoaffine reagents, which were synthesized in situ by the 5'-(32)P-labeled primers extension with photoactive analogues of dCTP containing in the exo-N-position of cytosine various perfluoroarylazide groups. The effect of structures of the sensitizers and photoactive reagents on the efficiency and selectivity of photolinking of primers to the enzyme and template, as well as formation of a number of other photomodification products was studied. It was shown that the sensitizers containing 10-chloro-9-methylanthracene and 3-methylperylene residues allow preparation of photolinks in such irradiation conditions when photomodification in their absence is not essentially observed.

Engagement of DNA polymerases during apoptosis.

DNA replicative and repair machinery was investigated by means of different techniques, including in vitro nuclear enzymatic assays, immunoelectron microscopy and confocal microscopy, in apoptotic cell lines such as HL-60 treated with methotrexate, P815 and K562 exposed to low temperatures and Friend cells exposed to ionizing radiation. The results showed a shift of DNA polymerase alpha and beta activities. DNA polymerase alpha, which in controls was found to be the principal replicative enzyme driving DNA synthesis, underwent, upon apoptosis, a large decrease of its activity being replaced by DNA polymerase beta which is believed to be associated with DNA repair. Such a modulation was concomitant with a topographical redistribution of both DNA polymerase alpha and the incorporation of BrdUrd throughout the nucleus. Taken together, these results indicate the occurrence of a dramatic response of the DNA machinery, through a possible common or at least similar behaviour when different cell lines are triggered to apoptosis. Although this possibility requires further investigation, these findings suggest an extreme attempt of the cell undergoing apoptosis to preserve its nuclear environment by switching on a repair/defence mechanism during fragmentation and chromatin margination.