A SINGLE N-2-ACETYLAMINOFLUORENE ADDUCT ALTERS THE FOOTPRINT OF T7 (EXO -) DNA-POLYMERASE BOUND TO A MODEL PRIMER-TEMPLATE JUNCTION

Bovine pancreatic deoxyribonuclease I (DNaseI) has been used to footpr int T7 (exo-) DNA polymerase bound to a model primer-template junction . The polymerase was blocked at a specific position either by the omis sion of dCTP from the reaction mix or by the presence of a N-(deoxygua nosin-8-yl)-2-acetylaminofluorene (dGuo-AAF) adduct. This lesion has b een shown to be a severe block for several DNA polymerases, both in in vitro primer elongation experiments, and during the in vivo replicati on of AAF-monomodified single-stranded vectors. The footprints obtaine d with unmodified primer-template DNA define two protected domains sep arated by an inter-region that remains sensitive to DNaseI, and severa l hypersensitive sites located on both strands. Binding of the polymer ase to AAF monomodified duplexes results in the same protection patter n as that obtained with the unmodified duplexes. However, the hypersen sitive sites either disappear or are dramatically reduced. The results suggest that the AAF lesion alters the correct positioning of the dup lex DNA within the polymerase cleft. (C) 1998 Elsevier Science B.V.