EFFECTS OF BASE SUBSTITUTIONS ON THE BINDING OF A DNA-BENDING PROTEIN

In order to investigate whether the 2-amino group of guanine, which li es in the minor groove of the B-form helix, can directly influence DNA flexibility and major groove recognition by proteins we have examined the properties of DNA molecules containing inosine and/or 2,6-diamino purine (DAP) residues. Appropriately substituted tyrT(A93) DNA fragmen ts were prepared by the polymerase chain reaction. Their mobility in n on-denaturing gels was affected, consistent with changed anisotropic f lexibility leading to increased curvature due to G --> I substitution and decreased curvature due to replacement of adenine with DAP. Band-s hift assays of FIS protein binding revealed facilitated interaction wi th inosine-containing DNA and markedly reduced binding to DAP-containi ng DNA, attributable to altered bendability. DNase footprinting experi ments confirmed that fewer sites would bind FIS in DAP-containing DNA at a given protein concentration, whereas higher levels of binding occ urred with inosine-containing molecules. Thus base substitutions which affect the placement and presence of the purine 2-amino group in the minor groove can affect both the intrinsic curvature and the bendabili ty of DNA. (C) 1995 Academic Press Limited