Effects of base substitutions on the binding of a DNA-bending protein.
In order to investigate whether the 2-amino group of guanine, which lies 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-diaminopurine (DAP) residues. Appropriately substituted tyrT(A93) DNA fragments were prepared by the polymerase chain reaction. Their mobility in non-denaturing gels was affected, consistent with changed anisotropic flexibility leading to increased curvature due to G-->I substitution and decreased curvature due to replacement of adenine with DAP. Band-shift assays of FIS protein binding revealed facilitated interaction with inosine-containing DNA and markedly reduced binding to DAP-containing DNA, attributable to altered bendability. DNase footprinting experiments confirmed that fewer sites would bind FIS in DAP-containing DNA at a given protein concentration, whereas higher levels of binding occurred 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 bendability of DNA.
D015075:2-Aminopurine / Q000031:analogs & derivatives; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D035021:Factor For Inversion Stimulation Protein; D006147:Guanine / Q000502:physiology; D007288:Inosine; D034982:Integration Host Factors; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D012365:RNA, Transfer, Tyr / Q000235:genetics
D002352:Carrier Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D035021:Factor For Inversion Stimulation Protein; D034982:Integration Host Factors; D012365:RNA, Transfer, Tyr; D015075:2-Aminopurine; C007300:2,6-diaminopurine; D007288:Inosine; D006147:Guanine