Structural distortions induced by integration host factor (IHF) at the H' site of phage lambda probed by (+)-CC-1065, pluramycin, and KMnO4 and by DNA cyclization studies.
Integration Host Factor (IHF) is a sequence-specific DNA-bending protein that is proposed to interact with DNA primarily through the minor groove. We have used various chemical probes [(+)-CC-1065, a minor-groove-specific agent that alkylates N3 of adenine and traps bends into the minor groove; pluramycin, a minor-major-groove threading intercalator that alkylates N7 of guanine; KMnO4, which reacts more strongly with bases in denatured DNA] to gain more information on the interaction of IHF with the H' site of phage lambda. In addition to the 13-bp core consensus recognition element present at all IHF binding sites, the H' site also has an upstream AT-rich element that increases the affinity of IHF for this site. Our results reveal new details of IHF-DNA interaction at this site. Results with (+)-CC-1065 modification suggest that IHF interacts with the adenines on the 3'-side of the AT-rich element and likely induces a minor-groove bend in its vicinity, which in turn stabilizes the interaction. Pluramycin modification experiments suggest the presence of both short- and long-range structural perturbations (possible DNA unwinding events) on either side of the IHF contact region. Although IHF is known to induce a large bend in DNA at the H' site, no separation of base pairs was detected when the bent DNA was probed with KMnO4. DNA cyclization studies indicate a large magnitude (approximately 180 degrees) for the IHF-induced bend at the H' site, consistent with > 140 degrees bend estimated by gel electrophoresis methods. These studies suggest that IHF-induced DNA bending is accompanied by the introduction of a DNA node, DNA unwinding, and/or by some other DNA distortion. An enhanced binding and stability of IHF was observed on small circular DNA.
Sun
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Department of Microbiology, University of Texas at Austin 78712, USA.
Hurley
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LH
Harshey
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D000225:Adenine / Q000737:chemistry; D000617:Aminoglycosides; D000900:Anti-Bacterial Agents / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D010582:Bacteriophage lambda / Q000737:chemistry*; D001483:Base Sequence; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry*; D018983:DNA Footprinting; D004270:DNA, Circular / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D000080890:Duocarmycins; D017665:Hydroxyl Radical; D007211:Indoles; D034982:Integration Host Factors; D007933:Leucomycins / Q000737:chemistry*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011196:Potassium Permanganate / Q000737:chemistry*
D000617:Aminoglycosides; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D000080890:Duocarmycins; D007211:Indoles; D034982:Integration Host Factors; D007933:Leucomycins; D015335:Molecular Probes; D011196:Potassium Permanganate; C003169:pluramycin; D017665:Hydroxyl Radical; C018782:CC 1065; D004247:DNA; D000225:Adenine