Structural distortions induced by integration host factor (IHF) at the H' site of phage λ probed by (+)-CC-1065, pluramycin, and KMnO 4 and by DNA cyclization studies

Daekyu Sun, Laurence H. Hurley, Rasika M. Harshey

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

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; KMnO 4, which reacts more strongly with bases in denatured DNA] to gain more information on the interaction of IHF with the H' site of phage λ. 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 KMnO 4. DNA cyclization studies indicate a large magnitude (approximately 180°) for the IHF-induced bend at the H' site, consistent with > 140° 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.

Original languageEnglish (US)
Pages (from-to)10815-10827
Number of pages13
JournalBiochemistry
Volume35
Issue number33
DOIs
StatePublished - Sep 4 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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