Laurence Hurley

PMID: 8718873;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.

PMID: 2966637;Abstract:

The recognition and repair of the helix-stabilizing and relatively nondistortive CC-1065-(N3-adenine)-DNA adduct by UVRABC nuclease has been investigated both in vivo with ΦX174 RFI DNA by a transfection assay and in vitro by a site-directed adduct in a 117 base pair fragment from M13mpl. CC-1065 is a potent antitumor antibiotic produced by Streptomyces zelensis which binds within the minor groove of DNA through N3 of adenine. In contrast to the helix-destabilizing and distortive modifications of DNA caused by ultraviolet light or N-acetoxy-2-(acetylamino)fluorene, CC-1065 increases the melting point of DNA and decreases the S1 nuclease activity. Using a viral DNA-Escherichia coli transfection system, we have found that the uvrA, uvrB, and uvrC genes, which code for the major excision repair proteins for UV- and NAAAF-induced DNA damage, are also involved in the repair of CC-1065-DNA adducts. In contrast, the uvrD gene product, which has been found to be involved in the repair of UV damage, has no effect in repairing CC-1065-DNA adducts. Purified UVRA, UVRB, and UVRC proteins must work in concert to incise the drug-modified ΦX174 RFI DNA. Using a site-directed and multiple CC-1065 modified (MspI-BstNI) 117 base pair fragment from M13mpl, we have found that UVRABC nuclease incises at the eighth phosphodiester bond on the 5′ side of the CC-1065-DNA adduct on the drug-modified strand. The enzymes do not cut the noncovalently modified strand. At low drug binding ratios, of the four CC-1065 binding sites identified in the (MspI-BstNI) 117 base pair fragment, GATTA*, GGAAA*, GATAA*, and TTTTA* (* indicates the covalently modified adenine), only the adduct at the high-affinity binding site, GATTA*, is incised by the UVRABC nucleases. No difference in the effect of CC-1065 on local DNA structure, as determined by the DNase I cleavage pattern, was evident among these sites. At high drug binding ratios, a fifth drug binding site, AGCTA*, is identified. At this concentration UVRABC nucleases are unable to incise any of these five CC-1065-DNA adducts. The DNA sequence and/or helix-stabilizing effect of multiple adducts may determine the recognition and/or incision of the drug-DNA adduct by UVRABC nuclease. These results are discussed in relation to the structure of the CC-1065-DNA adduct and the effect of drug binding on local DNA structure. © 1988 American Chemical Society.