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Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.

Nhp6A is an abundant non-histone chromatin-associated protein in Saccharomyces cerevisiae that contains a minor groove DNA binding motif called the HMG box. In this report, we show that Nhp6Ap binds to cisplatin intrastrand cross-links on duplex DNA with a 40-fold greater affinity than to unmodified DNA with the same sequence. Nevertheless, Nhp6Ap bound to cisplatinated DNA readily exchanges onto unmodified DNA. Phenanthroline-copper footprinting and two-dimensional NMR on complexes of wild-type and mutant Nhp6Ap with DNA were employed to probe the mode of binding to the cisplatin lesion. Recognition of the cisplatin adduct requires a surface-exposed phenylalanine on Nhp6Ap that promotes bending of DNA by inserting into the helix from the minor groove. We propose that Nhp6Ap targets the cisplatin adduct by means of intercalation by the phenylalanine and that it can bind in either orientation with respect to the DNA lesion. A methionine, which also inserts between base pairs and functions in target selection on unmodified DNA, plays no apparent role in recognition of the cisplatin lesion. Basic amino acids within the N-terminal arm of Nhp6Ap are required for high-affinity binding to the cisplatin adduct as well as to unmodified DNA. Cisplatin mediates its cytotoxicity by forming covalent adducts on DNA, and we find that Deltanhp6a/b mutants are hypersensitive to cisplatin in comparison with the wild-type strain. In contrast, Deltanhp6a/b mutants are slightly more resistant to hydrogen peroxide and ultraviolet irradiation. Therefore, Nhp6A/Bp appears to directly or indirectly function in yeast to enhance cellular resistance to cisplatin.

The mechanical response generated by binding of the nonspecific DNA-bending proteins HMGB1, NHP6A, and HU to single tethered 48.5 kb lambda-DNA molecules is investigated using DNA micromanipulation. As protein concentration is increased, the force needed to extend the DNA molecule increases, due to its compaction by protein-generated bending. Most significantly, we find that for each of HMGB1, NHP6A, and HU there is a well-defined protein concentration, not far above the binding threshold, above which the proteins do not spontaneously dissociate. In this regime, the amount of protein bound to the DNA, as assayed by the degree to which the DNA is compacted, is unperturbed either by replacing the surrounding protein solution with protein-free buffer or by straightening of the molecule by applied force. Thus, the stability of the protein-DNA complexes formed is dependent on the protein concentration during the binding. HU is distinguished by a switch to a DNA-stiffening function at the protein concentration where the formation of highly stable complexes occurs. Finally, introduction of competitor DNA fragments into the surrounding solution disassembles the stable DNA complexes with HMGB1, NHP6A, and HU within seconds. Since spontaneous dissociation of protein does not occur on a time scale of hours, we conclude that this rapid protein exchange in the presence of competitor DNA must occur only via "direct" DNA-DNA contact. We therefore observe that protein transport along DNA by direct transfers occurs even for proteins such as NHP6A and HU that have only one DNA-binding domain.

Fis is an abundant bacterial DNA binding protein that functions in many different reactions. We show here that Fis subunits rapidly exchange between dimers in solution by disulfide cross-linking mixtures of Fis mutants with different electrophoretic mobilities and by monitoring energy transfer between fluorescently labeled Fis subunits upon heterodimer formation. The effects of detergents and salt concentrations on subunit exchange imply that the dimer is predominantly stabilized by hydrophobic forces, consistent with the X-ray crystal structures. Specific and nonspecific DNA strongly inhibit Fis subunit exchange. In all crystal forms of Fis, the separation between the DNA recognition helices within the Fis dimer is too short to insert into adjacent major grooves on canonical B-DNA, implying that conformational changes within the Fis dimer and/or the DNA must occur upon binding. We therefore investigated the functional importance of dimer interface flexibility for Fis-DNA binding by studying the DNA binding properties of Fis mutants that were cross-linked at different positions in the dimer. Flexibility within the core dimer interface does not appear to be required for efficient DNA binding, Fis-DNA complex dissociation, or Fis-induced DNA bending. Moreover, FRET-based experiments provided no evidence for a change in the spatial relationship between the two helix-turn-helix motifs in the Fis dimer upon DNA binding. These results support a model in which the unusually short distance between DNA recognition helices on Fis is accommodated primarily through bending of the DNA.

The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites. Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid. The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome. All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses. A newly discovered site resides in the lac operon of Escherichia coli. The binding of Fis to this site may antagonize repression by the LacI repressor. These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.