Hydroxyl Radical Footprinting Experiments Research Articles

C-terminal deletion mutants of the FokI restriction endonuclease

We have constructed two C-terminal deletion mutants of the FokI restriction endonuclease by using the polymerasechain-reaction technique and expressed them in Escherichia coli. The two mutant proteins (MP) of 41 and 30 kDa, were purified to homogeneity and their DNA-binding properties were characterized. The 41-kDa MP specifically binds the DNA sequence, 5′- GGATG 3′- CCTAC , like the wild-type (wt) FokI, but does not cleave DNA. The 30-kDa MP does not bind DNA. The affinity of the 41-kDa MP for the DNA substrate is comparable to that of wt FokI. The 41-kDa MP interacts with its substrate like the wt FokI, as revealed by hydroxyl radical footprinting experiments. In the presence of a DNA substrate, the 41-kDa MP is cleaved by trypsin into a 30-kDa N-terminal fragment and an 11-kDa C-terminal fragment. Addition of the HPLC-purified 11-kDa C-terminal fragment to the 30-kDa MP restores its sequence-specific DNA-binding property. These results confirm that the N-terminal 41-kDa fragment of the FokI ENase constitutes the DNA recognition domain of the ENase.

Covalent binding of the carcinogen benzo[a]pyrene diol epoxide to Xenopus laevis 5 S DNA reconstituted into nucleosomes.

The sequence-specific interactions of bulky carcinogens with purified DNA might reasonably be expected to be altered when the DNA is organized into chromatin. We have approached this subject by studying the covalent binding of a potent carcinogen, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-I) to nucleosomal and free DNA, using a defined fragment of DNA derived from the 5'-end of the 5 S rRNA gene of Xenopus laevis, reconstituted into nucleosomes by salt exchange with unlabeled chicken mononucleosomes. Micrococcal nuclease and hydroxyl radical "footprinting" experiments demonstrated the formation of a uniquely positioned nucleosome covering the transcriptional start point of the gene. The reconstituted nucleosomes or control DNA samples were modified with BPDE-I. DNA was repurified from the nucleosomal and control modification reactions and then irradiated with laser light at 355 nm, causing strand breaks at the positions of adducts. Nucleosomal DNA exhibited a marked decrease in the level of carcinogen binding, especially in the central 80-90 base pairs of the nucleosomal region. Interestingly, although overall binding was inhibited about 2-fold, the sequence-specific pattern of binding to deoxyguanosine residues seen with purified DNA was maintained in the nucleosomal DNA.

Function of IHF in λ DNA Packaging: I. Identification of the Strong Binding Site for Integration Host Factor and the Locus for Intrinsic Bending in cosB

Integration host factor (IHF) plays an accessory role in λ DNA packaging. IHF affects the interaction of the λ DNA packaging protein, terminase, with cos, the site on λ. DNA at which terminase binds and introduces staggered nicks to generate cohesive ends of mature λ chromosomes cos includes cosB, the terminase binding site and cosN, the adjacent nicking site cosB includes multiple binding sites for gpNul, the small subunit of terminase, and an IHF binding site, I1. I1 contains two overlapping sequences, called I1A and I1B, that closely match the consensus sequence for IHF binding sites. The I1A sequence was determined to be the site of IHF binding by hydroxyl radical footprinting experiments. Comparison of the pattern of IHF-induced enhancements and diminishments at I1 with published patterns for IHF binding sites at the λ attachment site identifies I1A as the IHF binding site at I1. The conclusion that I1A is the IHF binding site was confirmed by studies with DNA mutant in I1A. The I1A- mutation, consisting of three adjacent base-pair changes in I1A, abolished IHF binding. In contrast to the I1A- mutation, a mutation in I1B, also consisting of three adjacent base-pair changes, caused a reduction in the affinity of IHF for I1A, and caused a reduction in the magnitude of the net intrinsic bending of cosλ.

Metalloregulated expression of the ars operon.

The plasmid-borne arsenical resistance (ars) operon encodes an arsenical-translocating ATPase and confers resistance to antimonials and arsenicals in Escherichia coli by extrusion of the toxic compounds from the cytosol. The trans-acting regulatory ArsR protein was shown to bind to a fragment of DNA containing the ars promoter. Hybrid formation of the ArsR protein with a ArsR-beta-lactamase chimeric protein suggested that the active form of the ArsR repressor is a dimer. From footprinting analysis the binding site was defined as a region of imperfect dyad symmetry just upstream of the -35 site. In vivo the operon was derepressed by oxyions of +III oxidation state of arsenic, antimony, and bismuth, as well as arsenate (As(V)), whereas in vitro ArsR protein-operator interaction was reduced by each of those compounds except arsenate, as determined by gel retardation and DNase I and hydroxyl radical footprinting experiments. This indicates that arsenate is not a true inducer and must be reduced to arsenite in vivo to induce. An operator mutant obtained by deletion of the in vitro ArsR-protected DNA sequence exhibited constitutive ars promoter activity, demonstrating that the binding site is the functional target for the ArsR repressor in vivo.

(+)-CC-1065 as a structural probe of Mu transposase-induced bending of DNA: overcoming limitations of hydroxyl-radical footprinting

Phage Mu transposase (A-protein) is primarily responsible for transposition of the Mu genome. The protein binds to six att sites, three at each end of Mu DNA. At most att sites interaction of a protein monomer with DNA is seen to occur over three minor and two consecutive major grooves and to result in bending up to about 90 degrees. To probe the directionality and locus of these A-protein-induced bends, we have used the antitumor antibiotic (+)-CC-1065 as a structural probe. As a consequence of binding within the minor groove, (+)-CC-1065 is able to alkylate N3 of adenine in a sequence selective manner. This selectivity is partially determined by conformational flexibility of the DNA sequence, and the covalent adduct has a bent DNA structure in which narrowing of the minor groove has occurred. Using this drug in experiments in which either gel retardation or DNA strand breakage are used to monitor the stability of the A-protein--DNA complex or the (+)-CC-1065 alkylation sites on DNA (att site L3), we have demonstrated that of the three minor grooves implicated in the interaction with A-protein, the peripheral two are 'open' or accessible to drug bonding following protein binding. These drug-bonding sites very likely represent binding at at least two A-protein-induced bending sites. Significantly, the locus of bending at these sites is spaced approximately two helical turns apart, and the bending is proposed to occur by narrowing of the minor groove of DNA. The intervening minor groove between these two peripheral sites is protected from (+)-CC-1065 alkylation. The results are discussed in reference to a proposed model for overall DNA bending in the A-protein att L3 site complex. This study illustrates the utility of (+)-CC-1065 as a probe for protein-induced bending of DNA, as well as for interactions of minor groove DNA bending proteins with DNA which may be masked in hydroxyl radical footprinting experiments.

Contacts between the LexA repressor--or its DNA-binding domain--and the backbone of the recA operator DNA.

Using hydroxyl radical footprinting and ethylation interference experiments, we have determined the backbone contacts made by the entire LexA repressor and its amino-terminal fragment with the recA operator DNA. These techniques reveal essentially the same contacts between both proteins and one side of the DNA helix if one assumes that the DNA stays in the normal B-conformation. This result is somewhat unexpected because protection of guanine bases against methylation suggested a somewhat twisted recognition surface. The backbone contacts revealed by both methods are symmetrically disposed with respect to the center of the operator, providing further evidence that the operator binds two LexA monomers. Each half-operator contains seven interfering phosphates. These phosphates are found on both sides of the 5'-CTGT sequence that is believed to be the principal recognition target. On the side close to the center of the operator are found two phosphates, whereas the other five are clustered on the side apart from the dyad axis. We are not aware of such an extended cluster of interfering phosphates for any other DNA-binding protein. A quantification of the hydroxyl radical footprints allowed us to compare further the affinity of the LexA repressor for the recA operator with that of its isolated DNA binding domain. We find an only 13-fold higher binding constant for LexA than for its amino-terminal domain, which is in good agreement with our earlier results for the uvrA operator using a completely different binding assay.