Howard A. Nash

Crystal Structure of an IHF-DNA Complex: A Protein-Induced DNA U-Turn

Integration host factor (IHF) is a small heterodimeric protein that specifically binds to DNA and functions as an architectural factor in many cellular processes in prokaryotes. Here, we report the crystal structure of IHF complexed with 35 bp of DNA. The DNA is wrapped around the protein and bent by >160 degrees, thus reversing the direction of the helix axis within a very short distance. Much of the bending occurs at two large kinks where the base stacking is interrupted by intercalation of a proline residue. IHF contacts the DNA exclusively via the phosphodiester backbone and the minor groove and relies heavily on indirect readout to recognize its binding sequence. One such readout involves a six-base A tract, providing evidence for the importance of a narrow minor groove.

E. coli integration host factor binds to specific sites in DNA

E. coli integration host factor (IHF) both participates directly in phage lambda site-specific recombination and regulates the expression of phage and bacterial genes. Using protection from nuclease and chemical attack as an assay, we examined the interaction of IHF with DNA. We found that IHF is a specific DNA binding protein that interacts with three distinct segments of attP, the recombination site carried by phage lambda. We also found that specific IHF binding sites are located in non-att DNA. Several non-att IHF binding sites that we have identified are adjacent to genes whose expression is altered in IHF mutants. From comparison of the sequences protected by IHF, we suggest that the critical determinant in specific IHF-DNA interaction is contained in the sequence T.PyAA...PuTTGaT.A.PuTT...PyAACtA.

The interaction of E. coli IHF protein with its specific binding sites

We have used two kinds of footprinting techniques, dimethylsulfate interference and hydroxyl radical protection, to explore the way that IHF recognizes its specific target sequences. Our results lead us to conclude that IHF recognizes DNA primarily through contacts with the minor groove, an unprecedented mode for a sequence-specific binding protein. We have also determined that, although IHF is a small protein that protects a large region of DNA, only a single IHF protomer is present at each binding site. IHF bends the DNA to which it binds. We have combined this fact plus our footprinting and stoichiometry data together with the crystal structure of a related protein, the nonspecific DNA binding protein HU, to propose a model for the way in which IHF binds to its DNA target.

Repair of topoisomerase I covalent complexes in the absence of the tyrosyl-DNA phosphodiesterase Tdp1

Accidental or drug-induced interruption of the breakage and reunion cycle of eukaryotic topoisomerase I (Top1) yields complexes in which the active site tyrosine of the enzyme is covalently linked to the 3′ end of broken DNA. The enzyme tyrosyl-DNA phosphodiesterase (Tdp1) hydrolyzes this protein-DNA link and thus functions in the repair of covalent complexes, but genetic studies in yeast show that alternative pathways of repair exist. Here, we have evaluated candidate genes for enzymes that might act in parallel to Tdp1 so as to generate free ends of DNA. Despite finding that the yeast Apn1 protein has a Tdp1-like biochemical activity, genetic inactivation of all known yeast apurinic endonucleases does not increase the sensitivity of a tdp1 mutant to direct induction of Top1 damage. In contrast, assays of growth in the presence of the Top1 poison camptothecin (CPT) indicate that the structure-specific nucleases dependent on RAD1 and MUS81 can contribute independently of TDP1 to repair, presumably by cutting off a segment of DNA along with the topoisomerase. However, cells in which all three enzymes are genetically inactivated are not as sensitive to the lethal effects of CPT as are cells defective in double-strand break repair. We show that the MRE11 gene is even more critical than the RAD52 gene for double-strand break repair of CPT lesions, and comparison of an mre11 mutant with a tdp1 rad1 mus81 triple mutant demonstrates that other enzymes complementary to Tdp1 remain to be discovered.

Pathways for repair of topoisomerase I covalent complexes in Saccharomyces cerevisiae.

Background The covalent linkage between DNA and the active site tyrosine of topoisomerase I can be stabilized by chemotherapeutic agents, adjacent DNA lesions, or mutational defects in the topoisomerase itself. Following collision with a replication fork, the covalent complex can be converted to a double‐strand break. Tdp1, an enzyme that can hydrolyse the bond between topoisomerase I and DNA, is thought to be involved in the repair of these lesions, but little is known about how such repair is accomplished.

Synapsis of attachment sites during lambda integrative recombination involves capture of a naked DNA by a protein-DNA complex

During lambda integration, Int recombinase must specifically bind to and cut attachment sites on both the viral and host chromosomes. We show here by foot-printing and by a novel cleavage assay that the bacterial attachment site, attB, cannot stably bind Int in competition with other DNAs. Instead, during recombination reactions, attB obtains its Int by collision with the intasome, a nucleoprotein assembly that forms on the viral attachment site, attP. Our cleavage assay also shows that the capture of attB by the attP intasome does not depend on DNA homology between the two sites; synapsis is governed solely by protein-protein and protein-DNA interactions.

Bending and supercoiling of DNA at the attachment site of bacteriophage λ

Integration of the DNA of bacteriophage lambda into the chromosome of E. coli depends on the formation of a complex nucleoprotein array at a specific locus on the phage genome, the attachment site. Recent work shows how bending of this DNA (induced by a specific DNA-binding protein), and strain in this DNA (induced by supercoiling) contribute to the formation of the nucleoprotein structure. Further, there are new insights into the way this structure directs critical events during recombination.

A Putative Cation Channel and Its Novel Regulator: Cross-Species Conservation of Effects on General Anesthesia

Volatile anesthetics like halothane and enflurane are of interest to clinicians and neuroscientists because of their ability to preferentially disrupt higher functions that make up the conscious state. All volatiles were once thought to act identically; if so, they should be affected equally by genetic variants. However, mutations in two distinct genes, one in Caenorhabditis and one in Drosophila, have been reported to produce much larger effects on the response to halothane than enflurane [1, 2]. To see whether this anesthesia signature is adventitious or fundamental, we have identified orthologs of each gene and determined the mutant phenotype within each species. The fly gene, narrow abdomen (na), encodes a putative ion channel whose sequence places it in a unique family; the nematode gene, unc-79, is identified here as encoding a large cytosolic protein that lacks obvious motifs. In Caenorhabditis, mutations that inactivate both of the na orthologs produce an Unc-79 phenotype; in Drosophila, mutations that inactivate the unc-79 ortholog produce an na phenotype. In each organism, studies of double mutants place the genes in the same pathway, and biochemical studies show that proteins of the UNC-79 family control NA protein levels by a posttranscriptional mechanism. Thus, the anesthetic signature reflects an evolutionarily conserved role for the na orthologs, implying its intimate involvement in drug action.

Interaction of Int Protein with Specific Sites on λ att DNA

We have studied the interaction of highly purified Int protein with DNA restriction fragments from the lambda phage attachment site (attP) region. Two different DNA sequences are protected by bound Int protein against partial digestion by either pancreatic DNAase or neocarzinostatin. One Int binding site includes the 15 bp common core sequence (the crossover region for site-specific recombination) plus several bases of sequence adjoining the core in both the P and P′ arms. The second Int-protected site occurs 70 bp to the right of the common core in the P′ arm, just at the distal end of the sequence encoding Int protein. The two Int binding sites are of comparable size, 30–35 bp, but do not share any extensive sequence homology. The interaction of Int with the two sites is distinctly different, as defined by the observation that only the site in the P′ arm and not the site at the common core region is protected by Int in the face of challenge by the polyanion heparin. Restriction fragments containing DNA from the bacterial attachment site (attB) region exhibit a different pattern of interaction with Int. In the absence of heparin, a smaller (15 bp) sequence, which includes the left half of the common core region and the common core-B arm juncture, is protected against nuclease digestion by Int protein. No sequences from this region are protected by Int in the presence of heparin.

Knotting of DNA caused by a genetic rearrangement. Evidence for a nucleosome-like structure in site-specific recombination of bacteriophage lambda.

Intramolecular recombination between two attachment sites on a circular substrate can invert one segment of the circle with respect to the other. We have studied the topological form of the products of such site-specific inversion as a function of two parameters of the substrate circle: the degree of supercoiling and the distance between the recombining sites. For both integrative and excisive recombination, supercoiled substrates produced knotted recombinants; the complexity of the knots reflects the distance separating the sites. This confirms and extends earlier observations and supports the hypothesis that random interwrapping of segments of the double-helical substrate persists during recombination. For integrative recombination, we find that even at conditions that should limit random interwrapping, absence of supercoiling and very short separation between attachment sites, only about one-half of the recombinant products are simple circles and the rest are knotted. Under the same conditions, excisive recombination yields only simple circular inverted recombinants. We propose that the excess knotting that characterizes integrative recombination reflects the requirement for wrapping of one attachment site, presumably attP, into a nucleosome-like structure. This hypothesis accounts for both the frequency of knots and the observation that the extra knots are trefoils rather than more complex forms.