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.

The covalent joining of topoisomerases to DNA is normally a transient step in the reaction cycle of these important enzymes. However, under a variety of circumstances, the covalent complex is converted to a long-lived or dead-end product that can result in chromosome breakage and cell death. We have discovered and partially purified an enzyme that specifically cleaves the chemical bond that joins the active site tyrosine of topoisomerases to the 3' end of DNA. The reaction products made by the purified enzyme on a variety of model substrates indicate that the enzyme cleanly hydrolyzes the tyrosine-DNA phosphodiester linkage, thereby liberating a DNA terminated with a 3' phosphate. The wide distribution of this phosphodiesterase in eukaryotes and its specificity for tyrosine linked to the 3' end but not the 5' end of DNA suggest that it plays a role in the repair of DNA trapped in complexes involving eukaryotic topoisomerase I.Topoisomerases are ubiquitous enzymes that play important roles in virtually all cells. The defining activity of these enzymes is their capacity to break DNA and, after an interval in which topological changes may occur, to reseal the break without the intervention of a high energy cofactor. Reversible breakage and reunion by topoisomerases is achieved through an intermediate in which a specific tyrosine residue of the enzyme is covalently joined to the DNA at the site of the break. The tyrosine-phosphodiester linkage is formed with one of two distinct polarities. Some enzymes join to the 5' end of the break, liberating a fragment terminated by a 3' hydroxyl; representatives include bacterial topoisomerase I as well as DNA gyrase and its relatives, the topoisomerase TI family. Other topoisomerase enzymes join to the 3' end of the break, liberating a fragment with a free 5' hydroxyl; representatives include the eukaryotic type I topoisomerases and the Int family of site-specific recombinases (for reviews, see refs. 1 and 2).The covalent intermediate between a topoisomerase and DNA is normally quite transient, i.e., strand rejoining is fast relative to strand cleavage. However, when a topoisomerase acts on DNA that contains imperfections (3, 4) or when specific inhibitors are present (5, 6), the rejoining step is slowed or blocked. This shifts the covalent complex from a transient intermediate toward a dead-end product; the associated long-lived break in the DNA backbone can have dire consequences for chromosome stability and cell survival (5,6). In this work, we describe an activity that hydrolyzes the covalent bond that joins topoisomerases to the 3' end of DNA. The specificity, robustness, and widespread distribution of this enzyme suggest that it could be an important element in the repair of DNA that has become stuck in covalent linkage with eukaryotic type I topoisomerases. MATERIALS AND METHODSDNA Substrates. Conventional oligonucleotides were constructed using an Applied Biosystems model 391 DNA synthesizer (Perkin-Elmer) or were purchased from Midland Certified Reagent (Midla...

We have quantitatively evaluated the affinity of a set of target sites for the integration host factor (IHF) protein of Escherichia coli by their performance as competitors in an electrophoretic mobility shift assay. We also determined how well each of these sites is filled by IHF in vivo. The data show that several natural sites have an affinity not much greater than that required for intracellular occupancy. The data also indicate that very little of the IHF in a cell is present as free protein available for binding, suggesting that binding to non‐specific targets dominates the operation of this system. The correlation between in vitro affinity and in vivo occupancy provides a ready means to assess the likely physiological significance of putative IHF sites. It also provides a general method to assess the importance of non‐specific interactions by DNA binding proteins inside a cell.

The MGC Project Team 1Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.