The bacterial transposon Tn7 utilizes four Tn7-encoded proteins, TnsA, TnsB, TnsC and TnsD, to make insertions at a speci®c site termed attTn7. This target is selected by the binding of TnsD to attTn7 in a sequence-speci®c manner, followed by the binding of TnsC and activation of the transposase. We show that TnsD binding to attTn7 induces a distortion at the 5¢ end of the binding site and TnsC contacts the region of attTn7 distorted by TnsD. Previous work has shown that a target site containing triplex DNA, instead of TnsD±attTn7, can recruit TnsABC and effect sitespeci®c insertion of Tn7. We propose that the DNA distortion imposed by TnsD on attTn7, like the altered DNA structure via triplex formation, serves as a signal to recruit TnsC. We also show that TnsD primarily contacts the major groove of DNA, whereas TnsC is a minor groove binding protein. The footprint of the TnsC±TnsD±attTn7 nucleoprotein complex includes and extends beyond the Tn7 insertion site, where TnsC forms a platform to receive and activate the transposase to carry out recombination.

Nucleosomes, the fundamental building blocks of chromatin, play an architectural role in ensuring the integrity of the genome and act as a regulator of transcription. Intrinsic properties of the underlying DNA sequence, such as flexibility and intrinsic bending, direct the formation of nucleosomes. We have earlier identified genomic nucleosome-positioning sequences with increased in vitro ability for nucleosome formation. One group of sequences bearing a 10-base pair consensus repeat sequence of TATAAACGCC had the highest reported nucleosome affinity from genomic material. Here, we report the intrinsic physical properties of this sequence and the structural details of the nucleosome it forms, as analyzed by footprinting techniques. The minor groove is buried toward the histone octamer at the AA steps and facing outwards at the CC steps. By cyclization kinetics, the overall helical repeat of the free DNA sequence was found to be 10.5 base pairs/turn. Our experiments also showed that this sequence is highly flexible, having a J-factor 25-fold higher than that of random sequence DNA. In addition, the data suggest that twist flexibility is an important determinant for translational nucleosome positioning, particularly over the dyad region.DNA packaging into nucleosomes, the basic repeating units of chromatin, involves the wrapping of 146 bp 1 of doublestranded DNA into almost two complete turns around the histone octamer. The histone proteins have been highly conserved through evolution and are designed to bind to virtually any DNA sequence within the nucleus. There are, however, several known sequences that show a considerably higher ability to bind the histone octamer compared with bulk DNA.About 90% of the DNA in an eukaryotic cell is complexed with histones to form chromatin fibers. This represents a tremendous obstacle to transcription, replication, and repair machinery that requires access to these DNA regions (1). The location of a nucleosome on the DNA sequence is determined by several factors. At the primary level of compaction, the DNA sequence itself is responsible for determining whether or not a nucleosome is positioned due to inherent intrinsic mechanical properties. In vivo, secondary effects, such as the interaction of DNA with non-histone proteins and other ligands, and boundary effects can determine the basic and higher order positioning of nucleosomes in chromatin (2).Several DNA sequence motifs have been studied in an effort to determine the organization of nucleosome-positioning signals at the level of primary DNA sequence. Travers and coworkers (3) investigated the sequence properties of the DNA in a library of nucleosomal DNA from chicken erythrocytes. They found that AA/TT dinucleotides were present where the minor groove was compressed and facing inward toward the histone octamer. Conversely, CG/CC dinucleotides were located where the minor groove was wider and facing outwards. These dinucleotides also showed a preferential distribution of 10 -11-bp periodicity, indicating the importance of ...

The cleavage by calicheamicin gamma 1I (CLM gamma 1I) of a nucleosome formed on the 5S RNA gene of Xenopus borealis was studied in vitro as a first step toward the understanding of CLM gamma 1I-chromatin interactions within the cell. The drug does not cleave in the region of the dyad axis of the nucleosome. Outside of this region, double-stranded cleavage occurs with a periodicity of 10-11 bp. The sites of cleavage correspond to DNA sequences facing outward in the nucleosome. The drug shows some sequence preference of cleavage within these accessible sites. The predominant cleavage event within this nucleosome occurs at -1 helical turn from the dyad axis. This site constitutes a "hot spot" for CLM gamma 1I cleavage within the 5S nucleosome. We observe an overall footprinting effect whereby the drug preferentially cleaves DNA located outside the nucleosome core (analogous to the linker DNA of chromatin) as compared to cleavage within the nucleosome core. We discuss the importance of accessibility, structural deformations of DNA within the nucleosome, and steric constraints posed by sequence, in the recognition and cleavage of nucleosomal DNA by calicheamicin.

The bacterial transposon, Tn7, inserts into a single site in the Escherichia coli chromosome termed attTn7 via the sequence-specific DNA binding of the target selector protein, TnsD. The target DNA sequence required for Tn7 transposition is located within the C-terminus of the glucosamine synthetase (glmS) gene, which is an essential, highly conserved gene found ubiquitously from bacteria to humans. Here, we show that Tn7 can transpose in vitro adjacent to two potential targets in the human genome: the gfpt-1 and gfpt-2 sequences, the human analogs of glmS. The frequency of transposition adjacent to the human gfpt-1 target is comparable with the E.coli glmS target; the human gfpt-2 target shows reduced transposition. The binding of TnsD to these sequences mirrors the transposition activity. In contrast to the human gfpt sequences, Tn7 does not transpose adjacent to the gfa-1 sequence, the glmS analog in Saccharomyces cerevisiae. We also report that a nucleosome core particle assembled on the human gfpt-1 sequence reduces Tn7 transposition by likely impairing the accessibility of target DNA to the Tns proteins. We discuss the implications of these findings for the potential use of Tn7 as a site-specific DNA delivery agent for gene therapy.