A knob-associated tandem repeat in maize capable of forming fold-back DNA segments: are chromosome knobs megatransposons?

A class of tandemly repeated DNA sequences (TR-1) of 350-bp unit length was isolated from the knob DNA of chromosome 9 of Zea mays L. Comparative fluorescence in situ hybridization revealed that TR-1 elements are also present in cytologically detectable knobs on other maize chromosomes in different proportions relative to the previously described 180-bp repeats. At least one knob on chromosome 4 is composed predominantly of the TR-1 repeat. In addition, several small clusters of the TR-1 and 180-bp repeats have been found in different chromosomes, some not located in obvious knob heterochromatin. Variation in restriction fragment fingerprints and copy number of the TR-1 elements was found among maize lines and among maize chromosomes. TR-1 tandem arrays up to 70 kilobases in length can be interspersed with stretches of 180-bp tandem repeat arrays. DNA sequence analysis and restriction mapping of one particular stretch of tandemly arranged TR-1 units indicate that these elements may be organized in the form of fold-back DNA segments. The TR-1 repeat shares two short segments of homology with the 180-bp repeat. The longest of these segments (31 bp; 64% identity) corresponds to the conserved region among 180-bp repeats. The polymorphism and complex structure of knob DNA suggest that, similar to the fold-back DNA-containing giant transposons in Drosophila, maize knob DNA may have some properties of transposable elements.     

Crystal structure of restriction endonuclease BglI bound to its interrupted DNA recognition sequence

The crystal structure of the type II restriction endonuclease BglI bound to DNA containing its specific recognition sequence has been determined at 2.2 A resolution. This is the first structure of a restriction endonuclease that recognizes and cleaves an interrupted DNA sequence, producing 3' overhanging ends. BglI is a homodimer that binds its specific DNA sequence with the minor groove facing the protein. Parts of the enzyme reach into both the major and minor grooves to contact the edges of the bases within the recognition half-sites. The arrangement of active site residues is strikingly similar to other restriction endonucleases, but the co-ordination of two calcium ions at the active site gives new insight into the catalytic mechanism. Surprisingly, the core of a BglI subunit displays a striking similarity to subunits of EcoRV and PvuII, but the dimer structure is dramatically different. The BglI-DNA complex demonstrates, for the first time, that a conserved subunit fold can dimerize in more than one way, resulting in different DNA cleavage patterns.     

Site-directed mutations in the vnd/NK-2 homeodomain. Basis of variations in structure and sequence-specific DNA binding

Secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2 gene from Drosophila melanogaster are described. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56, but more importantly position 56 in combination with position 52, plays an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles in structure and sequence specificity of individual atom-atom interactions are described.     

Distribution of labor among bZIP segments in the control of DNA affinity and specificity

BACKGROUND: The basic region-leucine zipper (bZIP) family of proteins use an atypically simple motif for DNA recognition, yet family members discriminate differently between target sites that differ only in half-site spacing. Two such sites are the cAMP-response element (CRE) and the AP-1 target site. Fos/Jun prefers the AP-1 site (ATGACTCAT), while CRE-BP1 prefers CRE (ATGACGTCAT), and GCN4 binds both sites with equal affinity. We therefore asked what determines the relative specificity for CRE and AP-1 sites in bZIP proteins. RESULTS: Here we show that CRE/AP-1 specificity in CRE-BP1 is encoded within the spacer and basic segments of the bZIP element. Of these two regions, the basic segment is the more important. This specificity is in part achieved at the expense of affinity. CONCLUSIONS: The small size and simplicity of the bZIP recognition helix was already unusual; our findings show that the information that determines the target site specificity of members of the bZIP family of proteins is even more condensed than expected. These results suggest that it may be possible to design surprisingly small proteins that bind DNA with high sequence specificity, although it may be more difficult to achieve high-affinity binding in small proteins.     

The subunit delta-subunit b domain of the Escherichia coli F1F0 ATPase. The B subunits interact with F1 as a dimer and through the delta subunit

The delta and b subunits are both involved in binding the F1 to the F0 part in the Escherichia coli ATP synthase (ECF1F0). The interaction of the purified delta subunit and the isolated hydrophilic domain of the b subunit (bsol) has been studied here. Purified delta binds to bsol weakly in solution, as indicated by NMR studies and protease protection experiments. On F1, i.e. in the presence of ECF1-delta, delta, and bsol interact strongly, and a complex of ECF1.bsol can be isolated by native gel electrophoresis. Both delta subunit and bsol are protected from trypsin cleavage in this complex. In contrast, the delta subunit is rapidly degraded by the protease when bound to ECF1 when bsol is absent. The interaction of bsol with ECF1 involves the C-terminal domain of delta as delta(1-134) cannot replace intact delta in the binding experiments. As purified, bsol is a stable dimer with 80% alpha helix. A monomeric form of bsol can be obtained by introducing the mutation A128D (Howitt, S. M., Rodgers, A. J.,W., Jeffrey, P. D., and Cox, G. B. (1996) J. Biol. Chem. 271, 7038-7042). Monomeric bsol has less alpha helix, i.e. only 58%, is much more sensitive to trypsin cleavage than dimer, and unfolds at much lower temperatures than the dimer in circular dichroism melting studies, indicating a less stable structure. The bsol dimer, but not monomer, binds to delta in ECF1. To examine whether subunit b is a monomor or dimer in intact ECF1F0, CuCl2 was used to induce cross-link formation in the mutants bS60C, bQ104C, bA128C, bG131C, and bS146C. With the exception of bS60C, CuCl2 treatment resulted in formation of b subunit dimers in all mutants. Cross-linking yield was independent of nucleotide conditions and did not affect ATPase activity. These results show the b subunit to be dimeric for a large portion of the C terminus, with residues 124-131 likely forming a pair of parallel alpha helices.     

DNA cleaving activities of 9-membered masked enediyne analogues possessing DNA intercalator and sugar moieties

The DNA cleaving properties of various enediyne analogues possessing sugar moieties and DNA-intercalators were investigated. The DNA cleaving experiments show that these hybrids analogues induced sequence-selective DNA cleavage and the simple sugars in the enediyne serve as a DNA recognition element for DNA cleavage.     

Symmetry and chirality in topoisomerase II-DNA crossover recognition

Several experimental data support the notion that the recognition of DNA crossovers play an important role in the multiple functions of topoisomerase II. Here, a theoretical analysis of the possible modes of assembly of yeast topoisomerase II with right and left-handed tight DNA crossovers is performed, using the crystal coordinates of the docking partners. The DNA crossovers are assumed to be clamped into the central hole of the enzyme. Taking into account the rules for building symmetric ternary complexes and the structural constraints imposed by DNA-DNA and protein-DNA interactions, this analysis shows that two geometric solutions could exist, depending on the chirality of the DNA crossovers. In the first one, the two DNA segments are symmetrically recognized by the enzyme while each single double helix binds asymmetrically the protein dimer. In the second one, each double helix is symmetrically recognized by the protein around its dyad axis, while the two DNA segments have their own binding modes. The finding of potential DNA-binding domains which could interact with the crossovers provides structural supports for each model. The structural similarity of a loop containing a cluster of conserved basic residues pointing into the central hole of topoisomerase II and the second DNA-binding site of histone H5 which binds DNA crossover is of particular interest. Each solution, which is consistent with different sets of experimental data found in the literature, could either correspond to different functions of the enzyme or different steps of the reaction. This work provides structural insights for better understanding the role of chirality and symmetry in topoisomerase II-DNA crossover recognition, suggests testable experiments to further elucidate the structure of ternary complexes, and raises new questions about the relationships between the mechanism of strand-passage and strand-exchange catalyzed by the enzyme.     

Three novel plasmid R6K proteins act in concert to distort DNA within the alpha and beta origins of DNA replication

Three novel R6K genes which are responsible for expression of DNA distortion polypeptides (DDP) were identified. The DDPs act in vivo in concert to induce similar stepwise DNA helix distortions within two long inverted repeats (alpha LIR and beta LIR), which are essential elements for the two distally located R6K alpha and beta DNA replication origins. DDP1 and DDP2 are encoded by two tandem genes located at the 5' end of alpha LIR, whereas a gene coding for DDP3 is located at the 3' end of beta LIR. DDP1 and DDP2 are required for primary DNA distortion within alpha LIR or beta LIR, while DDP3 is essential for generation of secondary DNA distortion in these LIR sequences. Creation of DNA distortion within alpha LIR depends on its specific interaction with DDP1 and on the presence of the R6K primase DNA-binding site. The possible relevance of these findings to R6K replication is discussed.