Structure of 11-deoxydaunomycin bound to DNA containing a phosphorothioate

The anthracyclines form an important family of cancer chemotherapeutic agents with a strong dependence of clinical properties on minor differences in chemical structure. We describe the X-ray crystallographic solution of the three-dimensional structure of the anthracycline 11-deoxydaunomycin plus d(CGTsACG). In this complex, two drug molecules bind to each hexamer duplex. Both the drug and the DNA are covalently modified in this complex in contrast with the three previously reported DNA-anthracycline complexes. In the 11-deoxydaunomycin complex the 11 hydroxyl group is absent and a phosphate oxygen at the TpA step has been replaced by a sulfur atom leading to a phosphorothioate with absolute stereochemistry R. Surprisingly, removal of a hydroxyl group from the 11 position does not alter the relative orientation of the intercalated chromophore. However, it appears that the phosphorothioate modification influenced the crystallization and caused the 11-deoxydaunomycin-d(CGTsACG) complex to crystallize into a different lattice (space group P2) with different lattice contacts and packing forces than the non-phosphorothioated DNA-anthracycline complexes (space group P4(1)2(1)2). In the minor groove of the DNA, the unexpected position of the amino-sugar of 11-deoxydaunomycin supports the hypothesis that in solution the position of the amino sugar is dynamic.     

Single-particle image reconstruction of a tetramer of HIV integrase bound to DNA

The HIV integrase enzyme (IN) catalyzes the initial DNA breaking and joining reactions that integrate viral DNA in the host chromosome. Structures for individual IN domains have been determined by X-ray crystallography and NMR spectroscopy, but the structure of the complete IN-DNA complex has remained elusive. Homogeneous complexes of IN tetramers were assembled on DNA three-way junction substrates designed to resemble integration intermediates. Electron microscopy and single-particle image analysis of these complexes yielded a three-dimensional reconstruction at approximately 27 A resolution. The map of the IN-DNA complex displays four lobes of density approximately 50 A in diameter. Three of the lobes form a roughly triangular base with a central channel approximately 20 A in diameter. The fourth lobe is centered between two lobes and extends approximately 40 A above the base. We propose that the central channel tethers the target DNA, and two of the lobes may bind the ends of the viral DNA. The asymmetry of the complex is a feature not incorporated in previous structural models and potentially provides the first view of an asymmetric reaction intermediate.     

Purification and characterization of DNA methyltransferases from Neisseria gonorrhoeae.

Three DNA methyltransferases, M.NgoAI, and M.NgoBI and M.NgoBII, free of any nuclease activities were isolated from Neisseria gonorrhoeae strains WR220 and MUG116 respectively. M.NgoAI recognizes the sequence 5' GGCC 3' and methylates the first 5' cytosine on both strands. M.NgoBI and M.NgoBII recognize 5' TCACC 3' and 5' GTAN5CTC 3' respectively. M.NgoBII methylates cytosine on only one strand to produce 5' GTAN5mCTC 3'.     

The crystal structure of Neisseria gonorrhoeae PriB reveals mechanistic differences among bacterial DNA replication restart pathways

Reactivation of repaired DNA replication forks is essential for complete duplication of bacterial genomes. However, not all bacteria encode homologs of the well-studied Escherichia coli DNA replication restart primosome proteins, suggesting that there might be distinct mechanistic differences among DNA replication restart pathways in diverse bacteria. Since reactivation of repaired DNA replication forks requires coordinated DNA and protein binding by DNA replication restart primosome proteins, we determined the crystal structure of Neisseria gonorrhoeae PriB at 2.7 Å resolution and investigated its ability to physically interact with DNA and PriA helicase. Comparison of the crystal structures of PriB from N. gonorrhoeae and E. coli reveals a well-conserved homodimeric structure consisting of two oligosaccharide/oligonucleotide-binding (OB) folds. In spite of their overall structural similarity, there is significant species variation in the type and distribution of surface amino acid residues. This correlates with striking differences in the affinity with which each PriB homolog binds single-stranded DNA and PriA helicase. These results provide evidence that mechanisms of DNA replication restart are not identical across diverse species and that these pathways have likely become specialized to meet the needs of individual organisms.     

Structure of the DNA repair helicase hel308 reveals DNA binding and autoinhibitory domains

Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas pre-binding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates.     

Crystal structure of flavocetin-A, a platelet glycoprotein Ib-binding protein, reveals a novel cyclic tetramer of C-type lectin-like heterodimers

Snake venom contains a number of the hemostatically active C-type lectin-like proteins, which affect the interaction between von Willebrand factor (vWF) and the platelet glycoprotein (GP) Ib or platelet receptor to inhibit/induce platelet activation. Flavocetin-A (FL-A) is a high-molecular mass C-type lectin-like protein (149 kDa) isolated from the habu snake venom. FL-A binds with high affinity to the platelet GP Ibalpha-subunit and functions as a strong inhibitor of vWF-dependent platelet aggregation. We have determined the X-ray crystal structure of FL-A and refined to 2.5 A resolution. This is a first elucidation of a three-dimensional structure of the platelet GP Ib-binding protein. The overall structure reveals that the molecule is a novel cyclic tetramer (alphabeta)(4) made up of four alphabeta-heterodimers related by a crystallographic 4-fold symmetry. The tetramerization is mediated by an interchain disulfide bridge between cysteine residues at the C-terminus of the alpha-subunit and at the N-terminus of the beta-subunit in the neighboring alphabeta-heterodimer. The high affinity of FL-A for the platelet GP Ib alpha-subunit could be explained by a cooperative-binding action through the multiple binding sites of the tetramer.     

Cloning and linkage analysis of Neisseria gonorrhoeae DNA methyltransferases.

We have cloned DNA methyltransferases (MTases) from various strains of Neisseria gonorrhoeae. Each of these clones represents a single specificity, indicating that the multiple gonococcal MTase specificities are encoded by monospecific MTases. The DNAs of five strains (FA5100, F62, MS11, Pgh3-2, and WR302) were digested with NheI, SpeI, or NheI plus SpeI and subjected to pulsed-field gel electrophoresis. The DNA MTase clones were used to probe Southern blots of these pulsed-field gels to determine whether the MTase genes are linked and whether there are strain-to-strain differences. The results indicate that none of these genes are closely linked, but variable hybridization patterns indicate that there exist restriction fragment length polymorphisms between the strains tested. Most of the chromosomal regions containing these restriction fragment length polymorphisms are clustered in regions containing gonococcal genes known or suspected to antigenically vary via genetic recombination.     

Mutation of the priA gene of Neisseria gonorrhoeae affects DNA transformation and DNA repair

In Escherichia coli, PriA is central to the restart of chromosomal replication when replication fork progression is disrupted and is also involved in homologous recombination and DNA repair. To investigate the role of PriA in recombination and repair in Neisseria gonorrhoeae, we identified, cloned, and insertionally inactivated the gonococcal priA homologue. The priA mutant showed a growth deficiency and decreased DNA repair capability and was completely for deficient in DNA transformation compared to the isogenic parental strain. The priA mutant was also more sensitive to the oxidative damaging agents H2O2 and cumene hydroperoxide compared to the parental strain. These phenotypes were complemented by supplying a functional copy of priA elsewhere in the chromosome. The N. gonorrhoeae priA mutant showed no alteration in the frequency of pilin antigenic variation. We conclude that PriA participates in DNA repair and DNA transformation processes but not in pilin antigenic variation.     

Structure of BamHI bound to nonspecific DNA: a model for DNA sliding

The central problem faced by DNA binding proteins is how to select the correct DNA sequence from the sea of nonspecific sequences in a cell. The problem is particularly acute for bacterial restriction enzymes because cleavage at an incorrect DNA site could be lethal. To understand the basis of this selectivity, we report here the crystal structure of endonuclease BamHI bound to noncognate DNA. We show that, despite only a single base pair change in the recognition sequence, the enzyme adopts an open configuration that is on the pathway between free and specifically bound forms of the enzyme. Surprisingly, the DNA drops out of the binding cleft with a total loss of base-specific and backbone contacts. Taken together, the structure provides a remarkable snapshot of an enzyme poised for linear diffusion (rather than cleavage) along the DNA.     

MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA

Molecular Biology - The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a...     

Structure of a designed dimeric zinc finger protein bound to DNA

Proteins that employ dimerization domains to bind cooperatively to DNA have a number of potential advantages over monomers with regards to gene regulation. Using a combination of structure-based design and phage display, a dimeric Cys(2)His(2) zinc finger protein has been created that binds cooperatively to DNA via an attached leucine zipper dimerization domain. This chimera, derived from components of Zif268 and GCN4, displayed excellent DNA-binding specificity, and we now report the 1.5 A resolution cocrystal structure of the Zif268-GCN4 homodimer bound to DNA. This structure shows how phage display has annealed the DNA binding and dimerization domains into a single functional unit. Moreover, this chimera provides a potential platform for the creation heterodimeric zinc finger proteins that can regulate a desired target gene through cooperative DNA recognition.     

Construction of miniplasmids from the 7.2-kb and 5.1-kb penicillinase-producing plasmids of Neisseria gonorrhoeae reveals two replication regions

Two replication regions have been identified on a 7.2-kb penicillinase-producing plasmid (pJD4) of Neisseria gonorrhoeae. Through construction of mini-plasmids, one replication region of pJD4 was located on a 1.5-kb fragment, designated region "a," that included the unique HindIII site of this plasmid. This region is absent from the 5.1-kb naturally occurring gonococcal penicillinase-producing plasmid (pJD5) which is considered to be a deletion-derivative of the 7.2-kb plasmid. A 1.5-kb fragment (region "b"), part of a 2.5-kb fragment essential for the replication of the 5.1-kb plasmid (pJD5), was found to be responsible for incompatibility. Incompatibility studies showed that in vitro-derived deletion-derivatives from pJD4 and pJD5 containing either region "a" or region "b" were compatible. The DNA sequence of part of region "a" showed that this region was A-T rich. It contained seven sets of A-T rich multiple direct repeats and two putative dnaA boxes, suggesting that the mechanism of replication of region "a" was similar to that of OriC in Escherichia coli.     

BstYI bound to noncognate DNA reveals a "hemispecific" complex: implications for DNA scanning

DNA recognition by proteins is essential for specific expression of genes in a living organism. En route to a target DNA site, a protein will often sample noncognate DNA sites through nonspecific protein-DNA interactions, resulting in a variety of conformationally different binding states. We present here the crystal structure of endonuclease BstYI bound to a noncognate DNA. Surprisingly, the structure reveals the enzyme in a "hemispecific" binding state on the pathway between nonspecific and specific recognition. A single base pair change in the DNA abolishes binding of only one monomer, with the second monomer bound specifically. We show that the enzyme binds essentially as a rigid body, and that one end of the DNA is accommodated loosely in the binding cleft while the other end is held tightly. Another intriguing feature of the structure is Ser172, which has a dual role in establishing nonspecific and specific contacts. Taken together, the structure provides a snapshot of an enzyme in a "paused" intermediate state that may be part of a more general mechanism of scanning DNA.