A structural analysis of the bent kinetoplast DNA from Crithidia fasciculata by high resolution chemical probing.

The chemical probes potassium permanganate (KMnO4) and diethylpyrocarbonate (DEPC) have been used to study the conformation of bent kinetoplast DNA from Crithidia fasciculata at different temperatures. Chemical reactivity data shows that the numerous short A-tracts of this bent DNA adopt a similar structure at 43 degrees C. This conformation appears to be very similar to the conformation of A-tracts in DNA exhibiting normal gel mobility. The A-tract structure detected by chemical probing is characterized by a high degree of base stacking on the thymine strand, and by an abrupt conformational change at the 3' end of the adenine strand. In general, no major alteration of this A-tract specific structure was detected between 4-53 degrees C. However, probing with KMnO4 revealed two unusual features of the C. fasciculata sequence that may contribute to the highly aberrant gel mobility of this DNA: 1) the B DNA/A-tract junction 5' dC/A3-6 3'. 5' dT3-6/G 3' is disproportionately represented and is conformationally distinct from other 5' end junctions, and 2) low temperature favors a novel strand-specific conformational distortion over a 20 base pair region of the bent kinetoplast DNA. Presence of the minor groove binding drug distamycin had little detectable effect on the A-tract conformation. However, distamycin did inhibit formation of the novel KMnO4 sensitive low temperature structure and partially eliminated the anomalous gel mobility of the kinetoplast DNA. Finally, we describe a simple and reproducible procedure for the production of an adenine-specific chemical DNA sequence ladder.     

Chemical biology on the genome

In this article I discuss studies towards understanding the structure and function of DNA in the context of genomes from the perspective of a chemist. The first area I describe concerns the studies that led to the invention and subsequent development of a method for sequencing DNA on a genome scale at high speed and low cost, now known as Solexa/Illumina sequencing. The second theme will feature the four-stranded DNA structure known as a G-quadruplex with a focus on its fundamental properties, its presence in cellular genomic DNA and the prospects for targeting such a structure in cels with small molecules. The final topic for discussion is naturally occurring chemically modified DNA bases with an emphasis on chemistry for decoding (or sequencing) such modifications in genomic DNA. The genome is a fruitful topic to be further elucidated by the creation and application of chemical approaches.     

DGGE analysis of 16S rDNA of ammonia-oxidizing bacteria in chemical–biological flocculation and chemical coagulation systems

Microbial community DNA was extracted from activated sludge samples taken from a chemical bioflocculation process and a chemical coagulation process in Shanghai, China. 16S rDNA of ammonia-oxidizing bacteria (AOB)was amplified by nested polymerase chain reaction and fingerprinted by denaturing gradient gel electrophoresis for microbial structure analysis. The Shannon diversity index of each sample was determined. The results indicated that the microbial structure of AOB in chemical bioflocculation process was comparable at two operational conditions. The ammonia-oxidizing bacterial communities were similar in three channels of the chemical bioflocculation process and in three serial tanks in the chemical coagulation process at the same condition. The diversity of microbial structures in the chemical bioflocculation process was higher than in the chemical coagulation process, in which the microbial structure was similar to that in the influent. Although the microbial study provides insights to the nitrification removal, higher microbial diversity of AOB does not necessarily mean higher ammonia oxidization. Molecular analysis should be combined with chemical assays to optimize operational conditions.     

DNA-dependent protein kinase catalytic subunit. Structural requirements for kinase activation by DNA ends.

DNA-dependent protein kinase (DNA-PK) is a DNA end-activated protein kinase composed of a catalytic subunit, DNA-PKcs, and a DNA binding subunit, Ku, that is involved in repair of DNA double-stranded breaks (DSBs). We have previously shown that DNA-PKcs interacts with single-stranded DNA (ssDNA) ends with a separate ssDNA binding site to be activated for its kinase activity. Here, the properties of the ssDNA binding site were examined by using DNA fragments with modified ssDNA extensions. DNA fragments with a wide range of ssDNA modifictations activated DNA-PKcs, indicating a relaxed specificity for the chemical structure of terminal nucleotides of a DSB. Methyl substitution of the phosphate backbone impaired kinase activation but not binding, indicating that interaction with the DNA backbone was involved in kinase activation. Experiments with RNA and RNA/DNA hybrid fragments suggested that the discrimination between RNA and DNA ends resides in the double-stranded DNA binding function of DNA-PKcs. DNA fragments exposing only one ssDNA end activated DNA-PKcs poorly, suggesting that DNA-PKcs distinguishes between DSBs and ssDNA breaks by simultaneous interaction with two ssDNA ends. These properties potentially explain how DNA-PKcs can be specifically activated by DSBs but still recognize the diverse chemical structures exposed when DSBs are introduced by ionizing radiation.     

An intramolecular DNA triplex is disrupted by point mutations associated with hereditary persistence of fetal hemoglobin.

Several investigators have suggested that secondary structures in DNA may be involved with physiologic gene regulatory processes in higher organisms. This hypothesis has been difficult to prove, however, since naturally occurring mutations that alter secondary DNA structures have not yet been identified. In this report, we describe a secondary DNA structure upstream from the human gamma-globin genes; this structure is formed in a homopyrimidine-homopurine tract and is stabilized by acidic pH and negative supercoiling of plasmid DNA. Since this structure is asymmetrically cleaved by S1 nuclease, it probably contains a single-stranded region and an intramolecular triplex. The single-stranded region is actually accessible for Watson-Crick base pairing with exogenous oligomers, a characteristic that permitted us to directly map the secondary DNA structure without additional chemical modifications of the supercoiled DNA. Five different point mutations just downstream from the single-stranded region are associated with hereditary persistence of fetal hemoglobin; four of these mutations dramatically reduce the stability of the secondary DNA structure, suggesting that these mutations alter formation of the intramolecular triplex by destabilizing critical Hoogsteen (triple-stranded) base pairs. These mutations may therefore represent a novel class of genetic defects that alter gene expression by changing the interaction of a critical regulatory molecule with a secondary DNA structure.     

HMG-D complexed to a bulge DNA: an NMR model

An NMR model is presented for the structure of HMG-D, one of the DROSOPHILA: counterparts of mammalian HMG1/2 proteins, bound to a particular distorted DNA structure, a dA(2) DNA bulge. The complex is in fast to intermediate exchange on the NMR chemical shift time scale and suffers substantial linebroadening for the majority of interfacial resonances. This essentially precludes determination of a high-resolution structure for the interface based on NMR data alone. However, by introducing a small number of additional constraints based on chemical shift and linewidth footprinting combined with analogies to known structures, an ensemble of model structures was generated using a computational strategy equivalent to that for a conventional NMR structure determination. We find that the base pair adjacent to the dA(2) bulge is not formed and that the protein recognizes this feature in forming the complex; intermolecular NOE enhancements are observed from the sidechain of Thr 33 to all four nucleotides of the DNA sequence step adjacent to the bulge. Our results form the first experimental demonstration that when binding to deformed DNA, non-sequence-specific HMG proteins recognize the junction between duplex and nonduplex DNA. Similarities and differences of the present structural model relative to other HMG-DNA complex structures are discussed.     

Hyperthermophiles and the problem of DNA instability

Rates of chemical decomposition of DNA at the optimal growth temperatures of hyperthermophiles seem incongruent with the requirements of accurate genome replication. The peculiar physiology, ecology and phylogeny of hyperthermophiles combine to suggest that these prokaryotes have solved a molecular problem (spontaneous loss of native DNA structure) of a magnitude that well-studied microorganisms do not face. The failure of DNA base composition to correlate with optimal growth temperature among hyperthermophiles provides indirect evidence that other mechanisms maintain their chromosomal DNA in the duplex form. Studies in vitro indicate that DNA primary structure is more difficult to maintain at extremely high temperature than is secondary structure, yet hyperthermophiles exhibit only modest levels of spontaneous mutation. Radiation sensitivity studies also indicate that hyperthermophiles repair their DNA efficiently in vivo , and underlying mechanisms are beginning to be examined. Several enzymes of DNA metabolism from hyperthermophilic archaea exhibit unusual biochemical features that may ultimately prove relevant to DNA repair. However, genomic sequencing results suggest that many DNA repair genes of hyperthermophilic archaea may not be recognized because they are not sufficiently related to those of well-studied organisms.     

The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles).

We have studied the structure and reactivities of two kinds of mismatched DNA sequences--unopposed bases, or bulges, and multiple mismatched pairs of bases. These were generated in a constant sequence environment, in relatively long DNA fragments, using a technique based on heteroduplex formation between sequences cloned into single-stranded M13 phage. The mismatched sequences were studied from two points of view, viz 1. The mobility of the fragments on gel electrophoresis in polyacrylamide was studied in order to examine possible bending of the DNA due to the presence of the mismatch defect. Such bending would constitute a global effect on the conformation of the molecule. 2. Sequences in and around the mismatches were studied using enzyme and chemical probes of DNA structure. This would reveal more local structural effects of the mismatched sequences. We observed that the structures of the bulges and the multiple mismatches appear to be fundamentally different. The bulged sequences exhibited a large gel retardation, consistent with a significant bending of the DNA at the bulge, and whose magnitude depends on the number of mismatched bases. The larger bulges were sensitive to cleavage by single-strand specific nucleases, and modified by diethyl pyrocarbonate (adenines) or osmium tetroxide (thymines) in a non-uniform way, suggesting that the bulges have a precise structure that leads to exposure of some, but not all, of the bases. In contrast the multiple mismatches ('bubbles') cause very much less bending of the DNA fragment in which they occur, and uniform patterns of chemical reactivity along the length of the mismatched sequences, suggesting a less well defined, and possibly flexible, structure. The precise structure of the bulges suggests that such features may be especially significant for recognition by proteins.     

Sequencing the DNA of recombinant chromosomes

With contemporary molecular cloning and DNA sequencing techniques, deriving the primary structure of higher cell genes is now routine. This publication reviews the chemical DNA sequencing method, and suggests strategies for sequencing recombinant DNA, whether arising naturally by chromosome rearrangement in vivo or created experimentally by gene splicing in vitro. One such strategy prepares end-labeled restriction fragments from an inserted or rearranged DNA region, for sequencing by the chemical method. Another maps the point at which the sequences of two related DNA regions diverge, and indicates which restriction endonucleases would be useful for sequencing across that point. These techniques can facilitate sequencing of DNA integrations, excisions, translocations, inversions, and introns in cloned chromosomal segments.     

Stability and strand asymmetry in the non-B DNA structure at the bcl-2 major breakpoint region

The t(14;18) translocation involving the Ig heavy chain locus and the BCL-2 gene is the single most common chromosomal translocation in human cancer. Recently we reported in vitro and in vivo chemical probing data indicating that the 150-bp major breakpoint region (Mbr), which contains three breakage subregions (hotspots) (known as peaks I, II, and III), has single-stranded character and hence a non-B DNA conformation. Although we could document the non-B DNA structure formation at the bcl-2 Mbr, the structural studies were limited to chemical probing. Therefore, in the present study, we used multiple methods including circular dichroism to detect the non-B DNA at the bcl-2 Mbr. We established a new gel shift method to detect the altered structure at neutral pH on shorter DNA fragments containing the bcl-2 Mbr and analyzed the fine structural features. We found that the single-stranded region in the non-B DNA structure observed is stable for days and is asymmetric with respect to the Watson and Crick strands. It could be detected by oligomer probing, a bisulfite modification assay, or a P1 nuclease assay. We provide evidence that two different non-B conformations exist at peak I in addition to the single one observed at peak III. Finally we used mutagenesis and base analogue incorporation to show that the non-B DNA structure formation requires Hoogsteen pairing. These findings place major constraints on the location and nature of the non-B conformations assumed at peaks I and III of the bcl-2 Mbr.     

Sulfur-containing Nucleic Acids: Synthesis,Chemical Reactions in Supramolecular Biopolymer Complexes,Biological Applications

The chemical behavior of sulfur-containing oligonucleotides and their reactivity in self-assembled nucleic acids (NA) and specific NA–protein complexes is considered. Reviewed are postsynthetic approaches that allow introducing sulfur-containing linkages at preselected positions of the sugar-phosphate backbone of DNA and between neighboring nucleobases, to incorporate disulfide bridges between complementary strands of double- and triple-stranded DNAs, in large catalytic RNA, etc. Special reference is given to the site-specific chemical modifications as a tool for elucidating the structure, folding, and function of biomolecules. Structure-directed chemical reactions are shown to be helpful in detecting point mutations in DNA, targeting the modifications on specific positions of NA, probing the molecular recognition in protein–DNA interfaces, studying the conformational dynamics of nucleic acids, and discriminating between different folding models.     

DNA repair inhibition: a possible mechanism of action of co-carcinogens

Co-carcinogens with a wide diversity of chemical structure have the common property of inhibiting DNA repair replication in normal human lymphocytes. This evidence suggests that inhibition of DNA repair may be an important factor in the mechanism of action of co-carcinogens.     

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.     

Dynamic structures of intact chicken erythrocyte chromatins as studied by 1H-31P cross-polarization NMR.

The dynamic properties of DNA in intact chicken erythrocyte cells, nuclei, nondigested chromatins, digested soluble chromatins, H1, H5-depleted soluble chromatins and nucleosome cores were investigated by means of single-pulse and 1H-31P cross-polarization NMR. The temperature dependence of the phosphorus chemical shift anisotropy was identical for the former three in the presence of 3 mM MgCl2, suggesting that the local higher order structure is identical for these chromatins. The intrinsic phosphorus chemical shift anisotropy of the nucleosome cores was -159 ppm. The chemical shift anisotropy of DNA in the chromatins can be further averaged by the motion of the linker DNA. The spin-lattice relaxation time in the rotating frame of the proton spins (T1p) of the nondigested chromatins was measured at various locking fields. The result was analyzed on the assumption of the isotropic motion to get a rough value of the correlation time of the motion efficient for the relaxation, which was eventually ascribed to the segmental motion of the linker DNA with restricted amplitude. The 30 nm filament structure induced by NaCl was shown to be dynamically different from that induced by MgCl2. Side-by-side compaction of 30-nm filaments was suggested to be induced in the MgCl2 concentration range higher than 0.3 mM. Biological significance of the dynamic structure was discussed in connection with the results obtained.     

Chemical and structural characterization of interstrand cross-links formed between abasic sites and adenine residues in duplex DNA

A new type of interstrand DNA–DNA cross-link between abasic (Ap) sites and 2′-deoxyadenosine (dA) residues was recently reported, but the chemical structure and properties of this lesion were not rigorously established. Here we characterized the nucleoside cross-link remnant released by enzymatic digestion of duplex DNA containing the dA-Ap cross-link. A synthetic standard was prepared for the putative nucleoside cross-link remnant 6 in which the anomeric carbon of the 2-deoxyribose residue was connected to the exocyclic N⁶-amino group of dA. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the synthetic material 6 matched the authentic cross-link remnant released by enzymatic digestion of cross-linked DNA. These findings establish the chemical structure of the dA-Ap cross-link released from duplex DNA and may provide methods for the detection of this lesion in cellular DNA. Both the nucleoside cross-link remnant 6 and the cross-link in duplex DNA were quite stable at pH 7 and 37°C, suggesting that the dA-Ap cross-link could be a persistent lesion with the potential to block the action of various DNA processing enzymes.     

Single strand DNA catenane synthesis using the formation of G-quadruplex structure

DNA is a good material for constructing nanostructures such as DNA origami. One of the challenges in this field is constructing a topologically complex structure. Here, we synthesized a DNA catenane through the formation of a G-quadruplex structure. The formation of the DNA catenane was investigated by gel electrophoresis. Interestingly, the synthesized DNA catenane was destroyed by heat treatment. Because conventional methods to construct DNA catenane include enzymatic ligation or chemical reactions, DNA is cyclized by covalent bond connection and never destroyed by heat treatment. To our knowledge, this is the first report of the synthesis of DNA catenane without using covalent bonds. Our novel way of synthesizing DNA catenane may be of use in easily recoverable DNA topological labeling.     

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.