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At least 343 records · Page 19Linked to original sources
PubMed1998

Targeting of human retrotransposon integration is directed by the specificity of the L1 endonuclease for regions of unusual DNA structure.

L1 elements are polyA retrotransposons which inhabit the human genome. Recent work has defined an endonuclease (L1 EN) encoded by the L1 element required for retrotransposition. We report the sequence specificity of this nicking endonuclease and the physical basis of its DNA recognition. L1 endonuclease is specific for the unusual DNA structural features found at the TpA junction of 5'(dTn-dAn) x 5'(dTn-dAn) tracts. Within the context of this sequence, substitutions which generate a pyrimidine-purine junction are tolerated, whereas purine-pyrimidine junctions greatly reduce or eliminate nicking activity. The A-tract conformation of the DNA substrate 5' of the nicked site is required for L1 EN nicking. Chemical or physical unwinding of the DNA helix enhances L1 endonuclease activity, while disruption of the adenine mobility associated with TpA junctions reduces it. Akin to the protein-DNA interactions of DNase I, L1 endonuclease DNA recognition is likely mediated by minor groove interactions. Unlike several of its homologues, however, L1 EN exhibits no AP endonuclease activity. Finally, we speculate on the implications of the specificity of the L1 endonuclease for the parasitic relationship between retroelements and the human genome.

Base Sequence↗
PubMed1998

Conformations of nicked and gapped DNA structures by NMR and molecular dynamic simulations in water.

We have analyzed and compared the molecular structures and dynamics of DNA duplexes containing a nick or a gap of one nucleotide where the base in front of the gap is a guanine. The continuous strand has the sequence 5'(CAGAGTCXCTGGCTC) where the residue X is absent for the nick, 14-mer, and where it is a G residue for the gap. Duplexes were formed with the two corresponding 7-mers. Neither of these is phosphorylated adjacent at the nick site, but it is a good model for a single strand break. For the nick structure, the quantitative NMR data show that the global conformation is very close to canonical B-form DNA, but it displays enhanced local flexibility. For the gap structure, we observe only one species in which the extra G is well stacked into the helix. The two half-helices around this residue also show a B-form conformation. As with the nick duplex, the adjacent G imino protons show enhanced exchange with solvent. The gap does not close completely. Using distance constraints, MD calculations show that the nick conformation is very close to a duplex with no lesion but is indeed more flexible in the central part. The gapped structure shows two families of conformations. One is close to B-DNA, the other is significantly kinked at the gap which reduces the size of the cavity. We observe a spine of hydration within the cavities, similar, but of different geometry in the two cases.

Base Composition↗
PubMed1994

Endonuclease VII of phage T4 nicks N-2-acetylaminofluorene-induced DNA structures in vitro.

We have tested in vitro the activity of T4 endonuclease VII on three different double-stranded oligonucleotides bearing a single N-2-acetylaminofluorene (AAF) adduct covalently bound to each of the three guanine residues located within the NarI site (G1G2CG3CC), a strong frameshift mutation hot spot in E. coli. With the oligonucleotides modified at G2 and G3 a specific cleavage pattern with T4 endonuclease VII was observed in the complementary strand while no cleavage was found in the adduct-bearing strand. On the other hand, when G1 was modified, only a very faint cleavage band was observed (< 1%). These differences in nicking among the three AAF-modified DNA substrates are discussed in terms of the polymorphic nature in adduct-induced DNA structures as previously shown. This "non-physiological" activity of a DNA resolvase is discussed in terms of a potential role for such enzymes in the induction of frameshift mutations.

2-Acetylaminofluorene↗
PubMed2001

Target DNA structure plays a critical role in Tn7 transposition.

The bacterial transposon Tn7 utilizes four Tn7-encoded proteins, TnsA, TnsB, TnsC and TnsD, to make insertions at a specific site termed attTn7. This target is selected by the binding of TnsD to attTn7 in a sequence-specific 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 site- specific 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.

Bacterial Proteins↗
PubMed1997

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases.

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure-specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.

Binding, Competitive↗
PubMed2001

A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression.

Germline mutations of the human BRCA2 gene confer susceptibility to breast cancer. Although the function of the BRCA2 protein remains to be determined, murine cells homozygous for BRCA2 inactivation display chromosomal aberrations. We have isolated a 2 MDa BRCA2-containing complex and identified a structural DNA binding component, designated as BRCA2-Associated Factor 35 (BRAF35). BRAF35 contains a nonspecific DNA binding HMG domain and a kinesin-like coiled coil domain. Similar to BRCA2, BRAF35 mRNA expression levels in mouse embryos are highest in proliferating tissues with high mitotic index. Strikingly, nuclear staining revealed a close association of BRAF35/BRCA2 complex with condensed chromatin coincident with histone H3 phosphorylation. Importantly, antibody microinjection experiments suggest a role for BRCA2/BRAF35 complex in modulation of cell cycle progression.

Amino Acid Sequence↗
PubMed1997

The DNA structures at the ends of eukaryotic chromosomes.

The sequence organisation of the telomeric regions is extremely similar for all eukaryotes examined to date. Subtelomeric areas may contain large sequence arrays of middle repetitive, complex elements that sometimes have similarities to retrotransposons. In between and within these complex sequences are short, satellite-like repeats. These areas contain very few genes and are thought to be organised into a heterochromatin-like domain. The terminal regions almost invariably consist of short, direct repeats. These repeats usually contain clusters of 2-4 G residues and the strand that contains these clusters (the G strand) always forms the extreme 3'-end of the chromosome. Thus, most telomeric repeats are clearly related to each other which in turn suggests a common evolutionary origin. A number of different structures can be formed by single-stranded telomeric G strand repeats and, as has been suggested recently, by the G strand. Since the main mechanism for the maintenance of telomeric repeats predicts the occurrence of single-stranded extensions of the G strand, the propensity of G-rich DNA to fold into alternative DNA structures may have implications for telomere biology.

Animals↗
PubMed1993

Constitutive binding of EBNA1 protein to the Epstein-Barr virus replication origin, oriP, with distortion of DNA structure during latent infection.

Replication of the circular, 170 kb genome of Epstein-Barr virus (EBV) during latent infection is performed by the cellular replication machinery under cell-cycle control. A single viral protein, EBNA1, directs the cellular replication apparatus to initiate replication within the genetically defined replication origin, oriP, at a cluster of four EBNA1 binding sites, referred to here as the physical origin of bidirectional replication, or OBR. A second cluster of EBNA1 binding sites within oriP, the 30 bp repeats, serves an essential role as a replication enhancer and also provides a distinct episome maintenance function that is unrelated to replication. We examined the functional elements of oriP for binding by EBNA1 and possibly other proteins in proliferating Raji cells by generating in vivo footprints using two reagents, dimethylsulfate (DMS) and KMnO4. We also employed deoxyribonuclease I (DNase I) with permeabilized cells. The in vivo and permeabilized cell footprints at the EBNA1 binding sites, particularly those obtained using DMS, gave strong evidence that all of these sites are bound by EBNA1 in asynchronously dividing cells. No consistent evidence was found to suggest binding by other proteins at any other sites within the functional regions of oriP. Thymines at symmetrical positions of the OBR within oriP were oxidized when cells were treated with permanganate, suggestive of bends or other distortions of DNA structure at these positions; binding of EBNA1 in vitro to total DNA from Raji cells induced reactivity to permanganate at identical positions. The simplest interpretation of the results, which were obtained using asynchronously dividing cells, is that EBNA1 binds to its sites at oriP and holds the OBR in a distorted conformation throughout most of the cell cycle, implying that replication is initiated by a cellular mechanism and is not limited by an availability of EBNA1 for binding to oriP.

Antigens, Viral↗
PubMed2000

DNA structure control by polycationic species: polyamine, cobalt ammines, and di-metallo transition metal chelates.

Many polycationic species bind to DNA and induce structural changes. The work reported here is the first phase of a program whose long-term aim is to create a class of simple and inexpensive sequence-selective compounds that will enable enhanced DNA structure control for a wide range of applications. Three classes of molecule have been included in this work: the polyamine spermine (charge: 4(+)) and spermidine (charge: 3(+)) (which are known to induce a wide range of DNA conformational changes but whose binding modes are still not well understood); cobalt (III) amine transition metal complexes as potential polyamine mimics and [Fe(H(2)O)(6)](3+); and the first member of a new class of di-metallo tris-chelated cylinders of helical structure (charge 4(+)). Temperature-dependent absorption, circular dichroism, linear dichroism, gel electrophoresis, and molecular modeling data are presented. The cobalt amines prove to be effective polyamine mimics, although their binding appears to be restricted to backbone and major groove. All the ligands stabilize the DNA, but the 4(+) di-iron tris-chelate does so comparatively weakly and seems to have a preference for single-stranded DNA. All the molecules studied bend the DNA, with the di-iron tris-chelate having a particularly dramatic effect even at very low drug load.

Chelating Agents↗
PubMed2001

In vivo acetylation of HMG1 protein enhances its binding affinity to distorted DNA structures.

The postsynthetic acetylation of HMG1 protein has been known for more than 20 years, but the effect of this modification on the properties of the protein has not been studied so far. Acetylated HMG1 was isolated from cells grown in the presence of sodium n-butyrate and identified as a monoacetylated protein, modified at lysine 2. Acetylated and parental forms of HMG1 were compared relative to their binding affinity to distorted DNA structures. By using mobility shift assay to determine the dissociation constants, we show that acetylation enhanced the ability of HMG1 to recognize UV light- or cisplatin-damaged DNA and four-way junctions. Since the modified lysine lies adjacent to the HMG1 DNA-binding domain, the results obtained were attributed to acetylation-induced conformational change in HMG1. The potential role of acetylation in modulating the interactions of HMG1 with both damaged DNA and other proteins is discussed.

Acetylation↗
PubMed1990

A note on sequence-dependence of DNA structure.

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.

Base Sequence↗
PubMed1992

A consensus repeat sequence from the human insulin gene linked polymorphic region adopts multiple quadriplex DNA structures in vitro.

A hypervariable region consisting of repeats of a 14 base pair (bp) consensus sequence ACAGGGGT(G/C)(T/C)GGGG is located 363 bp upstream of the human insulin gene. Different repeat numbers of this oligonucleotide give rise to a polymorphism, and so this region is commonly known as the insulin gene linked polymorphic region (ILPR). Here we present evidence, based on the mobility in non-denaturing polyacrylamide gels of two dissimilarly sized oligonucleotides containing the ILPR consensus sequence, that this sequence can adopt a number of quadriplex DNA structures in vitro.

Base Sequence↗
PubMed1988

Solving DNA structures by MERLOT.

MERLOT, a molecular replacement computer package, has been successfully used for the determination of initial phases for five different oligonucleotide structures. Two of these, CCGG and CGCGCGTTTTCGCGCG, with 316 and 323 non-H atoms respectively in the asymmetric unit, have been solved ab initio using the MERLOT package. The rest are re-examinations of previously solved DNA oligomer structures, ioCCGG, CGCGAATTCGCG and CGCGAATTbrCGCG, with 160, 486 and 488 non-H atoms in the asymmetric unit respectively. Problems involved in applying the molecular-replacement technique to DNA structures, and their possible solutions, are discussed.

Base Sequence↗
PubMed1998

DNA topoisomerase: the key enzyme that regulates DNA super structure.

DNA is a macromolecule carrying all genetic information and it must be packed in the cell nucleus. DNA is a double helix where one strand coils around the other. The helix is unwound and rewound every now and then to take out its information, which requires local alteration of the helical structure, resulting in the super-coiling of DNA. For replication, all the coils must be unwound at least once, and two daughter molecules are often catenated to each other. To solve these problems caused by the helical structure of DNA, topoisomerase activity introducing the transient breaking and rejoining of DNA strand is essential to perform each DNA transaction. In this article, we first review the mechanistic aspect of topoisomerase activity, and then discuss its basic clinical importance.

Adenosine Triphosphate↗
PubMed1998

DNA structure and fluctuations sensed from a 1.1ns molecular dynamics trajectory of a fully charged Zif268-DNA complex in water.

Molecular dynamics simulations of the zinc finger domain of protein Zif268, in a complex with a high affinity DNA sequence, yields a globally stable system with small yet significant readjustments with persistence time of the order of 1.1ns. The results confirm the quality of the standard GROMOS87 force field with a corrected solvent-to-solute interaction that does not affect the water-water SPC interactions nor the intra-molecular cohesive forces. Specificity determinants are discussed. The simulations of DNA alone, with the same force field, showed the important role played by the solvent and the symmetry of the counterion distribution. (Tapia & Velázquez, J. Am. Chem. Soc., 119, 5934, 1997) In the present work, this feature was retained when appropriate. The results for root mean square deviations and temperature B-factors illustrate the reliability of this approach. The structure of DNA is held by its interactions with the zinc finger protein. This behavior is not much affected by the slow whithering away of finger-1 from DNA. The factors contributing to the molecular stability found in GROMOS' potential energy function appear to be sufficient to yield stable fluctuation patterns when surrounding medium effects are properly included.

Computer Simulation↗
PubMed2006

Architecture with GIDEON, a program for design in structural DNA nanotechnology.

We present geometry based design strategies for DNA nanostructures. The strategies have been implemented with GIDEON-a graphical integrated development environment for oligonucleotides. GIDEON has a highly flexible graphical user interface that facilitates the development of simple yet precise models, and the evaluation of strains therein. Models are built on a simple model of undistorted B-DNA double-helical domains. Simple point and click manipulations of the model allow the minimization of strain in the phosphate-backbone linkages between these domains and the identification of any steric clashes that might occur as a result. Detailed analysis of 3D triangles yields clear predictions of the strains associated with triangles of different sizes. We have carried out experiments that confirm that 3D triangles form well only when their geometrical strain is less than 4% deviation from the estimated relaxed structure. Thus geometry-based techniques alone, without detailed energetic considerations, can be used to explain certain general trends in DNA structure formation. We have used GIDEON to build detailed models of double crossover and triple crossover molecules, evaluating the non-planarity associated with base tilt and junction misalignments. Computer modeling using a graphical user interface overcomes the limited precision of physical models for larger systems, and the limited interaction rate associated with earlier, command-line driven software.

Base Sequence↗