Mononucleotide repeat expansions with non-natural polymerase substrates.

Replicative strand slippage is a biological phenomenon, ubiquitous among different organisms. However, slippage events are also relevant to non-natural replication models utilizing synthetic polymerase substrates. Strand slippage may notably affect the outcome of the primer extension reaction with repetitive templates in the presence of non-natural nucleoside triphosphates.

Proteasomes and Several Aspects of Their Heterogeneity Relevant to Cancer.

The life of every organism is dependent on the fine-tuned mechanisms of protein synthesis and breakdown. The degradation of most intracellular proteins is performed by the ubiquitin proteasome system (UPS). Proteasomes are central elements of the UPS and represent large multisubunit protein complexes directly responsible for the protein degradation.

Biological consequences of structural and functional proteasome diversity.

Cell homeostasis and regulation of metabolic pathways are ensured by synthesis, proper folding and efficient degradation of a vast amount of proteins. Ubiquitin-proteasome system (UPS) degrades most intracellular proteins and thus, participates in regulation of cellular metabolism. Within the UPS, proteasomes are the elements that perform substrate cleavage.

Mass-spectrometry analysis of modifications at DNA termini induced by DNA polymerases.

Non-natural nucleotide substrates are widely used in the enzymatic synthesis of modified DNA. The terminal activity of polymerases in the presence of modified nucleotides is an important, but poorly characterized, aspect of enzymatic DNA synthesis. Here, we studied different types of polymerase activity at sequence ends using extendable and non-extendable synthetic models in the presence of the Cy5-dUTP analog Y.

The central domain of yeast transcription factor Rpn4 facilitates degradation of reporter protein in human cells.

Despite high interest in the cellular degradation machinery and protein degradation signals (degrons), few degrons with universal activity along species have been identified. It has been shown that fusion of a target protein with a degradation signal from mammalian ornithine decarboxylase (ODC) induces fast proteasomal degradation of the chimera in both mammalian and yeast cells. However, no degrons from yeast-encoded proteins capable to function in mammalian cells were identified so far.

Recognition of base pair inversions in duplex by chimeric (alpha,beta) triplex-forming oligonucleotides.

DNA recognition by triplex-forming oligonucleotides (TFOs) is usually limited by homopurine-homopyrimidine sequence in duplexes. Modifications of the third strand may overcome this limitation. Chimeric alpha-beta TFOs are expected to form triplex DNA upon binding to non-regular sequence duplexes.

Analysis of crystal structures of aspartic proteinases: on the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes.

To elucidate the role of amino acid residues adjacent to the catalytic site of pepsin-like enzymes, we analyzed and compared the crystal structures of these enzymes, their complexes with inhibitors, and zymogens in the active site area (a total of 82 structures). In addition to the water molecule (W1) located between the active carboxyls and playing a role of the nucleophile during catalytic reaction, another water molecule (W2) at the vicinity of the active groups was found to be completely conserved. This water molecule plays an essential role in formation of a chain of hydrogen-bonded residues between the active site flap and the active carboxyls on ligand binding.

Structural polymorphism of oligo(dC) with mixed alpha,beta-anomeric backbone.

Oligonucleotides with mixed alpha,beta-anomeric backbone have been proposed recently for the recognition of random DNA sequence via new triplex motif (Doronina and Behr, Chem. Soc. Reviews 26, 63-71 (1997)).

Zero-length protein-nucleic acid crosslinking by radical-generating coordination complexes as a probe for analysis of protein-DNA interactions in vitro and in vivo.

Redox-active coordination complexes such as 1,10-phenanthroline-Cu(II) (OP-Cu) and bleomycin-Fe(III) are commonly used as "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we report that under certain conditions these complexes may crosslink proteins to nucleic acids. In vitro experiments suggest that proteins are crosslinked to DNA by a mechanism similar to dimethyl sulfate-induced crosslinking.

Localization of histone H1 binding sites within the nucleosome by UV-induced H1-DNA crosslinking in vivo.

In our previous paper (Belikov et al., (1993), Nucl. Acids Res.

Nucleosome structural transition during chromatin unfolding is caused by conformational changes in nucleosomal DNA.

We have recently reported that certain core histone-DNA contacts are altered in nucleosomes during chromatin unfolding (Usachenko, S. I., Gavin I.

Uncurved-DNA signals are important for translational positioning of nucleosomes.

We describe here a possible mechanism for non-random arrangement of nucleosomes along genomic DNA by short stretches of uncurved DNA. Available biochemical data on nucleosome positioning on different DNA sequences were analyzed. A strong correlation between location of nucleosome borders and location of uncurved sequences along DNA was observed.

Sequential arrangement, not transcriptional activity, determines conformational stability of nucleosomes.

Gel electrophoresis in urea gradient was applied to study the unfolding effect of increasing concentrations of urea on the nucleosome structure. We showed that conformational stability of nucleosomes is determined by nucleotide sequence but not by transcriptional activity of DNA in chromatin.

The detection of B-form/A-form junction in a deoxyribonucleotide duplex.

The transition of the 14-meric deoxyoligonucleotide duplex d-(ACCCCCTTTTTTTG).d-(CAAAAAAAGGGGGT) from the B- to the A-conformation in water/trifluorethanol (TFE) solution was studied with the use of circular dichroism. An increase in the fraction of TFE induces a two-step B-A transition.

Mapping protein-DNA interactions with CIS-DDP: chromatin structure of promoter region of D. Melanogaster hsp 70 gene.

DNA-protein crosslinking by cis-dichlorodiammineplatinum (II) (cis-DDP) was applied to study chromatin structure in situ. Histone H1 (H5) is crosslinked to DNA in significant amounts whereas core histones remain practically unattached. "Protein image hybridization" experiments show that the 5'-region of the D metanogaster hsp 70 gene is free of histone H1 in both control nuclei and nuclei isolated from heat-shocked embryos.

Mapping of DNA-binding proteins along the yeast genome by UV-induced DNA-protein crosslinking.

UV-induced crosslinking of DNA-binding proteins to DNA in intact nuclei of Saccharomyces cerevisiae and subsequent 'protein image' hybridization were applied to map non-histone proteins along single-copy genes of yeast. We detected two polypeptides that most probably correspond to core subunits of yeast RNA-polymerase II in the coding region of transketolase gene (TKL2). Several non-histone proteins were also detected which bind to the upstream region of TKL2 gene, and to the intergenic spacer between calmodulin (CMD1) and beta-mannosyl transferase (ALG1) genes.

Alterations in nucleosome core structure in linker histone-depleted chromatin.

We have previously shown that the sequential arrangement of histone-DNA contacts is essentially the same in the nucleosomal core of sea urchin sperm nuclei, where chromatin is highly condensed and repressed, and in nuclei from lily bud sepals or yeast, where chromatin is highly active in transcription and replication and is significantly or completely depleted of histone H1. However, the difference in the strength of some histone-DNA contacts has not been understood or discussed. In this work, we demonstrate that some of these differences are due to a conformational change in the nucleosomal core.

Mechanism of Lac repressor switch-off: orientation of the Lac repressor DNA-binding domain is reversed upon inducer binding.

Lac repressor's DNA-binding domains contain helix-turn-helix motif which, though similar to those of phage lambda Cro protein, are oriented differently with respect to DNA: in the specific complexes with Lac operator, N termini of the repressor's subunits are facing inwards. We demonstrate that, in the presence of an inducer, the repressor's N termini cross-link to the operator's outermost nucleotides. We suggest that the inducer fixes the repressor's DNA-binding domains in the Cro-type configuration and thus garbles its recognition surface.

DNA sequencing by hybridization to oligonucleotide matrix. Calculation of continuous stacking hybridization efficiency.

In this paper we consider the efficiency of additional rounds of "continuous stacking" hybridization in DNA sequence reconstruction by hybridization with oligonucleotide matrix (SHOM). After the initial hybridization of target DNA with the matrix of oligonucleotides of fixed length L some additional hybridizations should be carried out in the presence of fluorescently labeled oligonucleotides of another length l. These additional oligonucleotides can hybridize in tandem with matrix tuples (continuous stacking hybridization) thus forming an extended duplex with the target DNA strand.

Identification of DNA binding proteins in vaccinia virus by DNA-protein crosslinking.

DNA binding proteins of vaccinia virus (VV) virions, strain LIVP, have been studied by their covalent crosslinking to DNA, using two-dimensional gel retardation electrophoresis of crosslinked DNA and proteins as well as the 'protein image' hybridization assay. Five proteins with molecular masses of 16, 25, 27, 41 and 54 kDa, respectively, associated with all analysed DNA sequences, including early and late genes and their promoters, have been identified.

Conformational changes in E. coli RNA polymerase during promoter recognition.

We analysed complexes formed during recognition of the lacUV5 promoter by E. coli RNA polymerase using formaldehyde as a DNA-protein and protein-protein cross-linking reagent. Most of the cross-linked complexes specific for the open complex (RPO) contain the beta' subunit of RNA polymerase cross-linked with promoter DNA in the regions: -50 to -49; -5 to -10; + 5 to +8 and +18 to +21.

Mapping and positioning DNA-binding proteins along genomic DNA. Structure of D. melanogaster ribosomal 'Alu-repeats' and 1.688 satellite chromatin.

Chromatin structure of so-called 'Alu-repeat' in D. melanogaster ribosomal non-transcribed spacer that contains sequences homologous to the promoter of ribosomal genes has been studied. Using the 'protein image' hybridization assay based on UV-light-induced DNA-protein crosslinking and 2-D gel retardation electrophoresis, two proteins of the molecular mass of 50 kD (rABP50) and 70 kD (rABP70), associated with 'Alu-repeat' DNA have been found.

Purification and crystallization of Shiga toxin from Shigella dysenteriae.

The protein toxin produced by Shigella dysenteriae consists of one enzymatically active A subunit of 293 amino acid residues and five B subunits of 69 amino acid residues that are involved with cell attachment. The holotoxin has been purified by blue Sepharose and chromatofocusing column chromatography. Two crystal forms of purified holotoxin have been grown by vapor diffusion.

Novel CDC2-related protein kinases produced in murine hematopoietic stem cells.

The polymerase chain reaction with degenerate primers was used for the amplification of cDNA encoding CDC2-related protein kinase (PK) sequences from murine hematopoietic stem cells. In total, nine different PK-encoding sequences were obtained. At least four of them encode previously unknown PKs.