R. V. Polozov

Non-random DNA fragmentation in next-generation sequencing

Next Generation Sequencing (NGS) technology is based on cutting DNA into small fragments, and their massive parallel sequencing. The multiple overlapping segments termed “reads” are assembled into a contiguous sequence. To reduce sequencing errors, every genome region should be sequenced several dozen times. This sequencing approach is based on the assumption that genomic DNA breaks are random and sequence-independent. However, previously we showed that for the sonicated restriction DNA fragments the rates of double-stranded breaks depend on the nucleotide sequence. In this work we analyzed genomic reads from NGS data and discovered that fragmentation methods based on the action of the hydrodynamic forces on DNA, produce similar bias. Consideration of this non-random DNA fragmentation may allow one to unravel what factors and to what extent influence the non-uniform coverage of various genomic regions.

Binding Regularities in Complexes of Transcription Factors with Operator DNA: Homeodomain Family

Abstract In order to disclose general regularities of binding in homeodomain-DNA complexes we considered five of them and extended the observed regularities over the entire homeodomain family. The five complexes have been selected by similarity of protein structures and patterns of contacting residues. Their long range interactions and interfaces were compared. The long-range stage of the recognition process was characterized by electrostatic potentials about 5 A away from molecular surfaces of protein or DNA. For proteins, clear positive potential is displayed only at the side contacting the DNA. The double-chained DNA molecule displays a rather strong negative potential, especially in their grooves. Thus, a functional role of electrostatics is a guiding of the protein into the DNA major groove, so the protein and DNA could form a loose non-specific complex. At the close-range stage, neutralization of the phosphate charges by positively charged residues is necessary for decreasing the strong electrostatic potential of DNA, allowing nucleotide bases to participate in the formation of protein-DNA atomic contacts in the interface. The recognizing ?-helix of protein was shown to form both invariant and variable groups of contacts with DNA by means of certain specific side groups. The invariant contacts included highly specific protein-DNA hydrogen bonds between asparagine and adenine, nonpolar contacts of hydrophobic amino acids serving as a stereochemical barrier for fixing the protein factor on DNA, and an interface cluster of water molecules providing local conformational mobility necessary for the dissociation process. There is a unique water molecule within the interface that is conservative and located at the interface center. Invariant contacts of the proteins are mostly formed with the TAAT motif of the promoter DNA forward strand. While the invariant contacts specify the family of homeodomains, the variable contacts that are formed with the reverse strand of DNA provide specificity of individual complexes within the homeodomain family.

Recognition rules for binding of Zn-Cys2His2 transcription factors to operator DNA

The molecules of Zn-finger transcription factors consist of several similar small protein units. We analyzed the crystal structures 46 basic units of 22 complexes of Zn-Cys2His2 family with the fragments of operator DNA. We showed that the recognition of DNA occurs via five protein contacts. The canonical binding positions of the recognizing α-helix were −1, 3, 6, and 7, which make contacts with the tetra-nucleotide sequence ZXYZ of the coding DNA strand; here the canonical binding triplet is underlined. The non-coding DNA strand forms only one contact at α-helix position 2. We have discovered that there is a single highly conservative contact His7α with the phosphate group of nucleotide Z, which precedes each triplet XYZ of the coding DNA chain. This particular contact is invariant for the all Zn-Cys2His2 family with high frequency of occurrence 83%, which we considered as an invariant recognition rule. We have also selected a previously unreported Zn-Cys2His2-Arg subfamily of 21 Zn-finger units bound with DNA triplets, which make two invariant contacts with residues Arg6α and His7α with the coding DNA chain. These contacts show frequency of occurrence 100 and 90%, and are invariant recognition rule. Three other variable protein-DNA contacts are formed mainly with the bases and specify the recognition patterns of individual factor units. The revealed recognition rules are inherent for the Zn-Cys2His2 family and Zn-Cys2His2-Arg subfamily of different taxonomic groups and can distinguish members of these families from any other family of transcription factors.

Recognition Rules for Binding of Homeodomains to Operator DNA

Abstract The spatial arrangement of interfaces between homeodomain transcription factors and operator DNA has been considered. We analyzed the binding contacts for a representative set of 22 complexes of homeodomain transcription factors with a double-stranded operator DNA in the region of the major groove. It was shown that the recognition of DNA by the recognizing α-helix of protein is governed by two contact groups. Invariant protein-DNA group of contacts includes six contacts, formed by atomic groups of coding and non-coding DNA chains with the groups of amino acids. The recognizing α-helix forms contacts by polar groups of residues Trp2 (NE1), Asn5, and Lys9 with the canonical sequence T1A2A3T4 of the coding DNA chain, and contacts by residues Lys0, Arg7 and Lys11 with the sequence A4X5X6X7 of a non-coding DNA chain, where X is any nucleotide. Variable protein-DNA group of contacts comprises two groups bound with the sequence T3A4X5X6 of the non-coding DNA-chain. These contacts are mainly with the bases and specify the binding pattern of individual homeodomains. The invariant contact group represents a recognition pattern for transcription factors of the homeodomain family: multiple adenine-asparagine contact and six position-specific phosphate contacts mainly with lysine or arginine. Within this group, we have found three most significant invariant contacts which allow deducing the recognition rules for homeodomains. These rules are inherent for different taxonomic groups of the homeodomain family and can distinguishing members of this family from any other family of transcription factors.

Ultrasonic Cleavage of DNA: Quantitative Analysis of Sequence Specificity

Looking for new means of assessing local conformational and dynamic heterogeneities in DNA structure, we have estimated the rates of phosphodiester bond cleavage in DNA fragments of known sequence caused by ultrasonic treatment. Among the 16 dinucleotide steps possible, those with 5′-ward cytosine [5′-d(CpN)-3′] are distinguished by significantly higher cleavage rates: CG > CA = CT > CC. The possible causes of this intriguing phenomenon are considered.

The entropic nature of protein thermal stabilization

We performed thermodynamic analysis of temperature-induced unfolding of mesophilic and thermophilic proteins. It was shown that the variability in protein thermostability associated with pH-dependent unfolding or linked to the substitution of amino acid residues on the protein surface is evidence of the governing role of the entropy factor. Numerical values of conformational components in enthalpy, entropy and free energy which characterize protein unfolding in the “gas phase” were obtained. Based on the calculated absolute values of entropy and free energy, a model of protein unfolding is proposed in which the driving force is the conformational entropy of native protein, as an energy of the heat motion (T·SNC) increasing with temperature and acting as an factor devaluating the energy of intramolecular weak bonds in the transition state.

Ultrasonic cleavage of DNA in complexes with Ag(I), Cu(II), Hg(II)

We investigated a phenomenon of ultrasonic cleavage of DNA complexed with transition metal cations Ag(I), Cu(II) and Hg(II). We found the statistically significant dependence of relative intensity of cleavage on cation type and concentration. Each cation may cause two different types of distortion in the DNA double-helix depending on whether it binds to major or minor DNA groove. The intensity of ultrasonic cleavage decreases where the cation binds to the major DNA groove; the intensity of cleavage increases where the cation binds to the minor DNA groove and disturbs the hydrogen bonds of complementary base pairs or where it intercalates between bases. Both types of DNA distortion can affect the intensity of N↔S intercon-version of deoxyribose.

Thermodynamics of globular proteins

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the ‘gas phase’. It was shown that specific values of the energy of weak intramolecular bonds (∆Hint), conformational free energy (∆Gconf) and entropy (∆Sconf) are the same for proteins with molecular weight 7–25 kDa. Using the energy value (∆Hint) and the proposed approach for estimation of the conformational entropy of native protein (SNC), numerical values of the absolute free energy (GNC) were obtained.

DNA-Based Nanostructures: Changes of Mechanical Properties of DNA upon Ligand Binding

The formation of DNA-based nanostructures involves the binding of different kinds of ligands to DNA as well as the interaction of DNA molecules with each other. Complex formation between ligand and DNA can alter physicochemical properties of the DNA molecule. In the present work, the accessibility of DNA-ligand complexes to cleavage by DNase I are considered, and the exact algorithms for analysis of diagrams of DNase I footprinting for ligand-DNA complexes are obtained. Changes of mechanical properties of the DNA upon ligand binding are also demonstrated by the cleavage patterns generated upon ultrasound irradiation of cis-platin-DNA complexes. Propagation of the mechanical perturbations along DNA in the presence of bound ligands is considered in terms of a string model with a heterogeneity corresponding to the position of a bound ligand on DNA. This model can reproduce qualitatively the cleavage patterns obtained upon ultrasound irradiation of cis-platin-DNA complexes.

Quantitative Analysis of Electrophoresis Data - Application to Sequence-Specific Ultrasonic Cleavage of DNA

The complete genomes of many different species are now being revealed in ever increasing pace. The impressive progress made in genome sequencing was largely attributed to development of high resolution denaturing polyacrylamide gel electrophoresis (PAGE). Next-generation sequencing platforms use new powerful technologies, providing gigabases of genetic information in a single run (Farias-Hesson et al., 2010). Nevertheless, scientific research often deals with situations when one needs to change the experimental conditions or the data analysis protocols, but commercial available devices and programs don’t give such an opportunity. We have faced this problem during the research focused on the phenomenon of sequence specific ultrasonic cleavage of double-stranded (ds) DNA (Grokhovsky, 2006). The observed sequence dependence of DNA cleavage efficiency was quite surprising. It seems that sequence-specificity of ultrasonic cleavage reflects the local variations in DNA structural dynamics. Thus, ultrasound may provide a basis for developing a new method for studying sequence effects on local structural dynamics of DNA fragments.