E. A. Kubareva

Controlling the enzymatic activity of a restriction enzyme by light

For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest “photoswitch effect,” i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in Vmax rather than Km.

Functionalisation of calcium phosphate nanoparticles by oligonucleotides and their application for gene silencing

In molecular biology, the production of proteins can be effectively inhibited by introducing specific oligonucleotides into a living cell (gene silencing or antisense strategy; important for gene therapy). Calcium phosphate nanoparticles can serve as carriers for biomolecules in such therapeutic applications due to their high biocompatibility and biodegradability. Stable colloids were prepared by coating the inorganic nanoparticles with single- and double-stranded oligonucleotides. The dispersions were analysed by dynamic light scattering, zeta potential measurements, transmission electron microscopy, and scanning electron microscopy. Particles with a diameter of about 100 nm were obtained under optimized conditions. The efficiency of such nanoparticles to specifically inhibit protein synthesis was tested on HeLa-EGFP cells whose green fluorescence was turned off by the coated nanoparticles (gene silencing with siRNA). If siRNA was incorporated into the calcium phosphate particle and thereby protected from intracellular degradation, the transfection efficiency was significantly increased. The dispersions were stable and could be stored at 4 °C without loss of activity for several weeks, making them available as biochemical reagents.

PspGI, a Type II Restriction Endonuclease from the Extreme Thermophile Pyrococcus sp.: Structural and Functional Studies to Investigate an Evolutionary Relationship with Several Mesophilic Restriction Enzymes

We present here the first detailed biochemical analysis of an archaeal restriction enzyme. PspGI shows sequence similarity to SsoII, EcoRII, NgoMIV and Cfr10I, which recognize related DNA sequences. We demonstrate here that PspGI, like SsoII and unlike EcoRII or NgoMIV and Cfr10I, interacts with and cleaves DNA as a homodimer and is not stimulated by simultaneous binding to two recognition sites. PspGI and SsoII differ in their basic biochemical properties, viz. stability against chemical denaturation and proteolytic digestion, DNA binding and the pH, MgCl(2) and salt-dependence of their DNA cleavage activity. In contrast, the results of mutational analyses and cross-link experiments show that PspGI and SsoII have a very similar DNA binding site and catalytic center as NgoMIV and Cfr10I (whose crystal structures are known), and presumably also as EcoRII, in spite of the fact that these enzymes, which all recognize variants of the sequence -/CC-GG- (/ denotes the site of cleavage), are representatives of different subgroups of type II restriction endonucleases. A sequence comparison of all known restriction endonuclease sequences, furthermore, suggests that several enzymes recognizing other DNA sequences also share amino acid sequence similarities with PspGI, SsoII and EcoRII in the region of the presumptive active site. These results are discussed in an evolutionary context.

In vivo and in vitro analysis of 6S RNA-templated short transcripts in Bacillus subtilis

By differential high-throughput RNA sequencing (dRNA-seq) we have identified “product RNAs” (pRNAs) as short as 8-12 nucleotides that are synthesized by Bacillus subtilis RNA polymerase (RNAP) in vivo using the regulatory 6S-1 RNA as template. The dRNA-seq data were confirmed by in vitro transcription experiments and Northern blotting. In our libraries, we were unable to detect statistically meaningful numbers of reads potentially representing pRNAs derived from 6S-2 RNA. However, pRNAs could be synthesized in vitro from 6S-2 RNA as template by the B. subtilis σA RNAP. 6S-1 pRNA levels are low during exponential, increase in stationary, and burst during outgrowth from stationary phase, demonstrating that pRNA synthesis is a conserved regulatory mechanism, but a more dynamic and fine-tuning process than previously thought. Most pRNAs have a length of 8-15 nt, very few up to 24 nt. The average length of pRNAs tended to increase from stationary to outgrowth conditions. Synthesis of pRNA is initiated at C40 of 6S-1 RNA and U41 of 6S-2 RNA, yielding pRNAs with a 5’-terminal G or A residue, respectively. A B. subtilis 6S-1 RNA mutant strain encoding a pRNA with a 5’-terminal A residue showed the same relative distribution of ~ 14-nt pRNAs between the different growth states, but generally displayed lower pRNA levels than the reference strain encoding wild-type 6S-1 RNA. A ~ two-fold lower affinity of the C40U mutant 6S-1 RNA towards σA RNAP may have contributed to this reduction in pRNA levels. We infer that 6S-1 pRNA synthesis, although evolutionarily optimized for initiation with a +1G residue, is not primarily regulated at the transcription initiation level via growth phase-dependent variations in the cellular GTP pool.

DNA Polymerases β and λ Bypass Thymine Glycol in Gapped DNA Structures

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

Thymidine glycol: the effect on DNA molecular structure and enzymatic processing

Thymine glycol (Tg) in DNA is a biologically active oxidative damage caused by ionizing radiation or oxidative stress. Due to chirality of C5 and C6 atoms, Tg exists as a mixture of two pairs of cis and trans diastereomers: 5R cis-trans pair (5R,6S; 5R,6R) and 5S cis-trans pair (5S,6R; 5S,6S). Of all the modified pyrimidine lesions that have been studied to date, only thymine glycol represents a strong block to high-fidelity DNA polymerases in vitro and is lethal in vivo. Here we describe the preparation of thymine glycol-containing oligonucleotides and the influence of the oxidized residue on the structure of DNA in different sequence contexts, thymine glycol being paired with either adenine or guanine. The effect of thymine glycol on biochemical processing of DNA, such as biosynthesis, transcription and repair in vitro and in vivo, is also reviewed. Special attention is paid to stereochemistry and 5R cis-trans epimerization of Tg, and their relation to the structure of DNA double helix and enzyme-mediated DNA processing. Described here are the comparative structure and properties of other forms of pyrimidine base oxidation, as well as the role of Tg in tandem lesions.

Cross-linking of SsoII restriction endonuclease to cognate and non-cognate DNAs

Specific and non‐specific interactions SsoII restriction endonuclease (R·SsoII) were probed by the method of covalent attachment to modified DNA containing an active monosubstituted pyrophosphate internucleotide bond instead of a phosphodiester one. R·SsoII with six N‐terminal His residues was shown to be cross‐linked to duplexes with this type of modification, either containing or not the recognition sequence. Competition experiments with covalent attachment of R·SsoII to activated DNAs demonstrated the similar affinity of the enzyme to cognate and non‐cognate DNAs in the absence of cofactor, Mg2+ ions.

A model of restriction endonuclease MvaI in complex with DNA: A template for interpretation of experimental data and a guide for specificity engineering

R.MvaI is a Type II restriction enzyme (REase), which specifically recognizes the pentanucleotide DNA sequence 5′‐CCWGG‐3′ (W indicates A or T). It belongs to a family of enzymes, which recognize related sequences, including 5′‐CCSGG‐3′ (S indicates G or C) in the case of R.BcnI, or 5′‐CCNGG‐3′ (where N indicates any nucleoside) in the case of R.ScrFI. REases from this family hydrolyze the phosphodiester bond in the DNA between the 2nd and 3rd base in both strands, thereby generating a double strand break with 5′‐protruding single nucleotides. So far, no crystal structures of REases with similar cleavage patterns have been solved. Characterization of sequence‐structure‐function relationships in this family would facilitate understanding of evolution of sequence specificity among REases and could aid in engineering of enzymes with new specificities. However, sequences of R.MvaI or its homologs show no significant similarity to any proteins with known structures, thus precluding straightforward comparative modeling. We used a fold recognition approach to identify a remote relationship between R.MvaI and the structure of DNA repair enzyme MutH, which belongs to the PD‐(D/E)XK superfamily together with many other REases. We constructed a homology model of R.MvaI and used it to predict functionally important amino acid residues and the mode of interaction with the DNA. In particular, we predict that only one active site of R.MvaI interacts with the DNA target at a time, and the cleavage of both strands (5′‐CCAGG‐3′ and 5′‐CCTGG‐3′) is achieved by two independent catalytic events. The model is in good agreement with the available experimental data and will serve as a template for further analyses of R.MvaI, R.BcnI, R.ScrFI and other related enzymes. Proteins 2007.

Specific conjugation of DNA binding proteins to DNA templates through thiol–disulfide exchange

The double‐stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence‐specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems.

Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs—commonalities and differences

Bacterial 6S RNAs bind to the housekeeping RNA polymerase (σ(A)-RNAP in Bacillus subtilis) to regulate transcription in a growth phase-dependent manner. B. subtilis expresses two 6S RNAs, 6S-1 and 6S-2 RNA, with different expression profiles. We show in vitro that 6S-2 RNA shares hallmark features with 6S-1 RNA: Both (1) are able to serve as templates for pRNA transcription; (2) bind with comparable affinity to σ(A)-RNAP; (3) are able to specifically inhibit transcription from DNA promoters, and (4) can form stable 6S RNA:pRNA hybrid structures that (5) abolish binding to σ(A)-RNAP. However, pRNAs of equal length dissociate faster from 6S-2 than 6S-1 RNA, owing to the higher A,U-content of 6S-2 pRNAs. This could have two mechanistic implications: (1) Short 6S-2 pRNAs (<10 nt) dissociate faster instead of being elongated to longer pRNAs, which could make it more difficult for 6S-2 RNA-stalled RNAP molecules to escape from the sequestration; and (2) relative to 6S-1 RNA, 6S-2 pRNAs of equal length will dissociate more rapidly from 6S-2 RNA after RNAP release, which could affect pRNA turnover or the kinetics of 6S-2 RNA binding to a new RNAP molecule. As 6S-2 pRNAs have not yet been detected in vivo, we considered that cellular RNAP release from 6S-2 RNA might occur via 6S-1 RNA displacing 6S-2 RNA from the enzyme, either in the absence of pRNA transcription or upon synthesis of very short 6S-2 pRNAs (∼ 5-mers, which would escape detection by deep sequencing). However, binding competition experiments argued against these possibilities.