Anna Maciaszek
Polish Academy of Sciences
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Featured researches published by Anna Maciaszek.
FEBS Letters | 1998
Maria Koziołkiewicz; Anna Maciaszek; Wojciech J. Stec; Dmitry G. Semizarov; Lyubov S. Victorova; Alexander A. Krayevsky
Phosphorothioate analogues of oligonucleotides (PS‐oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [RP] configuration, while the presence of the linkage(s) of the [SP] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3′‐end. The presence of [SP] linkage at this position strongly reduces the enzyme activity while the [RP] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.
Bioorganic Chemistry | 2014
Paulina Bartos; Anna Maciaszek; Anna Rosinska; Elzbieta Sochacka; Barbara Nawrot
The newly discovered S-geranylated 2-thiouridines (geS2U) (Dumelin et al., 2012) and 2-selenouridines (Se2U) were recently shown to be synthesized by a single enzyme (selenouridine synthase, SelU) through two distinct pathways using the same 2-thiouridine substrate (S2U); however, no clear catalytic mechanism was proposed. We suggest that S-geranyl-2-thiouridine is an intermediate of the SelU-catalyzed conversion of S2U to Se2U. The successful chemical transformation of S2U→geS2U→Se2U is demonstrated here as an initial approximation of the intracellular pathway. The structure of Se2U was confirmed by spectroscopic methods, which included, for the first time, (77)Se NMR data (δ 354ppm).
Journal of Biological Chemistry | 2010
Paul A. Rowley; Aashiq H. Kachroo; Chien Hui Ma; Anna Maciaszek; Piotr Guga; Makkuni Jayaram
The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3′-5′ phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5′-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.
Nucleic Acids Research | 2010
Aashiq H. Kachroo; Chien Hui Ma; Paul A. Rowley; Anna Maciaszek; Piotr Guga; Makkuni Jayaram
Two conserved catalytic arginines, Arg-173 and Arg-292, of the tyrosine site-specific recombinase Cre are essential for the transesterification steps of strand cleavage and joining in native DNA substrates containing scissile phosphate groups. The active site tyrosine (Tyr-324) provides the nucleophile for the cleavage reaction, and forms a covalent 3′-phosphotyrosyl intermediate. The 5′-hydroxyl group formed during cleavage provides the nucleophile for the joining reaction between DNA partners, yielding strand exchange. Previous work showed that substitution of the scissile phosphate (P) by methylphosphonate (MeP) permits strand cleavage by a Cre variant lacking Arg-292. We now demonstrate that MeP activation and cleavage are not blocked by substitution of Arg-173 or even simultaneous substitutions of Arg-173 and Arg-292 by alanine. Furthermore, Cre(R173A) and Cre(R292A) are competent in strand joining, Cre(R173A) being less efficient. No joining activity is detected with Cre(R173A, R292A). Consistent with their ability to cleave and join strands, Cre(R173A) and Cre(R292A) can promote recombination between two MeP-full-site DNA partners. These findings shed light on the overall contribution of active site electrostatics, and tease apart distinctive contributions of the individual arginines, to the chemical steps of recombination. They have general implications in active site mechanisms that promote important phosphoryl transfer reactions in nucleic acids.
Phosphorus Sulfur and Silicon and The Related Elements | 2008
Wojciech J. Stec; Anna Maciaszek; Piotr Guga
Selenium-labelled macromolecules can be effectively analyzed by X-ray crystallography due to the phenomenon of multi-wavelength anomalous dispersion (MAD). The originally developed nucleoside 2-selena-1,3,2-oxathiaphospholane derivatives allow for synthesis of fully modified, stereodefined oligomers (up to approximately 15-mers) with any combination of internucleotide phosphoroselenoate linkages of R P or S P absolute configuration as well as unmodified phosphate bonds, which, in general, cannot be achieved using enzymatic methods.
Nucleic Acids Research | 2015
Paul A. Rowley; Aashiq H. Kachroo; Chien Hui Ma; Anna Maciaszek; Piotr Guga; Makkuni Jayaram
Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5′ to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.
RSC Advances | 2018
Agnieszka Tomaszewska-Antczak; Katarzyna Jastrzębska; Anna Maciaszek; Barbara Mikołajczyk; Piotr Guga
Enantiomerically pure, protected acyclic nucleosides of the GNA type (glycol nucleic acids) (GN′), obtained from (R)-(+)- and (S)-(−)-glycidols and the four canonical DNA nucleobases (Ade, Cyt, Gua and Thy), were transformed into 3′-O-DMT-protected 2-thio-4,4-pentamethylene-1,3,2-oxathiaphospholane derivatives (OTP-GN′) containing a second stereogenic center at the phosphorus atom. These monomers were chromatographically separated into P-diastereoisomers, which were further used for the synthesis of P-stereodefined “dinucleoside” phosphorothioates GNPST (GN = GA, GC, GG, GT). The absolute configuration at the phosphorus atom for all eight GNPST was established enzymatically and verified chemically, and correlated with chromatographic mobility of the OTP-GN′ monomers on silica gel. The GNPS units (derived from (R)-(+)-glycidol) were introduced into self-complementary PS-(DNA/GNA) octamers of preselected, uniform absolute configuration at P-atoms. Thermal dissociation experiments showed that the thermodynamic stability of the duplexes depends on the stereochemistry of the phosphorus centers and relative arrangement of the GN units in the oligonucleotide strands. These results correlate with the changes of conformation assessed from circular dichroism spectra.
Archive | 2016
Piotr Guga; Anna Maciaszek
This chapter covers selected papers published in 2014 on chemical synthesis and biological application of modified nucleotides, including data on prodrugs and antiviral compounds of this class. Nucleotides are the building blocks of nucleic acids and, either alone or conjugated with other biomolecules, play fundamental roles in biological processes. Their analogues are also expected to be useful tools for tuning of natural biochemical pathways (including medical applications) and for the investigation of intracellular nucleotide-related processes at a molecular level. There is also a developing field of fluorescently labelled nucleotide-like probes for precise monitoring of bioprocesses at nanomolar levels.
Molecular Pharmacology | 2001
Luba Benimetskaya; Paul S. Miller; Simon Benimetsky; Anna Maciaszek; Piotr Guga; Serge L. Beaucage; Andrzej Wilk; Andrzej Grajkowski; Anthony L. Halperin; C. A. Stein
Antisense & Nucleic Acid Drug Development | 1997
Wojciech J. Stec; Czesław S. Cierniewski; Andrzej Okruszek; Anna Kobylańska; Zofia Pawłowska; Maria Koziołkiewicz; Elżbieta Pluskota; Anna Maciaszek; Beata Rębowska; Marta Stasiak