Biomedical subjects

O I Lavrik

Publications and source records attributed to O I Lavrik.

At least 19 recordsLinked to original sources

PubMed2003

A new binary system for photosensitized labeling of DNA polymerases in nuclear extract.

A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3;-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5;-phosphate) at the 5;-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase beta labeling.

Animals↗

PubMed2003

Interaction of flap endonuclease-1 and replication protein A with photoreactive intermediates of DNA repair.

A new method for enzymatic synthesis of radioactive DNA flapped structures containing a photoreactive dCMP moiety at a branch point with 4-(4-azido-2,3,5,6-tetrafluorobenzylidene-hydrazinocarbonyl)butylcarbamoyl group attached at exo-N-position of cytosine was developed. The formation of complexes of flap endonuclease-1 (FEN-1) with flapped DNA was shown by photoaffinity modification and gel retardation assays. The substrate properties of the flapped structures with different flap lengths were studied in the reaction of endonuclease cleavage catalyzed by FEN-1. It was demonstrated that inhibition of FEN-1 activity by replication protein A (RPA) depends on the length of the single-stranded part of the flapped substrate. A significant inhibition of cleavage was observed when the flap length was sufficient for effective RPA binding, while for structures with short single-stranded part the efficiency of cleavage was independent of the presence of RPA. FEN-1 and RPA were modified by photoaffinity labeling using flap structures with single-stranded parts consisting of 8 and 21 nucleotides. Products of DNA photoattachment to FEN-1 were observed in both cases, while the covalent adducts with RPA were obtained only with the 21-nucleotide-long flap. Photoaffinity modification demonstrated that FEN-1 and RPA compete for the binding of the flapped substrates with long single-stranded parts.

Base Sequence↗

PubMed2002

Highly efficient labeling of DNA polymerases by a binary system of photoaffinity reagents.

A binary system of photoaffinity reagents was proposed earlier for highly efficient labeling of DNA polymerases by 5;-[32P]DNA primers. In the present study we demonstrate the feasibility of this approach to increase the efficiency of DNA polymerase labeling. A photoactive 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was incorporated at the 3;-end of 5;-[32P]DNA primers synthesized by DNA polymerase beta or Tte in the presence of one of the dTTP analogs--FAB-4-dUTP, FAB-9-dUTP, or FAB-4-ddUTP. The reaction mixture was irradiated by light with wavelength of 334-365 nm (direct labeling) or 365-450 nm in the presence of photosensitizer, one of dTTP analogs containing a pyrene moiety, Pyr-6-dUTP or Pyr-8-dUTP. In the case of the binary system of photoaffinity reagents, a FAB group is activated by energy transfer from sensitizer localized in the dNTP-binding site of DNA polymerase in the triple complex, comprised by reagent, DNA polymerase, and Pyr-6(8)-dUTP. Direct activation of the FAB group under these conditions is negligible. The most efficient photolabeling of DNA polymerases was observed with a primer containing a FAB-4-dUMP group at the 3;-end, and Pyr-6-dUTP as a photosensitizer. Using 10-fold molar excess of photoreagent to DNA polymerase beta, the labeling efficiency was shown to achieve 60%, which is 2-fold higher than the efficiency of the direct DNA polymerase labeling under harsher conditions (334-365 nm).

DNA Polymerase beta↗

PubMed2001

A binary system of photoreagents for high-efficiency labeling of DNA polymerases.

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.

DNA Primers↗

PubMed2001

Replication protein A modulates its interface with the primed DNA template during RNA-DNA primer elongation in replicating SV40 chromosomes.

The eukaryal single-stranded DNA binding protein replication protein A (RPA) binds short oligonucleotides with high affinity but exhibits low cooperativity in binding longer templates, opposite to prokaryal counterparts. This discrepancy could reflect the smaller size of the replicative template portion availed to RPA. According to current models, this portion accommodates an RNA-DNA primer (RDP) of <40 nt (nested discontinuity) or a several-fold longer Okazaki fragment (initiation zone). Previous in situ UV-crosslinking revealed that RPA also interacts with nascent DNA, especially growing RDPs. Here we compare nascent SV40 DNA chains UV-crosslinked to the middle and large RPA subunits and use the data to re-examine the two models. The middle subunit interacted with the nascent chains after a few DNA residues were added to the RNA primer while the large subunit became accessible after extension by several more. Upon RDP maturation, the middle subunit disengaged while the large subunit remained accessible during further limited extension. A corresponding shift in preference in favor of the large subunit has been reported for purified RPA and synthetic gapped duplexes upon reduction of the gap from 19 to 9 nt. Combined, these facts support the proposal that the mature RDP faces downstream a correspondingly small gap, possibly created by removal of the RNA primer moiety from an adjacent, previously synthesized RDP (nested discontinuity) but insufficient for continuous elongation of the RDP into an Okazaki fragment (initiation zone).

Animals↗

PubMed2001

DNA polymerase beta -mediated long patch base excision repair. Poly(ADP-ribose)polymerase-1 stimulates strand displacement DNA synthesis.

Recently, photoaffinity labeling experiments with mouse cell extracts suggested that PARP-1 functions as a surveillance protein for a stalled BER intermediate. To further understand the role of PARP-1 in BER, we examined the DNA synthesis and flap excision steps in long patch BER using a reconstituted system containing a 34-base pair BER substrate and five purified human enzymes: uracil-DNA glycosylase, apurinic/apyrimidinic endonuclease, DNA polymerase beta, flap endonuclease-1 (FEN-1), and PARP-1. PARP-1 stimulates strand displacement DNA synthesis by DNA polymerase beta in this system; this stimulation is dependent on the presence of FEN-1. PARP-1 and FEN-1, therefore, cooperate to activate long patch BER. The results are discussed in the context of a model for BER sub-pathway choice, illustrating a dual role for PARP-1 as a surveillance protein for a stalled BER intermediate and an activating factor for long patch BER DNA synthesis.

Amino Acid Substitution↗

PubMed2001

Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair.

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.

Animals↗

PubMed2001

Determination of tRNA(Phe) nucleotides contacting the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase by photoaffinity crosslinking.

The nucleotides of tRNA(Phe) interacting with the subunits of Thermus thermophilus phenylalanyl-tRNA synthetase (the alpha(2)beta(2) heterotetramer) have been determined by photoaffinity crosslinking of randomly s(4)U-monosubstituted tRNA(Phe) transcripts which retain aminoacylation parameters closely similar to those of the native tRNA(Phe). The thiolated transcripts have been fractionated by affinity electrophoresis and separately crosslinked to the enzyme. Sites of crosslinking to the beta subunit have been identified at positions 33 and 39 and crosslinking sites to the alpha subunit have been localized at positions 20, 45 and 47, using alkaline hydrolysis analysis of the crosslinked proteinase K-treated tRNAs. An additional crosslink to the beta subunit, not identified in the full-length crosslinked tRNAs, has been deduced to occur at position 12, based on the analysis of an unusual (fast migrating) crosslinked product. Nucleotide s(4)U8 of native tRNA(Phe) has been shown to form a minor crosslink to the alpha subunit. Four of the seven crosslinking sites, namely nucleotides 8, 12, 20 and 39, are among those shown to be protected against cleavage by iodine in footprinting experiments; in contrast, only nucleotide 12 is among the contact sites defined in the crystal structure. The data of independent biochemical approaches strongly suggest conformational flexibility of the complex under functional conditions, thus reflecting the importance of macromolecular dynamics for the interaction.

Binding Sites↗

PubMed2001

Polarity of human replication protein A binding to DNA.

Replication protein A (RPA), the nuclear single-stranded DNA binding protein is involved in DNA replication, nucleotide excision repair (NER) and homologous recombination. It is a stable heterotrimer consisting of subunits with molecular masses of 70, 32 and 14 kDa (p70, p32 and p14, respectively). Gapped DNA structures are common intermediates during DNA replication and NER. To analyze the interaction of RPA and its subunits with gapped DNA we designed structures containing 9 and 30 nucleotide gaps with a photoreactive arylazido group at the 3'-end of the upstream oligonucleotide or at the 5'-end of the downstream oligonucleotide. UV crosslinking and subsequent analysis showed that the p70 subunit mainly interacts with the 5'-end of DNA irrespective of DNA structure, while the subunit orientation towards the 3'-end of DNA in the gap structures strongly depends on the gap size. The results are compared with the data obtained previously with the primer-template systems containing 5'- or 3'-protruding DNA strands. Our results suggest a model of polar RPA binding to the gapped DNA.

Azides↗

PubMed2001

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs.

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5'-flap strands of the branched DNA structure and possesses 5'-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3'-end of the primer were synthesized in situ by the action of DNA polymerase beta using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5'-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5'- and 3'-ends of the nick or with primer-template systems containing 5'- or 3'-protruding DNA strands was shown. Formation of a structure with the 5'-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3'-end and the apurine/apyrimidine site at the 5'-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.

Animals↗

PubMed2001

Investigation of the dNTP-binding site of HIV-1 reverse transcriptase using photoreactive analogs of dNTP.

The interaction of dNTPs with the active site of HIV-1 reverse transcriptase (HIV RT) has been investigated. The kinetic parameters of primer elongation catalyzed by wild-type HIV-1 RT and two of its mutants with substitutions for Tyr115 using dTTP and two of its photoreactive analogs were determined. The substitution for Tyr115 with alanine or tryptophan resulted in an increase in K(m) values of dTTP and its analogs. Wild-type RT and its mutants were photoaffinity modified using photoreactive primer synthesized in situ. The modification was made in two variants: direct photocross-linking under UV irradiation and photosensitized modification using Pyr-dUTP as a sensitizer. The use of the sensitizer decreased the number of modification products and increased selective labeling of the catalytic subunit of both the mutant and wild-type RT.

Alanine↗

PubMed2000

Study of interaction of human replication factor A with DNA using new photoreactive analogs of dTTP.

Replication factor A (RPA) is a protein that binds single-stranded DNA in eukaryotic cells; it participates in replication, repair, and recombination of DNA. RPA is composed of three subunits with molecular masses 70 (p70), 32 (p32), and 14 kD (p14). The photoaffinity labeling method was used to study the interaction of RPA with the 3;-end of duplex DNA containing extended 5;-end of a single strand. We have synthesized dTTP analogs containing photoreactive 2,3,5,6-tetrafluoro-4-azidobenzoyl group attached to the 5th position of the uracil residue with linkers of variable length (9, 11, and 13 atom chains). Using these analogs and dTTP analog containing the same photoreactive residue attached to the 5th position of the uracil residue with a 4-atom linker, a number of oligonucleotide primers carrying a single photoreactive group on the 3;-end were enzymatically synthesized. Using the complex of the photoreactive primers with DNA template containing extended 19-base 5;-end, human RPA was photoaffinity modified. The primers were covalently bound to the p70 and p32 subunits of RPA and the p14 subunit was not labeled by the primers. The data are discussed considering the previously suggested model of interaction of RPA with DNA during replication.

Base Sequence↗

PubMed2000

Highly selective affinity labeling of DNA-polymerase from Thermus thermophilus B35 by a binary system of photoreactive agents.

The thermostable DNA-polymerase from Thermus thermophilus B35 (Tte-polymerase) was affinity labeled by a binary system of photoreagents comprising base-substituted TTP analogs. The 5;-[32P]-labeled primer was elongated by Tte-polymerase in the presence of a TTP analog containing the photoreactive 2,3,5, 6-tetrafluoro-4-azidobenzoyl group (FAB-4-dUTP). Then the reaction mixture was UV-irradiated (365-450 nm) in the presence or the absence of a photosensitizer (TTP analog containing a pyrene moiety, Pyr-dUTP). The initial rate of the Pyr-dUTP-sensitized photomodification was almost 10-fold higher than the rate of direct photomodification (in the absence of Pyr-dUTP); in the case of the sensitized modification, the product of covalent cross-linking of the photoreactive primer with Tte-polymerase was apparently homogenous according to the data of electrophoresis. The enzyme was protected from the photosensitized modification by dNTP. To confirm the selectivity of the photosensitized modification of Tte-polymerase, another DNA-binding protein (human replication factor A, RPA) was added to the reaction mixture. In the presence of the photosensitizer (Pyr-dUTP), RPA was not labeled and only Tte-polymerase was modified, whereas in the case of direct modification, Tte-polymerase and the p32 and p70 subunits of RPA were labeled. The suggested method enables highly selective affinity modification of DNA-polymerases.

Affinity Labels↗

PubMed2000

[A photoreactive analogue of 2'3'-dideoxyuridine 5'-triphosphate: preparation and use for photoaffinity modification of human replication protein A].

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2',3'-dideoxyuridine 5'-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2'-deoxyuridine 5'-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase beta and to terminate DNA synthesis. A 5'-32P-labeled primer with a photoreactive group at the 3'-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.

DNA-Binding Proteins↗

PubMed2000

Phenylalanyl-tRNA synthetase interacts with DNA: studies on activity using deoxyribooligonucleotides.

Phenylalanyl-tRNA synthetase from Thermus thermophilus complexes with short (up to 30 nucleotide length) single-stranded DNA fragments more efficiently than with double-stranded fragments. The complexing between DNA and the protein significantly increases with deoxyribooligonucleotide longer than 20 nucleotides. Using affinity labeling, the binding site of DNA was located near the interface of the alpha- and beta-subunits. The binding sites of DNA and tRNAPhe do not overlap. Phenylalanyl-tRNA synthetase from E. coli also binds DNA.

DNA↗

PubMed2000

Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates.

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.

Cations, Divalent↗

PubMed2000

Non-canonical functions of aminoacyl-tRNA synthetases.

The basic function of aminoacyl-tRNA synthetases (aaRSs) is an activation of the amino acids and their transfer to specific RNAs. In addition to this key role in protein biosynthesis, the enzymes of this group participate in other cell processes. aaRSs regulate the expression of some genes, not only their own. The regulation is carried out on the level of transcription, processing of mRNA, and translation. They catalyze the synthesis of dinucleoside oligophosphates and thus indirectly influence many other cell functions. Tyrosyl-tRNA synthetase was shown to have cytokine activities. Some aaRSs interact specifically with other proteins. Thus, aminoacyl-tRNA synthetases are involved in the regulation of the multiple processes of living cells. In this review we summarize the available data on unusual activities of aaRSs.

Amino Acyl-tRNA Synthetases↗

PubMed2000

Interaction of T. thermophilus phenylalanyl-tRNA synthetase with the 3'-terminal nucleotide of tRNAPhe.

The interaction of Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) with the 3;-terminal nucleotide of tRNAPhe has been studied by affinity labeling to solve the problem arising from X-ray crystallographic study: the binding sites of phenylalanine and the 3;-terminal nucleotide base were revealed to be identical in the crystal structures of PheRS complexed with the substrates. tRNAPhe derivatives containing a photoreactive 4-thiouridine (tRNAPhe-s4U-76) or 6-thioguanosine residue (tRNAPhe-s6G-76) in the 3;-end have been prepared using terminal tRNA nucleotidyl transferase. Kinetic measurements of aminoacylation provide evidence for a functional role of base-specific interactions of the 3;-terminal adenosine in productive interaction of tRNAPhe with the enzyme: tRNAPhe-s4U-76 cannot be aminoacylated; the replacement of A-76 with s6G results in a 370-fold reduction of catalytic efficiency of aminoacylation mainly due to decreased Vmax value. Relative cross-linking of the s6G-substituted tRNA to the alpha-subunit (69% of the total yield of the cross-linked alpha- and beta-subunits) is two times higher as compared to the cross-linking of tRNAPhe-s4U-76. The dialdehyde derivative, tRNAPhe-Aox-76, with periodate-oxidized 3;-terminal ribose is cross-linked with the same selectivity to the alpha-subunit as tRNAPhe-s6G-76. The results suggest specific binding of the 3;-terminal nucleotide of tRNAPhe by the catalytic subunit of PheRS in the absence of other substrates. Comparative analysis of the cross-linked products in the absence and in the presence of small substrates revealed ATP and aminoacyl-adenylate to effect the interaction of the tRNAPhe acceptor end with PheRS. The correct positioning of the 3;-terminal nucleotide of tRNAPhe corresponding to the structure of the productive complex with PheRS is therefore promoted only in the presence of all three substrates.

Affinity Labels↗