Poly(ADP-ribose) Polymerase-1 Inhibits Strand-Displacement Synthesis of DNA Catalyzed by DNA Polymerase β

Poly(ADP-ribose) polymerase-1 (PARP-1), a eucaryotic nuclear DNA-binding protein that is activated by breaks in DNA chains, may be involved in the base excision repair (BER) because DNAs containing single-stranded gaps and breaks are intermediates of BER. The effect of PARP-1 on the DNA synthesis catalyzed in vitro by DNA polymerase beta (pol beta) was studied using analogs of DNA substrates produced during BER and imitating intermediates of the short patch and long patch subpathways of BER. Oligonucleotide duplexes of 34 bp that contained a mononucleotide gap or a single-strand break with tetrahydrofuran phosphate or phosphate at the 5;-end of the downstream oligonucleotide were taken as DNA substrates. The efficiency of DNA synthesis was determined at various ratios of pol beta and PARP-1. The efficiency of gap filling was decreased in the presence of PARP-1, but strand-displacement DNA synthesis was inhibited significantly stronger, which seemed to be due to competition between PARP-1 and pol beta for DNA. In the presence of NAD+ and single-strand breaks in DNA, PARP-1 catalyzes the synthesis of poly(ADP-ribose) covalently attached to the enzyme, and this automodification is thought to provide for dissociation of PARP-1 from DNA. The effect of PARP-1 automodification on inhibition of DNA synthesis was studied, and efficiency of mononucleotide gap filling was shown to be restored, but strand-displacement synthesis did not revert to the level observed in the absence of PARP-1. PARP-1 is suggested to regulate the interaction between pol beta and DNA, in particular, via its own automodification.     

Role of Low-Molecular-Weight Substrates in Functional Binding of the tRNAPhe Acceptor End by Phenylalanyl-tRNA Synthetase

The functional roles of phenylalanine and ATP in productive binding of the tRNA(Phe) acceptor end have been studied by photoaffinity labeling (cross-linking) of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNA(Phe) analogs containing the s(4)U residue in different positions of the 3'-terminal single-stranded sequence. Human and E. coli tRNA(Phe)s used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study. Destabilization of the complex with human tRNA(Phe) caused by replacement of three recognition elements decreases selectivity of labeling of the alpha- and beta-subunits responsible for the binding of adjacent nucleotides of the CCA-end. Phenylalanine affects the positioning of the base and ribose moieties of the 76th nucleotide, and the recorded effects do not depend on structural differences between bacterial and eukaryotic tRNA(Phe)s. Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s(4)U76-substituted analog of human tRNA(Phe) (tRNA(Phe)-s(4)U76) than with E. coli tRNA(Phe)-s(4)U76: in the first case the labeling of the alpha-subunits is inhibited more effectively; the labeling of the beta-subunits is inhibited in the first case and increased in the second case. The findings analyzed with respect to available structural data on the enzyme complexes with individual substrates suggest that the binding of phenylalanine induces a local rearrangement in the active site and directly controls positioning of the tRNA(Phe) 3'-terminal nucleotide. The effect of ATP on the acceptor end positioning is caused by global structural changes in the complex, which modulate the conformation of the acceptor arm. The rearrangement of the acceptor end induced by small substrates results in reorientation of the 3'-OH-group of the terminal ribose from the catalytic subunit onto the noncatalytic one, and this may explain the unusual stereospecificity of aminoacylation in this system.     

Photoreactive dTTP analogues as substrates for thermostable DNA polymerase from Thermus thermophilus B35

Substrate properties of several dTTP analogues bearing a photoreactive 2-nitro-5-azidobenzoyl (NAB) group attached at position 5 of uracil through linkers of various lengths, dTTP-NAB-x-dUTP (where x = 2, 4, 7-13 is the number of atoms in the linker), were studied. All the analogues are substrates for thermostable Thermus thermophilus B35 DNA polymerase in the elongation reaction of the 5'-32P-labeled primer-template complex. The kinetic parameters of some of the analogues were determined and compared with those of natural dTTP. It was shown that an increase in the linker length results in a higher efficiency of the analogue. The incorporation of NAB-x-dUMP residues into the 3'-primer end did not impede a further elongation of the chain in the presence of natural dNTP.     

c-FLIPR, a new regulator of death receptor-induced apoptosis

c-FLIPs (c-FLICE inhibitory proteins) play an essential role in regulation of death receptor-induced apoptosis. Multiple splice variants of c-FLIP have been described on the mRNA level; so far only two of them, c-FLIP(L) and c-FLIP(S,) had been found to be expressed at the protein level. In this report, we reveal the endogenous expression of a third isoform of c-FLIP. We demonstrate its presence in a number of T and B cell lines as well as in primary human T cells. We identified this isoform as c-FLIP(R), a death effector domain-only splice variant previously identified on the mRNA level. Impor-/tantly, c-FLIP(R) is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex upon CD95 stimulation. Several properties of c-FLIP(R) are similar to c-FLIP(S): both isoforms have a short half-life, a similar pattern of expression during activation of primary human T cells, and are strongly induced in T cells upon CD3/CD28 costimulation. Taken together, our data demonstrate endogenous expression of c-FLIP(R) and similar roles of c-FLIP(R) and c-FLIP(S) isoforms in death receptor-mediated apoptosis.     

Preparation of photoreactive oligonucleotide duplexes and their application for photoaffinity modification of DNA-binding proteins]

To introduce photoreactive dNTP residues to the 3'-end of a mononucleotide gap, base-substituted photoreactive deoxynucleoside triphosphate derivatives, (5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-trans-3-aminopropenyl-1]- and 5-(N-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]- trans-3-aminopropenyl-1)-2'-deoxyuridine 5'-triphosphates, were used as substrates in the DNA polymerase beta-catalyzed reaction. The resulting nick, containing a modified base at the 3'-end, was sealed by T4 phage DNA ligase. This approach enables the preparation of DNA duplexes bearing photoreactive groups at predetermined position(s) of the nucleotide chain. Using the generated photoreactive DNA duplexes, the photoaffinity modifications of DNA polymerase beta and human replicative protein A (hRPA) were carried out. It was shown that DNA polymerase beta and hRPA subunits were modified with the photoreactive double-stranded DNA considerably less effectively than by the nicked DNA. In the case of double-stranded DNA, the hRPA p70 subunit was preferentially labeled, implying a crucial role of this subunit in the protein-DNA interaction.     

Protein–Protein Interactions in DNA Base Excision Repair

The system of base excision repair (BER) ensures correction of the most abundant DNA damages in mammalian cells and plays an important role in maintaining genome stability. Enzymes and protein factors participate in the multistage BER in a coordinated fashion, which ensures repair efficiency. The suggested coordination mechanisms are based on formation of protein complexes stabilized via either direct or indirect DNA-mediated interactions. The results of investigation of direct interactions of the proteins participating in BER with each other and with other proteins are outlined in this review. The known protein partners and sites responsible for their interaction are presented for the main participants as well as quantitative characteristics of their affinity. Information on the mechanisms of regulation of protein–protein interactions mediated by DNA intermediates and posttranslational modification is presented. It can be suggested based on all available data that the multiprotein complexes are formed on chromatin independent of the DNA damage with the help of key regulators of the BER process – scaffold protein XRCC1 and poly(ADP-ribose) polymerase 1. The composition of multiprotein complexes changes dynamically depending on the DNA damage and the stage of BER process.     

Express method of isolation of mammalian phenylalanine-tRNA-synthetase and preparation of monoclonal antibodies against this enzyme

A rapid and efficient procedure for isolating homogeneous beef liver phenylalanyl-tRNA synthetase (EC.6.1.1) was developed that enables to purify the enzyme 5000 fold and to achieve the activity of 8 e.a.u. per mg of protein. The molecular mass of the native enzyme was estimated to be 260 kDa, for alpha subunit - 59 kDa, and for beta - 72 kDa. Two cellular clones were derived by means of hybridization of immunised splenocytes with myeloma cells. They secrete monoclonal antibodies, designated P6 and P1 2, that bind to human placental and bovine liver phenylalanyl-tRNA synthetases but not to the same enzymes from E. coli and T. thermophilus. P6 and P1 2 antibodies do not affect the aminoacylation capacity of human or bovine phenylalanyl-tRNA synthetases. By immunoblotting, it was shown that P6 antibodies recognize the alpha subunit of the enzyme.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase