Phenylalanyl-tRNA synthetase from E. coli MRE-600: analysis of the active site distribution on the enzyme subunits by affinity labelling

Affinity labelling has been employed to localize the substrate-binding sites on the enzyme subunits of phenylalanyl-tRNA synthetase (L-phenylalanine:tRNAPhe-ligase, EC 6.1.1.20) of Escherichia coli MRE-600 (alpha 2 beta 2-type). N-Chlorambucilylphenylalanyl-tRNA, N-bromoacetylphenylalanyl-tRNA, tRNAPhe containing an azido group at the eighth position of the molecule (S4U), tRNAPhe containing azido groups at different points of the molecule, p-azidoanilidate of phenylalanine, adenosine 5'-trimethaphosphate and N-bromoacetyl-L-phenylalaninyladenylate were used in experiments. It has been shown that tRNA-binding sites are formed on heavy beta-subunits of the enzyme. Phenylalanyl-tRNA is also localized on beta-subunits, while the aminoacyl moiety of aminoacyl-tRNA is localized near the contact region of subunits. The phenylalanine-binding site is located on light alpha-subunits of the enzyme. Adenosine 5'-trimethaphosphate and the analogue of phenylalanyladenylate modify both types of enzyme subunits. In our opinion, the catalytic center of tRNA aminoacylation is formed in the contact region of subunits, and the aminoacyl moiety is transferred into tRNA (from the alpha- into beta-subunit in the region of their contact).     

The eukaryotic replication complex and its affinity modification analysis

Replication of eukaryotic DNA is driven by a protein complex, in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases alpha, delta, and epsilon involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is in an interplay with DNA replication. The function of the entire multicomponent system is regulated by protein--nucleic acid and protein--protein interactions. The eukaryotic replication complex was not isolated as a stable supramolecular structure, suggesting its dynamic organization. Hence X-ray analysis and other instrumental techniques are hardly suitable for studying this system. An alternative approach is affinity modification. Its most promising version involves in situ generation of photoreactive DNA replication intermediates. The review considers the recent progress in photoaffinity modification studies of DNA polymerases, eukaryotic replication factors, and their interactions with DNA replication intermediates.     

Reagents for Modification of Protein–Nucleic Acid Complexes: III.1Site-Specific Photomodification of Elongation Complex of DNA Polymerase β with Arylazide Derivatives of Primers Sensitized with Fluorescent ATP γ-Amides

ATP -amides containing in -N-position 1-methylpyrene, 9-methylanthracene, 10-chloro-9-methyl-anthracene, and 3-methylperylene residues were synthesized and characterized. These compounds were used as sensitizers of site-specific photomodification of the reconstituted elongating complex of the mammalian DNA polymerase . The photomodification was carried out with the use of photoaffinity reagents, which were synthesized in situby the 5"-³²P-labeled primers extension with photoreactive analogues of dCTP containing in the exo-N-position of cytosine various perfluoroarylazide groups. The effect of structures of the sensitizers and photoreactive reagents on the efficiency and selectivity of photocrosslinking of primers to the enzyme and template, as well as formation of a number of other photomodification products was studied. It was shown that the sensitizers containing 10-chloro-9-methylanthracene and 3-methylperylene residues allow one to obtain photocrosslinks under such irradiation conditions when photomodification in their absence is not essentially observed.     

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 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 , 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).     

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.     

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.     

Hit clustering can improve virtual fragment screening: CDK2 and PARP1 case studies

Virtual fragment screening could be a promising alternative to existing experimental screening techniques. However, reliable methods of in silico fragment screening are yet to be established and validated. In order to develop such an approach we first checked how successful the existing molecular docking methods can be in predicting fragment binding affinities and poses. Using our Lead Finder docking software the RMSD of the binding energy prediction was observed to be 1.35 kcal/mol(-1) on a set of 26 experimentally characterized fragment inhibitors, and the RMSD of the predicted binding pose from the experimental one was <1.5 ?. Then, we explored docking of 68 fragments obtained from 39 drug molecules for which co-crystal structures were available from the PDB. It appeared that fragments that participate in oriented non-covalent interactions, such as hydrogen bonds and metal coordination, could be correctly docked in 70-80% of cases suggesting the potential success of rediscovering of corresponding drugs by in silico fragment approach. Based on these findings we've developed a virtual fragment screening technique which involved structural filtration of protein-ligand complexes for specific interactions and subsequent clustering in order to minimize the number of preferable starting fragment candidates. Application of this method led to 2 millimolar-scale fragment PARP1 inhibitors with a new scaffold.