Inna A. Vasil'eva

Quantitative characterization of protein–protein complexes involved in base excision DNA repair

Base Excision Repair (BER) efficiently corrects the most common types of DNA damage in mammalian cells. Step-by-step coordination of BER is facilitated by multiple interactions between enzymes and accessory proteins involved. Here we characterize quantitatively a number of complexes formed by DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), poly(ADP-ribose) polymerase 1 (PARP1), X-ray repair cross-complementing protein 1 (XRCC1) and tyrosyl-DNA phosphodiesterase 1 (TDP1), using fluorescence- and light scattering-based techniques. Direct physical interactions between the APE1-Polβ, APE1-TDP1, APE1-PARP1 and Polβ-TDP1 pairs have been detected and characterized for the first time. The combined results provide strong evidence that the most stable complex is formed between XRCC1 and Polβ. Model DNA intermediates of BER are shown to induce significant rearrangement of the Polβ complexes with XRCC1 and PARP1, while having no detectable influence on the protein–protein binding affinities. The strength of APE1 interaction with Polβ, XRCC1 and PARP1 is revealed to be modulated by BER intermediates to different extents, depending on the type of DNA damage. The affinity of APE1 for Polβ is higher in the complex with abasic site-containing DNA than after the APE1-catalyzed incision. Our findings advance understanding of the molecular mechanisms underlying coordination and regulation of the BER process.

Effect of Nucleotide Replacements in tRNAPhe on Positioning of the Acceptor End in the Complex with Phenylalanyl-tRNA Synthetase

The effect of replacement of tRNAPhe recognition elements on positioning of the 3′-terminal nucleotide in the complex with phenylalanyl-tRNA synthetase (PheRS) from T. thermophilus in the absence or presence of phenylalanine and/or ATP has been studied by photoaffinity labeling with s4U76-substituted analogs of wild type and mutant tRNAPhe. The double mutation G34C/A35U shows the strongest disorientation in the absence of low-molecular-weight substrates and sharply decreases the protein labeling, which suggests an initiating role of the anticodon in generation of contacts responsible for the acceptor end positioning. Efficiency of photo-crosslinking with the α- and β-subunits in the presence of individual substrates is more sensitive to nucleotide replacements in the anticodon (G34 by A or A36 by C) than to changes in the general structure of tRNAPhe (as a result of replacement of the tertiary pair G19-C56 by U19-G56 or of U20 by A). The degree of disorders in the 3′-terminal nucleotide positioning in the presence of both substrates correlates with decrease in the turnover number of aminoacylation due to corresponding mutations. The findings suggest that specific interactions of the enzyme with the anticodon mainly promote the establishment (controlled by phenylalanine) of contacts responsible for binding of the CCA-end and terminal nucleotide in the productive complex, and the general conformation of tRNAPhe determines, first of all, the acceptor stem positioning (controlled by ATP). The main recognition elements of tRNAPhe, which optimize its initial binding with PheRS, are also involved in generation of the catalytically active complex providing functional conformation of the acceptor arm.

Dynamic light scattering study of base excision DNA repair proteins and their complexes

Base excision repair (BER) involves many enzymes acting in a coordinated fashion at the most common types of DNA damage. The coordination is facilitated by interactions between the enzymes and accessory proteins, X-ray repair cross-complementing protein 1 (XRCC1) and poly(ADP-ribose) polymerase 1 (PARP1). Here we use dynamic light scattering (DLS) technique to determine the hydrodynamic sizes of several BER enzymes and proteins, DNA polymerase β (Polβ), apurinic/apyrimidinic endonuclease 1 (APE1), tyrosyl-DNA phosphodiesterase 1 (TDP1), XRCC1 and PARP1, present alone or in the equimolar mixtures with each other. From the DLS data combined with glutaraldehyde cross-linking experiments and previous quantitative binding data the oligomeric states of BER proteins and their complexes are estimated. All the proteins have been proposed to form homodimers upon their self-association. The most probable oligomerization state of the binary complexes formed by PARP1 with various proteins is a heterotetramer. The oligomerization state of the binary complexes formed by XRCC1 varies from heterodimer to heterotetramer, depending on the partner. The DLS technique is applied for the first time to measure the hydrodynamic sizes of PARP1 molecules covalently bound with poly(ADP-ribose) (PAR) synthesized upon the automodification reaction. PARP1 has been detected to form huge conglomerates stabilized by Mg2+ coordinated bonds with PAR polymers.

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 tRNAPhe acceptor end have been studied by photoaffinity labeling (cross-linking) of T. thermophilus phenylalanyl-tRNA synthetase (PheRS) with tRNAPhe analogs containing the s4U residue in different positions of the 3′-terminal single-stranded sequence. Human and E. coli tRNAPhes used as basic structures differ by efficiency of the binding and aminoacylation with the enzyme under study. Destabilization of the complex with human tRNAPhe caused by replacement of three recognition elements decreases selectivity of labeling of the α- and β-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 tRNAPhes. Both in the absence and presence of phenylalanine, ATP more effectively inhibits the PheRS labeling with the s4U76-substituted analog of human tRNAPhe (tRNAPhe-s4U76) than with E. coli tRNAPhe-s4U76: in the first case the labeling of the α-subunits is inhibited more effectively; the labeling of the β-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 tRNAPhe 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.