Effect of Human XRCC1 Protein Oxidation on the Functional Activity of Its Complexes with the Key Enzymes of DNA Base Excision Repair.

Base excision repair (BER) ensures correction of most abundant DNA lesions in mammals. The efficiency of this multistep DNA repair process that can occur via different pathways depends on the coordinated action of enzymes catalyzing its individual steps. The scaffold XRCC1 (X-ray repair cross-complementing protein 1) protein plays an important coordinating role in the repair of damaged bases and apurinic/apyrimidinic (AP) sites via short-patch (SP) BER pathway, as well as in the repair of single-strand DNA breaks. In this study, we demonstrated for the first time in vitro formation of the ternary XRCC1 complex with the key enzymes of SP BER - DNA polymerase ß (Polß) and DNA ligase IIIα (LigIIIα) - using the fluorescence-based technique. It was found that Polß directly interacts with LigIIIα, but their complex is less stable than the XRCC1-Polß and XRCC1-LigIIIα complexes. The effect of XRCC1 oxidation and composition of the multiprotein complex on the efficiency of DNA synthesis and DNA ligation during DNA repair has been explored. We found that formation of the disulfide bond between Cys12 and Cys20 residues as a result of XRCC1 oxidation (previously shown to modulate the protein affinity for Polß), affects the yield of the final product of SP BER and of non-ligated DNA intermediates (substrates of long-patch BER). The effect of XRCC1 oxidation on the final product yield depended on the presence of AP endonuclease 1. Together with the data from our previous work, the results of this study suggest an important role of XRCC1 oxidation in the fine regulation of formation of BER complexes and their functional activity.

Role of Oxidation of XRCC1 Protein in Regulation of Mammalian DNA Repair Process.

The influence of XRCC1 protein oxidation on the modification of proteins catalyzed by poly(ADP-ribose)polymerases (PARP1 and PARP2) was studied for the first time. XRCC1, PARP1, and PARP2, functioning as scaffold proteins, are responsible for coordination of multistep repair of most abundant DNA lesions. We showed that the XRCC1 oxidation reduces the efficiency of its ADP-ribosylation and the protein affinity for poly(ADP-ribose). The ADP-ribose modification of various XRCC1 forms is enhanced in the presence of DNA polymerase ß (Polß), capable of forming a stable complex with XRCC1. Oxidation suppresses the inhibitory effect of XRCC1 and its complex with Polß on the automodification of PARP1 and PARP2, which may enhance the efficiency of repair. The results of this study indicate that the oxidation of XRCC1 plays a role in fine regulation of poly(ADP-ribosyl)ation levels of proteins and their coordinating functions in DNA repair.

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.

Y-Box-Binding Protein 1 Stimulates Abasic Site Cleavage.

Apurinic/apyrimidinic (AP) sites are among the most frequent DNA lesions. The first step in the AP site repair involves the magnesium-dependent enzyme AP endonuclease 1 (APE1) that catalyzes hydrolytic cleavage of the DNA phosphodiester bond at the 5' side of the AP site, thereby generating a single-strand DNA break flanked by the 3'-OH and 5'-deoxyribose phosphate (dRP) groups. Increased APE1 activity in cancer cells might correlate with tumor chemoresistance to DNA-damaging treatment. It has been previously shown that the multifunctional oncoprotein Y-box-binding protein 1 (YB-1) interacts with APE1 and inhibits APE1-catalyzed hydrolysis of AP sites in single-stranded DNAs. In this work, we demonstrated that YB-1 stabilizes the APE1 complex with double-stranded DNAs containing the AP sites and stimulates cleavage of these AP sites at low magnesium concentrations.

[Codases: fifty years after].

Aminoacyl-tRNA synthetases (codases) catalyze aminoacylation of a particular tRNA with the corresponding amino acid at the first step of protein biosynthesis. The review considers universal structure-functional characteristics of the largest family of enzymes partitioned into two classes. Modes of tRNA binding and recognition, and additional editing activity, which are responsible for the fidelity of aminoacyl-tRNA synthesis, are discussed. The aaRSs catalytic cores are highly relevant to the ancient metabolic reactions, namely, amino acids and cofactors biosynthesis. Thus, the biosynthetic machinery for producing amino acids had a profound effect on almost every aspect of aminoacylation reaction. The review also deals with secondary functions of synthetases in various processes of cell metabolism. Certain of these functions have to do with complex pathophysiological mechanisms involved in disease production. Their investigation may help to develop new diagnostic techniques and therapies.

Interaction of human phenylalanyl-tRNA synthetase with specific tRNA according to thiophosphate footprinting.

The interaction of human cytoplasmic phenylalanyl-tRNA synthetase (an enzyme with yet unknown 3D-structure) with homologous tRNA(Phe) under functional conditions was studied by footprinting based on iodine cleavage of thiophosphate-substituted tRNA transcripts. Most tRNA(Phe) nucleotides recognized by the enzyme in the anticodon (G34), anticodon stem (G30-C40, A31-U39), and D-loop (G20) have effectively or moderately protected phosphates. Other important specificity elements (A35 and A36) were found to form weak nonspecific contacts. The D-stem, T-arm, and acceptor stem are also among continuous contacts of the tRNA(Phe) backbone with the enzyme, thus suggesting the presence of additional recognition elements in these regions. The data indicate that mechanisms of interaction between phenylalanyl-tRNA synthetases and specific tRNAs are different in prokaryotes and eukaryotes.

Interaction of aminoacyl-tRNA synthetases with tRNA: general principles and distinguishing characteristics of the high-molecular-weight substrate recognition.

This review summarizes results of numerous (mainly functional) studies that have been accumulated over recent years on the problem of tRNA recognition by aminoacyl-tRNA synthetases. Development and employment of approaches that use synthetic mutant and chimeric tRNAs have demonstrated general principles underlying highly specific interaction in different systems. The specificity of interaction is determined by a certain number of nucleotides and structural elements of tRNA (constituting the set of recognition elements or specificity determinants), which are characteristic of each pair. Crystallographic structures available for many systems provide the details of the molecular basis of selective interaction. Diversity and identity of biochemical functions of the recognition elements make substantial contribution to the specificity of such interactions.

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.

Effect of nucleotide replacements in tRNAPhe on positioning of the acceptor end in the complex with phenylalanyl-tRNA synthetase.

The effect of replacement of tRNA(Phe) 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 s(4)U76-substituted analogs of wild type and mutant tRNA(Phe). 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 alpha- and beta-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 tRNA(Phe) (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 tRNA(Phe) determines, first of all, the acceptor stem positioning (controlled by ATP). The main recognition elements of tRNA(Phe), which optimize its initial binding with PheRS, are also involved in generation of the catalytically active complex providing functional conformation of the acceptor arm.

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.

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.

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.

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.

Affinity modification of phenylalanyl-tRNA synthetase from Thermus thermophilus by tRNAPhe transcripts containing 4-thiouridine.

Photoreactive derivatives of tRNAPhe containing residues of 4-thiouridine (s4U) were synthesized by the transcription system of T7 RNA polymerase. Complete substitution of s4U for 16 uridine residues ([16s4U]-tRNAPhe) caused a 14-fold decrease in the catalytic efficiency of aminoacylation of the tRNAPhe transcript by phenylalanyl-tRNA synthetase from T. thermophilus. [1s4U]-tRNAPhe obtained by random incorporation of s4U residues with further isolation of s4U-monosubstituted RNA molecules on an affinity gel has the same kinetic parameters in aminoacylation as the tRNAPhe transcript. The s4U-containing tRNAPhe transcripts were shown to bind covalently to phenylalanyl-tRNA synthetase, and the specificity of modification was demonstrated. The modification stoichiometry determined in this work suggests that the enzyme is a functional dimer. The modification labels both alpha- and beta-subunits of the enzyme, which has an oligomeric structure of alpha2beta2, and forms "cross-linking" products of subunits upon modification with [16s4U]-tRNAPhe. The prevalence of modification of the alpha-subunit suggests that tRNA has contacts with the enzyme, which have not been deciphered previously by X-ray analysis.

Localization of the binding site for the 3'-terminal sequence of tRNAPhe in subunits of phenylalanyl-tRNA synthetase from Thermus thermophilus.

A photoreactive tRNAPhe derivative containing a 4-thiouridine residue at the 3'-end (tRNAPhe-s4U-75) was prepared by tRNA nucleotidyltransferase-mediated incorporation of s4UMP into a tRNAPhe transcript lacking the 3'-terminal dinucleotide. The resulting tRNAPhe-s4U-75 was covalently bound to phenylalanyl-tRNA synthetase from Thermus thermophilus, and all criteria of an affinity modification were met. The main products of modification displaying various electrophoretic mobilities were formed by binding tRNAPhe-s4U-75 to the beta-subunit (major) of the enzyme. These data suggest that the nucleotide found at position 75 of tRNAPhe interacts with the beta-subunit of phenylalanyl-tRNA synthetase.

A peculiarity of the reaction of tRNA aminoacylation catalyzed by phenylalanyl-tRNA synthetase from the extreme thermophile Thermus thermophilus.

It was confirmed unambiguously that the anomalously high plateau in the tRNA aminoacylation reaction catalyzed by Thermus thermophilus phenylalanyl-tRNA synthetase is a result of enzymatic synthesis of tRNA bearing two bound phenylalanyl residues (bisphenylalanyl-tRNA). The efficiency of bisphenylalanyl-tRNA formation was shown to be quite low: the second phenylalanyl residue is attached to tRNA approximately 50 times more slowly than the first one. The thermophilic synthetase can aminoacylate twice not only T. thermophilus tRNAPhe but also Escherichia coli tRNAPhe and E. coli tRNAPhe transcript, indicating that the presence of modified nucleotides is not necessary for tRNAPhe overcharging. Bisphenylalanyl-tRNA is stable in acidic solution, but it decomposes in alkaline medium yielding finally tRNA and free phenylalanine. Under these conditions phenylalanine is released from bisphenylalanyl-tRNA with almost the same rate as from monophenylalanyl-tRNA. In the presence of the enzyme the rate of bisphenylalanyl-tRNA deacylation increases. Aminoacylated tRNAPhe isolated from T. thermophilus living cells was observed to contain no detectable bisphenylalanyl-tRNA under normal growth of culture. A possible mechanism of bisphenylalanyl-tRNA synthesis is discussed.

Covalent complex of phenylalanyl-tRNA synthetase with 4-thiouridine-substituted tRNA(Phe) gene transcript retains aminoacylation activity.

s4U-containing transcripts of tRNA(Phe) gene have been prepared by complete substitution of 16 U residues or by random incorporation of s4U residues followed by affinity electrophoresis isolation of s4U-monosubstituted tRNA transcripts. Both analogs have been cross-linked to Thermus thermophilus phenylalanyl-tRNA synthetase (PheRS) and the specificity of the cross-linking has been demonstrated. Functional activity of the covalent complex of PheRS with the s4U-monosubstituted transcript has been shown by aminoacylation of 60% of the enzyme-cross-linked tRNA. This is the first instance in which biological activity of aminoacyl-tRNA synthetase and cross-linked tRNA in a specific complex has been revealed.

Recognition of tRNAPhe by phenylalanyl-tRNA synthetase of Thermus thermophilus.

The tRNA(Phe) nucleotides required for recognition by phenylalanyl-tRNA synthetase of Thermus thermophilus have been determined using Escherichia coli tRNA(Phe) transcripts with various mutations. The anticodon nucleotides are shown to be the most important recognition elements. The discriminator nucleotide, A73, involved in the recognition set of yeast, E. coli and human phenylalanyl-tRNA synthetases contributes only slightly to tRNA(Phe) recognition by Th. thermophilus phenylalanyl-tRNA synthetase. Nucleotide 20 and some tertiary nucleotides, including the conserved G19.C56 base pair, are proposed to participate in stabilization of the precise tRNA conformation required for efficient aminoacylation. The role of the 3'-CCA terminus, common to all tRNAs, in the specific interaction of tRNA with phenylalanyl-tRNA synthetase is discussed.

[The role of the 3'-CCA sequence in the interaction of tRNA(Phe) from E. coli and Thermus thermophilus with homologous phenylalanyl-tRNA-synthetases]. / Rol' 3'-CCA-posledovatel'nosti vo vzaimodeistvii tRNA(Phe) iz E. coli i Thermus thermophilus s gomologichnymi fenilalanil-tRNK-sintetazami.

The 3'-CCA end of tRNA(Phe) from E. coli and Thermus thermophilus was modified by stepwise degradation and ligation of the shortened tRNA with different trinucleotides (pUpUpA, (pA)3, (pC)3, (pU)3). Kinetic parameters for the aminoacylation reaction of modified tRNAs have been determined. The role of the 3'-terminal trinucleotide of tRNA(Phe) in tRNA binding and aminoacylation by phenylalanyl-tRNA synthetases from E. coli and Thermus thermophilus is postulated.

Alterations at the 3'-CCA end of Escherichia coli and Thermus thermophilus tRNA(Phe) do not abolish their acceptor activity.

The 3'-CCA end of tRNA(Phe) from Escherichia coli and Thermus thermophilus was changed to AAA, CCC, UUU and UUA by the stepwise degradation procedure of the 3'-CCA end of tRNA(Phe) followed by the ligation with oligoribonucleotides. Substrate activity of tRNA(UUAPhe) and tRNA(CCCPhe) in tRNA aminoacylation was shown. tRNA(AAAPhe) is a bad substrate for E. coli and Th. thermophilus phenylalanyl-tRNA synthetases. tRNA(UUUPhe) has no detectable activity in tRNA aminoacylation. Therefore the nature of the 3'-end of tRNA(Phe) plays an important role in tRNA binding and its substrate efficiency. Nevertheless the CCA sequence at the 3'-end of tRNA(Phe) does not seem to be an absolute requirement for tRNA aminoacylation.