The crystal structure of the binary complex tRNA(Asp)-aspartyl tRNA synthetase from yeast was solved with the use of multiple isomorphous replacement to 3 angstrom resolution. The dimeric synthetase, a member of class II aminoacyl tRNA synthetases (aaRS's) exhibits the characteristic signature motifs conserved in eight aaRS's. These three sequence motifs are contained in the catalytic site domain, built around an antiparallel beta sheet, and flanked by three alpha helices that form the pocket in which adenosine triphosphate (ATP) and the CCA end of tRNA bind. The tRNA(Asp) molecule approaches the synthetase from the variable loop side. The two major contact areas are with the acceptor end and the anticodon stem and loop. In both sites the protein interacts with the tRNA from the major groove side. The correlation between aaRS class II and the initial site of aminoacylation at 3'-OH can be explained by the structure. The molecular association leads to the following features: (i) the backbone of the GCCA single-stranded portion of the acceptor end exhibits a regular helical conformation; (ii) the loop between residues 320 and 342 in motif 2 interacts with the acceptor stem in the major groove and is in contact with the discriminator base G and the first base pair UA; and (iii) the anticodon loop undergoes a large conformational change in order to bind the protein. The conformation of the tRNA molecule in the complex is dictated more by the interaction with the protein than by its own sequence.

The crystal structures of the various complexes formed by yeast aspartyl‐tRNA synthetase (AspRS) and its substrates provide snapshots of the active site corresponding to different steps of the aminoacylation reaction. Native crystals of the binary complex tRNA‐AspRS were soaked in solutions containing the two other substrates, ATP (or its analog AMPPcP) and aspartic acid. When all substrates are present in the crystal, this leads to the formation of the aspartyl‐adenylate and/or the aspartyl‐tRNA. A class II‐specific pathway for the aminoacylation reaction is proposed which explains the known functional differences between the two classes while preserving a common framework. Extended signature sequences characteristic of class II aaRS (motifs 2 and 3) constitute the basic functional unit. The ATP molecule adopts a bent conformation, stabilized by the invariant Arg531 of motif 3 and a magnesium ion coordinated to the pyrophosphate group and to two class‐invariant acidic residues. The aspartic acid substrate is positioned by a class II invariant acidic residue, Asp342, interacting with the amino group and by amino acids conserved in the aspartyl synthetase family. The amino acids in contact with the substrates have been probed by site‐directed mutagenesis for their functional implication.

The DNA damage-dependent poly(ADP-ribose) polymerases-1 and -2 (PARP-1 and PARP-2) are survival factors that share overlapping functions in the detection, signaling and repair of DNA strand breaks resulting from genotoxic lesions in mammalian cells. Here we show that PARP-1 and PARP-2 subnuclear distributions partially overlap, with both proteins accumulating within the nucleolus independently of each other. PARP-2 is enriched within the whole nucleolus and partially colocalizes with the nucleolar factor nucleophosmin/B23. We have identified a nuclear localization signal and a nucleolar localization signal within the N-terminal domain of PARP-2. PARP-2, like PARP-1, interacts with B23 through its N-terminal DNA binding domain. This association is constitutive and does not depend on either PARP activity or ribosomal transcription, but is prevented by mutation of the nucleolar localization signal of PARP-2. PARP-1 and PARP-2, together with B23, are delocalized from the nucleolus upon RNA polymerase I inhibition whereas the nucleolar accumulation of all three proteins is only moderately affected upon oxidative or alkylated DNA damage. Finally, we show that murine fibroblasts deficient in PARP-1 or PARP-2 are not affected in the transcription of ribosomal RNAs. Taken together, these results suggest that the biological role of PARP-1 and PARP-2 within the nucleolus relies on functional nucleolar transcription, without any obvious implication of either PARP on this major nucleolar process.