Poly(ADP-ribosyl)ation (PARylation) is posttranslational modification of proteins by linear or branched chains of ADP-ribose units, originating from NAD+. The central enzyme for PAR production in cells and the main target of poly(ADP-ribosyl)ation during DNA damage is poly(ADP-ribose) polymerase 1 (PARP1). PARP1 ability to function as a catalytic and acceptor protein simultaneously made a considerable contribution to accumulation of contradictory data. This topic is directly related to other questions, such as the stoichiometry of PARP1 molecules in auto-modification reaction, direction of the chain growth during PAR elongation and functional coupling of PARP1 with PARylation targets. Besides DNA damage necessary for the folding of catalytically active PARP1, other mechanisms appear to be required for the relevant intensity and specificity of PARylation reaction. Indeed, in recent years, PARP research has been enriched by the discovery of novel PARP1 interaction partners modulating its enzymatic activity. Understanding the details of PARP1 catalytic mechanism and its regulation is especially important in light of PARP-targeted therapy and may significantly aid to PARP inhibitors drug design. In this review we summarize old and up-to-date literature to clarify several points concerning PARylation mechanism and discuss different ways for regulation of PAR synthesis by accessory proteins reported thus far.

The mode of interactions with tRNA explains the absolute necessity for the (alphabeta)2 architecture of PheRS. The interactions of tRNAPhe with PheRS and particularly with the coiled-coil domain of the alpha subunit result in conformational changes in TPsiC and D loops seen by comparison with uncomplexed yeast tRNAPhe. The tRNAPhe is a newly recognized type of RNA molecule specifically interacting with the RBD fold. In addition, a new type of anticodon-binding domain emerges in the aaRS family. The uniqueness of PheRS in charging 2'OH of tRNA is dictated by the size of its adenine-binding pocket and by the local conformation of the tRNA's CCA end.

Graphical Abstract Highlights d FUS is directed to DNA damage sites by binding PAR synthesized by PARP-1 d FUS then forms large compartments in which damaged DNA is concentrated d PARG dissociates damaged DNA compartments by hydrolyzing PAR d Then FUS shuttles from the nucleus to the cytoplasm Correspondence lavrik@niboch.nsc.ru (O.I.L.), david.pastre@univ-evry.fr (D.P.) In Brief Using a single-molecule approach, Singatulina et al. reconstitute the DNA repair system leading to the specific recruitment of FUS, an mRNA-binding protein related to liquid-liquid phase separation biology, to DNA damage sites, thus revealing the capacity of FUS to form dynamic compartments in which damaged DNA is concentrated. SUMMARY PARP-1 synthesizes long poly(ADP-ribose) chains (PAR) at DNA damage sites to recruit DNA repair factors. Among proteins relocated on damaged DNA, the RNA-binding protein FUS is one of the most abundant, raising the issue about its involvement in DNA repair. Here, we reconstituted the PARP-1/ PAR/DNA system in vitro and analyzed at the single-molecule level the role of FUS. We demonstrate successively the dissociation of FUS from mRNA, its recruitment at DNA damage sites through its binding to PAR, and the assembly of damaged DNA-rich compartments. PARG, an enzyme family that hydrolyzes PAR, is sufficient to dissociate damaged DNArich compartments in vitro and initiates the nucleocytoplasmic shuttling of FUS in cells. We anticipate that, consistent with previous models, FUS facilitates DNA repair through the transient compartmentalization of DNA damage sites. The nucleocytoplasmic shuttling of FUS after the PARG-mediated compartment dissociation may participate in the formation of cytoplasmic FUS aggregates.

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs.

G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro conditions, human RPA is able to bind and unfold G-quadruplex structures formed from a 21mer human telomeric sequence. Analyses by native gel electrophoresis, cross-linking and fluorescence resonance energy transfer indicate the formation of both 1:1 and 2:1 complexes in which G-quadruplexes are unfolded. In addition, quadruplex opening by hRPA is much faster than observed with the complementary DNA, demonstrating that this protein efficiently unfolds G-quartets. A two-step mechanism accounting for the binding of hRPA to G-quadruplexes is proposed. These data point to the involvement of hRPA in regulation of telomere maintenance.