The C-Terminal Domain of Y-Box Binding Protein 1 Exhibits Structure-Specific Binding to Poly(ADP-Ribose), Which Regulates PARP1 Activity

Abstract

Y-box-binding protein 1 (YB-1) is a multifunctional protein involved in the regulation of gene expression. Recent studies showed that in addition to its role in the RNA and DNA metabolism, YB-1 is involved in the regulation of PARP1 activity, which catalyzes poly(ADP-ribose) [PAR] synthesis under genotoxic stress through auto-poly(ADP-ribosyl)ation or protein trans-poly(ADP-ribosyl)ation. Nonetheless, the exact mechanism by which YB-1 regulates PAR synthesis remains to be determined. YB-1 contains a disordered Ala/Pro-rich N-terminal domain, a cold shock domain, and an intrinsically disordered C-terminal domain (CTD) carrying four clusters of positively charged amino acid residues. Here, we examined the functional role of the disordered CTD of YB-1 in PAR binding and in the regulation of PARP1-driven PAR synthesis in vitro. We demonstrated that the rate of PARP1-dependent synthesis of PAR is higher in the presence of YB-1 and is tightly controlled by the interaction between YB-1 CTD and PAR. Moreover, YB-1 acts as an effective cofactor in the PAR synthesis catalyzed by the PARP1 point mutants that generate various PAR polymeric structures, namely, short hypo- or hyperbranched polymers. We showed that either a decrease in chain length or an increase in branching frequency of PAR affect its binding affinity for YB-1 and YB-1–mediated stimulation of PARP1 enzymatic activity. These results provide important insight into the mechanism underlying the regulation of PARP1 activity by PAR-binding proteins containing disordered regions with clusters of positively charged amino acid residues, suggesting that YB-1 CTD-like domains may be considered PAR “readers” just as other known PAR-binding modules.

Poly ADP-Ribose Signaling is Dysregulated in Huntington Disease

“…In order to test whether huntingtin may bind PAR chains of various sizes, we included purified huntingtin protein in in vitro PARP1 reactions, and visualized the reactions by atomic force microscopy. In the absence of huntingtin, we observed auto-PARylated PARP1 structures consistent with previous reports [79–81] ( Fig 5C ). In contrast, the presence of huntingtin protein in the PARP1 reactions resulted in large rosette structures consistent with huntingtin protein bound to PAR chains ( Fig 5C ).…”

FUS Microphase Separation: Regulation by Nucleic Acid Polymers and DNA Repair Proteins

“…To gain insight into the impact of other nucleic-acid-binding proteins on the nucleic-acid-induced assembly of FUS into high-order structures, we chose replication protein A (RPA), Y-box binding protein 1 (YB-1), apurinic/apyrimidinic endonuclease 1 (APE1), DNA polymerase β (Polβ), X-ray repair cross-complementing 1 (XRCC1), and PARP1. RPA and YB-1 most often bind ssDNA and RNA, respectively, and have also been reported to interact with PAR [ 42 , 43 , 44 , 45 ]. Polβ and PARP1 participate in base excision repair/DNA single-strand break repair and can interact with dsDNA [ 23 , 46 ]; in particular, the damaged 30 bp DNA containing one nucleotide gap (dsDNA, Table 1 ) is a base excision repair DNA intermediate and a substrate for Polβ’s gap-filling activity and for PARP1 binding [ 23 , 46 , 47 ].…”

“…We also tested whether PAR exerted a similar effect on the phase behavior of YB-1, which is prone to aggregation and has PAR-binding activity ( Table 3 ) [ 43 , 45 ]. The addition of various concentrations of PAR to YB-1 yielded only small particles with an R h of approximately 22–26 nm ( Table 3 ).…”

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

“…All proteins used in this study were purified essentially as described [6, 32, 46, 81–84]. Streptococcus pyogenes Cas9 and its mutant forms nCas9 D10A, nCas9 H840A (nickases) and dCas9 (catalytically inactive Cas9 harbouring the double D10A/H840A mutation) were overproduced and purified from E. coli BL21(DE3) using the expression plasmids pMJ806, pMJ825, pMJ826 and pMJ841 (a gift from Dr. Jennifer Doudna; Addgene plasmids #39312, http://n2t.net/addgene:39312, RRID:Addgene_39312; #39315, http://n2t.net/addgene:39315, RRID:Addgene_39315; #39316, http://n2t.net/addgene:39316, RRID:Addgene_39316; #39318, http://n2t.net/addgene:39318; RRID:Addgene_39318).…”

“…Human PARP1 and murine PARP2 were produced in Sf9 insect cells using the expression plasmids kindly provided by Dr. Valérie Schreiber (University of Strasbourg, France). The human PARP1 G972R mutant was expressed in E. coli BL21(DE3) using the pET-32a-based expression plasmid [46]. Human RPA was expressed in E. coli BL21(DE3) using the expression plasmid kindly provided by Dr. Marc S. Wold (University of Iowa, USA).…”