ABSTRACT
Biomolecular condensates are nonmembrane cellular compartments whose formation in many cases involves phase separation (PS). Despite much research interest in this mechanism of macromolecular self-organization, the concept of PS as applied to a live cell faces certain challenges. In this review, we discuss a basic model of PS and the role of site-specific interactions and percolation in cellular PS-related events. Using a multivalent poly(ADP-ribose) molecule as an example, which has high PS-driving potential due to its structural features, we consider how site-specific interactions and network formation are involved in the formation of phase-separated cellular condensates.
Subject(s)
Poly Adenosine Diphosphate Ribose , Macromolecular SubstancesABSTRACT
Condensates are biomolecular assemblies that concentrate biomolecules without the help of membranes. They are morphologically highly versatile and may emerge via distinct mechanisms. Nucleic acids-DNA, RNA and poly(ADP-ribose) (PAR) play special roles in the process of condensate organization. These polymeric scaffolds provide multiple specific and nonspecific interactions during nucleation and 'development' of macromolecular assemblages. In this review, we focus on condensates formed with PAR. We discuss to what extent the literature supports the phase separation origin of these structures. Special attention is paid to similarities and differences between PAR and RNA in the process of dynamic restructuring of condensates during their functioning.
Subject(s)
Nucleic Acids , Poly Adenosine Diphosphate Ribose , RNA/genetics , RNA/chemistry , Biomolecular Condensates , Macromolecular SubstancesABSTRACT
Poly(ADP-ribosyl)ation is a post-translational modification of proteins that performs an essential regulatory function in the cellular response to DNA damage. The key enzyme synthesizing poly(ADP-ribose) (PAR) in the cells is poly(ADP-ribose) polymerase 1 (PARP1). Understanding the mechanisms of the PARP1 activity regulation within the cells is necessary for development of the PARP1-targeted antitumor therapy. This review is devoted to the studies of the role of the RNA-binding protein YB-1 in the PARP1-catalyzed PARylation. The mechanisms of PARP1 activity stimulation by YB-1 protein can possibly be extended to other RNA-binding proteins involved in the maintenance of the genome stability.
Subject(s)
Poly ADP Ribosylation , Poly(ADP-ribose) Polymerases , Catalysis , DNA Damage , Poly Adenosine Diphosphate Ribose , Poly(ADP-ribose) Polymerases/metabolism , Proteins/metabolismABSTRACT
Y-box-binding protein 1 (YB-1) is a multifunctional positively charged protein that interacts with DNA or RNA and poly(ADP-ribose) (PAR). YB-1 is poly(ADP-ribosyl)ated and stimulates poly(ADP-ribose) polymerase 1 (PARP1) activity. Here, we studied the mechanism of YB-1-dependent PAR synthesis by PARP1 in vitro using biochemical and atomic force microscopy assays. PAR synthesis activity of PARP1 is known to be facilitated by co-factors such as Mg2+. However, in contrast to an Mg2+-dependent reaction, the activation of PARP1 by YB-1 is accompanied by overall up-regulation of protein PARylation and shortening of the PAR polymer. Therefore, YB-1 and cation co-factors stimulated PAR synthesis in divergent ways. PARP1 autoPARylation in the presence of YB-1 as well as trans-PARylation of YB-1 are greatly affected by the type of damaged DNA, suggesting that PARP1 activation depends on the formation of a PARP1-YB-1-DNA ternary complex. An unstructured C-terminal part of YB-1 involved in an interaction with PAR behaves similarly to full-length YB-1, indicating that both DNA and PAR binding are involved in the stimulation of PARP1 activity by YB-1. Thus, YB-1 is likely linked to the regulation of PARylation events in cells via an interaction with PAR and damaged DNA.
Subject(s)
Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Protein Processing, Post-Translational , Y-Box-Binding Protein 1/metabolism , DNA/chemistry , DNA/genetics , DNA/metabolism , DNA Damage , Fluorescence Polarization , HeLa Cells , Humans , Magnesium/metabolism , Microscopy, Atomic Force , Nucleic Acid Conformation , Nucleosomes/genetics , Nucleosomes/metabolism , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly (ADP-Ribose) Polymerase-1/genetics , Protein Binding , Protein Multimerization , Y-Box-Binding Protein 1/chemistry , Y-Box-Binding Protein 1/geneticsABSTRACT
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.
Subject(s)
Adenosine Diphosphate Ribose/metabolism , DNA Repair , DNA/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly Adenosine Diphosphate Ribose/biosynthesis , Protein Processing, Post-Translational , Animals , Catalytic Domain , DNA/chemistry , DNA/metabolism , DNA Damage , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Poly (ADP-Ribose) Polymerase-1/chemistry , Poly (ADP-Ribose) Polymerase-1/genetics , Poly ADP Ribosylation , Protein Binding , Protein Folding , Protein MultimerizationABSTRACT
Y-box-binding protein 1 (YB-1) is a multifunctional cellular factor overexpressed in tumors resistant to chemotherapy. An intrinsically disordered structure together with a high positive charge peculiar to YB-1 allows this protein to function in almost all cellular events related to nucleic acids including RNA, DNA and poly(ADP-ribose) (PAR). In the present study we show that YB-1 acts as a potent poly(ADP-ribose) polymerase 1 (PARP1) cofactor that can reduce the efficiency of PARP1 inhibitors. Similarly to that of histones or polyamines, stimulatory effect of YB-1 on the activity of PARP1 was significantly higher than the activator potential of Mg2+ and was independent of the presence of EDTA. The C-terminal domain of YB-1 proved to be indispensable for PARP1 stimulation. We also found that functional interactions of YB-1 and PARP1 can be mediated and regulated by poly(ADP-ribose).
ABSTRACT
Base excision repair (BER) is a flagship DNA repair system responsible for maintaining genome integrity. Apart from basal enzymes, this system involves several accessory factors essential for coordination and regulation of DNA processing during substrate channeling. Y-box-binding protein 1 (YB-1), a multifunctional factor that can interact with DNA, RNA, poly(ADP-ribose) and plenty of proteins including DNA repair enzymes, is increasingly considered as a non-canonical protein of BER. Here we provide quantitative characterization of YB-1 physical interactions with key BER factors such as PARP1, PARP2, APE1, NEIL1 and pol ß and comparison of the full-length YB-1 and its C-terminally truncated nuclear form in regard to their binding affinities for BER proteins. Data on functional interactions reveal strong stimulation of PARP1 autopoly(ADP-ribosyl)ation and inhibition of poly(ADP-ribose) degradation by PARG in the presence of YB-1. Moreover, YB-1 is shown to stimulate AP lyase activity of NEIL1 and to inhibit dRP lyase activity of pol ß on model DNA duplex structure. We also demonstrate for the first time YB-1 poly(ADP-ribosyl)ation in the presence of RNA.
Subject(s)
DNA Repair/physiology , Y-Box-Binding Protein 1/metabolism , Animals , DNA Damage , DNA Glycosylases/metabolism , DNA Polymerase beta/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/genetics , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Humans , Mice , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Y-Box-Binding Protein 1/chemistry , Y-Box-Binding Protein 1/geneticsABSTRACT
Multifunctional Y-box binding protein 1 (YB-1) is actively studied as one of the components of cellular response to genotoxic stress. However, the precise role of YB-1 in the process of DNA repair is still obscure. In the present work we report for the first time new posttranslational modification of YB-1 - poly(ADP-ribosyl)ation, catalyzed by one of the main regulatory enzymes of DNA repair - poly(ADP-ribose)polymerase 1 (PARP1) in the presence of model DNA substrate carrying multiple DNA lesions. Therefore, poly(ADP-ribosyl)ation of YB-1 catalyzed with PARP1, can be stimulated by damaged DNA. The observed property of YB-1 underlines its ability to participate in the DNA repair by its involvement in the regulatory cascades of DNA repair.
