Studying Structure and Functions of Nucleosomes with Atomic Force Microscopy.

Chromatin is an epigenetic platform for implementation of DNA-dependent processes. Nucleosome, as a basic level of chromatin compaction, largely determines its properties and structure. In the study of nucleosomes structure and functions physicochemical tools are actively used, such as magnetic and optical "tweezers", "DNA curtains", nuclear magnetic resonance, X-ray crystallography, and cryogenic electron microscopy, as well as optical methods based on Förster resonance energy transfer. Despite the fact that these approaches make it possible to determine a wide range of structural and functional characteristics of chromatin and nucleosomes with high spatial and time resolution, atomic force microscopy (AFM) complements the capabilities of these methods. The results of structural studies of nucleosome focusing on the AFM method development are presented in this review. The possibilities of AFM are considered in the context of application of other physicochemical approaches.

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

CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

The HPF1-dependent histone PARylation catalyzed by PARP2 is specifically stimulated by an incised AP site-containing BER DNA intermediate.

Poly(ADP-ribose) polymerase 1 (PARP1) and PARP2 are DNA-dependent poly(ADP-ribose)transferases localized in nucleus. They have a significant homology in the C-terminal catalytic domain structure but differ in their N-terminal DNA-binding parts. The structural difference has an impact on the interaction of PARP1 and PARP2 with DNA and their DNA-dependent activation. Here, we compare the interaction of PARP1 and PARP2 with free 147 bp nucleosomal DNA and its nucleosome-associated variant (NCP) that contain in one strand a 1-nucleotide gap with 5'-dRP (imitating the intermediate of Base Excision Repair) or no specific damage. The affinity of PARP2 for the DNA strongly depends on the gap presence and to a lesser extent on the association with nucleosomes, while PARP1 interacts primarily with blunt ends of all DNAs and with a lower affinity with the single-strand break. The activities of PARP1 and PARP2 in the autoPARylation reaction and heteromodification of histones are distinctly stimulated by HPF1, depending on the gap presence in activating DNA. The most significant HPF1-induced stimulation of the histone modification in the presence of gapped NCP is a peculiar feature of PARP2. We propose a specific regulatory role of PARP2 in the process of DNA repair in the context of chromatin.

Inhibitory Effects of 7-Methylguanine and Its Metabolite 8-Hydroxy-7-Methylguanine on Human Poly(ADP-Ribose) Polymerase 1.

Previously, we have found that a nucleic acid metabolite, 7-methylguanine (7mGua), produced in the body can have an inhibitory effect on the poly(ADP-ribose) polymerase 1 (PARP1) enzyme, an important pharmacological target in anticancer therapy. In this work, using an original method of analysis of PARP1 activity based on monitoring fluorescence anisotropy, we studied inhibitory properties of 7mGua and its metabolite, 8-hydroxy-7-methylguanine (8h7mGua). Both compounds inhibited PARP1 enzymatic activity in a dose-dependent manner, however, 8h7mGua was shown to be a stronger inhibitor. The IC50 values for 8h7mGua at different concentrations of the NAD+ substrate were found to be 4 times lower, on average, than those for 7mGua. The more efficient binding of 8h7mGua in the PARP1 active site is explained by the presence of an additional hydrogen bond with the Glu988 catalytic residue. Experimental and computational studies did not reveal the effect of 7mGua and 8h7mGua on the activity of other DNA repair enzymes, indicating selectivity of their inhibitory action.

Study of Interaction of the PARP Family DNA-Dependent Proteins with Nucleosomes Containing DNA Intermediates of the Initial Stages of BER Process.

Reaction of (ADP-ribosyl)ation catalyzed by DNA-dependent proteins of the poly(ADP-ribose)polymerase (PARP) family, PARP1, PARP2, and PARP3, comprises the cellular response to DNA damage. These proteins are involved in the base excision repair (BER) process. Despite the extensive research, it remains unknown how PARPs are involved in the regulation of the BER process and how the roles are distributed between the DNA-dependent members of the PARP family. Here, we investigated the interaction of the PARP's family DNA-dependent proteins with nucleosome core particles containing DNA intermediates of the initial stages of BER. To do that, the nucleosomes containing damage in the vicinity of one of the DNA duplex blunt ends were reconstituted based on the Widom's Clone 603 DNA sequence. Dissociation constants of the PARP complexes with nucleosomes bearing DNA contained uracil (Native), apurine/apyrimidine site (AP site), or a single-nucleotide gap with 5'-dRp fragment (Gap) were determined. It was shown that the affinity of the proteins for the nucleosomes increased in the row: PARP3<

Dual function of HPF1 in the modulation of PARP1 and PARP2 activities.

Poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerases (PARPs) is one of the immediate cellular responses to DNA damage. The histone PARylation factor 1 (HPF1) discovered recently to form a joint active site with PARP1 and PARP2 was shown to limit the PARylation activity of PARPs and stimulate their NAD+-hydrolase activity. Here we demonstrate that HPF1 can stimulate the DNA-dependent and DNA-independent autoPARylation of PARP1 and PARP2 as well as the heteroPARylation of histones in the complex with nucleosome. The stimulatory action is detected in a defined range of HPF1 and NAD+ concentrations at which no HPF1-dependent enhancement in the hydrolytic NAD+ consumption occurs. PARP2, comparing with PARP1, is more efficiently stimulated by HPF1 in the autoPARylation reaction and is more active in the heteroPARylation of histones than in the automodification, suggesting a specific role of PARP2 in the ADP-ribosylation-dependent modulation of chromatin structure. Possible role of the dual function of HPF1 in the maintaining PARP activity is discussed.

Regulation of Poly(ADP-Ribose) Polymerase 1 Activity by Y-Box-Binding Protein 1.

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.

A Single-Molecule Atomic Force Microscopy Study of PARP1 and PARP2 Recognition of Base Excision Repair DNA Intermediates.

Nuclear poly(ADP-ribose) polymerases 1 and 2 (PARP1 and PARP2) catalyze the synthesis of poly(ADP-ribose) (PAR) and use NAD+ as a substrate for the polymer synthesis. Both PARP1 and PARP2 are involved in DNA damage response pathways and function as sensors of DNA breaks, including temporary single-strand breaks formed during DNA repair. Consistently, with a role in DNA repair, PARP activation requires its binding to a damaged DNA site, which initiates PAR synthesis. Here we use atomic force microscopy to characterize at the single-molecule level the interaction of PARP1 and PARP2 with long DNA substrates containing a single damage site and representing intermediates of the short-patch base excision repair (BER) pathway. We demonstrated that PARP1 has higher affinity for early intermediates of BER than PARP2, whereas both PARPs efficiently interact with the nick and may contribute to regulation of the final ligation step. The binding of a DNA repair intermediate by PARPs involved a PARP monomer or dimer depending on the type of DNA damage. PARP dimerization influences the affinity of these proteins to DNA and affects their enzymatic activity: the dimeric form is more effective in PAR synthesis in the case of PARP2 but is less effective in the case of PARP1. PARP2 suppresses PAR synthesis catalyzed by PARP1 after single-strand breaks formation. Our study suggests that the functions of PARP1 and PARP2 overlap in BER after a site cleavage and provides evidence for a role of PARP2 in the regulation of PARP1 activity.

Poly(ADP-ribosyl)ation and DNA repair synthesis in the extracts of naked mole rat, mouse, and human cells.

DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5'-terminal phosphate by PARP3 during the DNA synthesis. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.

Design, Synthesis and Molecular Modeling Study of Conjugates of ADP and Morpholino Nucleosides as A Novel Class of Inhibitors of PARP-1, PARP-2 and PARP-3.

We report on the design, synthesis and molecular modeling study of conjugates of adenosine diphosphate (ADP) and morpholino nucleosides as potential selective inhibitors of poly(ADP-ribose)polymerases-1, 2 and 3. Sixteen dinucleoside pyrophosphates containing natural heterocyclic bases as well as 5-haloganeted pyrimidines, and mimicking a main substrate of these enzymes, nicotinamide adenine dinucleotide (NAD+)-molecule, have been synthesized in a high yield. Morpholino nucleosides have been tethered to the ß-phosphate of ADP via a phosphoester or phosphoramide bond. Screening of the inhibiting properties of these derivatives on the autopoly(ADP-ribosyl)ation of PARP-1 and PARP-2 has shown that the effect depends upon the type of nucleobase as well as on the linkage between ADP and morpholino nucleoside. The 5-iodination of uracil and the introduction of the P-N bond in NAD+-mimetics have shown to increase inhibition properties. Structural modeling suggested that the P-N bond can stabilize the pyrophosphate group in active conformation due to the formation of an intramolecular hydrogen bond. The most active NAD+ analog against PARP-1 contained 5-iodouracil 2'-aminomethylmorpholino nucleoside with IC50 126 ± 6 µM, while in the case of PARP-2 it was adenine 2'-aminomethylmorpholino nucleoside (IC50 63 ± 10 µM). In silico analysis revealed that thymine and uracil-based NAD+ analogs were recognized as the NAD+-analog that targets the nicotinamide binding site. On the contrary, the adenine 2'-aminomethylmorpholino nucleoside-based NAD+ analogs were predicted to identify as PAR-analogs that target the acceptor binding site of PARP-2, representing a novel molecular mechanism for selective PARP inhibition. This discovery opens a new avenue for the rational design of PARP-1/2 specific inhibitors.

Characterization of DNA ADP-ribosyltransferase activities of PARP2 and PARP3: new insights into DNA ADP-ribosylation.

Poly(ADP-ribose) polymerases (PARPs) act as DNA break sensors and catalyze the synthesis of polymers of ADP-ribose (PAR) covalently attached to acceptor proteins at DNA damage sites. It has been demonstrated that both mammalian PARP1 and PARP2 PARylate double-strand break termini in DNA oligonucleotide duplexes in vitro. Here, we show that mammalian PARP2 and PARP3 can PARylate and mono(ADP-ribosyl)ate (MARylate), respectively, 5'- and 3'-terminal phosphate residues at double- and single-strand break termini of a DNA molecule containing multiple strand breaks. PARP3-catalyzed DNA MARylation can be considered a new type of reversible post-replicative DNA modification. According to DNA substrate specificity of PARP3 and PARP2, we propose a putative mechanistic model of PARP-catalyzed strand break-oriented ADP-ribosylation of DNA termini. Notably, PARP-mediated DNA ADP-ribosylation can be more effective than PARPs' auto-ADP-ribosylation depending on the DNA substrates and reaction conditions used. Finally, we show an effective PARP3- or PARP2-catalyzed ADP-ribosylation of high-molecular-weight (∼3-kb) DNA molecules, PARP-mediated DNA PARylation in cell-free extracts and a persisting signal of anti-PAR antibodies in a serially purified genomic DNA from bleomycin-treated poly(ADP-ribose) glycohydrolase-depleted HeLa cells. These results suggest that certain types of complex DNA breaks can be effectively ADP-ribosylated by PARPs in cellular response to DNA damage.

A versatile strategy for the design and synthesis of novel ADP conjugates and their evaluation as potential poly(ADP-ribose) polymerase 1 inhibitors.

A versatile strategy for the synthesis of [Formula: see text] mimetics was developed, involving an efficient pyrophosphate linkage formation in key conjugates containing a functional amino group which acts as useful reactive anchor for further derivatization. These [Formula: see text] mimetics consist of ADP conjugated through a diphosphate chain to an extended aliphatic linker bearing an aromatic acid residue. A number of conjugates containing aromatic carboxylic acids were found to inhibit poly(ADP-ribose) synthesis catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1). A new class of potential PARP-1 inhibitors mimicking [Formula: see text], a substrate in the PARP-1 catalyzed reaction, was proposed.

Y-box-binding protein 1 as a non-canonical factor of base excision repair.

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.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro.

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.

Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages.

Interaction of PARP-2 with AP site containing DNA.

In eukaryotes the stability of genome is provided by functioning of DNA repair systems. One of the main DNA repair pathways in eukaryotes is the base excision repair (BER). This system requires precise regulation for correct functioning. Two members of the PARP family - PARP-1 and PARP-2, which can be activated by DNA damage - are widely considered as regulators of DNA repair processes, including BER. In contrast to PARP-1, the role of PARP-2 in BER has not been extensively studied yet. Since AP site is one of the most frequent type of DNA damage and a key intermediate of BER at the stage preceding formation of DNA breaks, in this paper we focused on the characterization of PARP-2 interaction with AP site-containing DNAs. We demonstrated that PARP-2, like PARP-1, can interact with the intact AP site via Schiff base formation, in spite of crucial difference in the structure of the DNA binding domains of these PARPs. By cross-linking of PARPs to AP DNA, we determined that the N-terminal domains of both PARPs are involved in formation of cross-links with AP DNA. We have also confirmed that DNA binding by PARP-2, in contrast to PARP-1, is not modulated by autoPARylation. PARP-2, like PARP-1, can inhibit the activity of APE1 by binding to AP site, but, in contrast to PARP-1, this inhibitory influence is hardly regulated by PAR synthesis. At the same time, 5'-dRP lyase activity of both PARPs is comparable, although being much weaker than that of Pol ß, which is considered as the main 5'-dRP lyase of the BER process.

Interaction of PARP-2 with DNA structures mimicking DNA repair intermediates and consequences on activity of base excision repair proteins.

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Among the 17 members of the PARP family, PARP-1 and PARP-2 are described as enzymes whose catalytic activity is stimulated by some types of DNA damages. Whereas the role of PARP-1 in response to DNA damage has been widely illustrated, the contribution of another DNA-dependent PARP, PARP-2, is less documented. To find out specific DNA targets of PARP-2 we evaluated by EMSA Kd values of PARP-2-DNA complexes for several DNA structures mimicking intermediates of different DNA metabolizing processes. In addition, we tested these DNA as activators of PARP-1 and PARP-2 in poly(ADP-ribose) synthesis. Like PARP-1, PARP-2 doesn't show correlation between activation efficiency and Kd values for DNA. PARP-2 displayed the highest affinity for flap-containing DNA, but was more efficiently activated by 5'-overhang DNA. Evaluating the influence of PARP-1 and PARP-2 on DNA repair synthesis catalyzed by DNA polymerase ß revealed that both PARPs inhibit DNA polymerase ß activity. However, unlike PARP-1, poly(ADP-ribosyl)ation of PARP-2 does not result in restoration of DNA synthesis efficiency. Similarly, both PARPs proteins inhibited FEN1 activity, but only activation of PARP-1, not PARP-2, could restore FEN1 activity, and only when PARP-2 was not present. Taken together, our data show that PARP-2 can directly regulate BER proteins but also can modulate the influence of PARP-1 on these BER proteins, by decreasing its poly(ADP-ribosyl)ation activity.