Maria V. Sukhanova

Human base excision repair enzymes apurinic/apyrimidinic endonuclease1 (APE1), DNA polymerase β and poly(ADP-ribose) polymerase 1: interplay between strand-displacement DNA synthesis and proofreading exonuclease activity

We examined interactions between base excision repair (BER) DNA intermediates and purified human BER enzymes, DNA polymerase β (pol β), apurinic/apyrimidinic endonuclease (APE1) and poly(ADP-ribose) polymerase-1 (PARP-1). Studies under steady-state conditions with purified BER enzymes and BER substrates have already demonstrated interplay between these BER enzymes that is sensitive to the respective concentrations of each enzyme. Therefore, in this study, using conditions of enzyme excess over substrate DNA, we further examine the question of interplay between BER enzymes on BER intermediates. The results reveal several important differences compared with data obtained using steady-state assays. Excess PARP-1 antagonizes the action of pol β, producing a complete block of long patch BER strand-displacement DNA synthesis. Surprisingly, an excess of APE1 stimulates strand-displacement DNA synthesis by pol β, but this effect is blocked by PARP-1. The APE1 exonuclease function appears to be modulated by the other BER proteins. Excess APE1 over pol β may allow APE1 to perform both exonuclease function and stimulation of strand-displacement DNA synthesis by pol β. This enables pol β to mediate long patch sub-pathway. These results indicate that differences in the stoichiometry of BER enzymes may regulate BER.

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.

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.

Poly(ADP-ribose) Polymerase 1 Modulates Interaction of the Nucleotide Excision Repair Factor XPC-RAD23B with DNA via Poly(ADP-ribosyl)ation

Background: Poly(ADP-ribosyl)ation of DNA repair proteins is essential for the regulation of DNA repair processes. Results: Both subunits of the nucleotide excision repair factor XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. Conclusion: PARP1 influences the interaction of XPC-RAD23B with DNA via PAR synthesis. Significance: This study provides direct evidence for XPC-RAD23B belonging to the targets of poly(ADP-ribosyl)ation catalyzed by PARP1. Poly(ADP-ribosyl)ation is a reversible post-translational modification that plays an essential role in many cellular processes, including regulation of DNA repair. Cellular DNA damage response by the synthesis of poly(ADP-ribose) (PAR) is mediated mainly by poly(ADP-ribose) polymerase 1 (PARP1). The XPC-RAD23B complex is one of the key factors of nucleotide excision repair participating in the primary DNA damage recognition. By using several biochemical approaches, we have analyzed the influence of PARP1 and PAR synthesis on the interaction of XPC-RAD23B with damaged DNA. Free PAR binds to XPC-RAD23B with an affinity that depends on the length of the poly(ADP-ribose) strand and competes with DNA for protein binding. Using 32P-labeled NAD+ and immunoblotting, we also demonstrate that both subunits of the XPC-RAD23B are poly(ADP-ribosyl)ated by PARP1. The efficiency of XPC-RAD23B PARylation depends on DNA structure and increases after UV irradiation of DNA. Therefore, our study clearly shows that XPC-RAD23B is a target of poly(ADP-ribosyl)ation catalyzed by PARP1, which can be regarded as a universal regulator of DNA repair processes.

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 β 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 β 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)polymerase 1 stimulates the AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1

Poly(ADP-ribose)polymerase 1 (PARP1), the key DNA repair regulator, physically and functionally interacts with tyrosyl-DNA phosphodiesterase 1 (TDP1) and stimulates its abasic site cleaving activity. These interactions allow suggesting PARP1 contribution in the alternative Tdp1 related repair pathway.

Influence of Usnic Acid and its Derivatives on the Activity of Mammalian Poly(ADP-ribose)polymerase 1 and DNA Polymerase β

The influence of a number of usnic acid derivatives on auto(polyADP-ribosyl)ation catalyzed by PARP1 and DNA synthesis catalyzed by DNA polymerase β was studied. The derivatives of usnic acid containing aromatic substituents were shown to be moderate inhibitors of PARP1. The presence of both usnic acid tricyclic structure and aromatic substituent at any position of the molecule is a key factor for the inhibitory action. In the case of DNA polymerase β, no relationship between the structure and inhibitory properties has been found with the only exception. Derivatives with modified ring A showed mild activation of DNA synthesis catalyzed by DNA polymerase β.

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

Replication protein A as a modulator of the poly(ADP-ribose)polymerase 1 activity

\hbox {NAD}^{+}