Base Excision Repair Intermediates Research Articles

Unrepaired base excision repair intermediates in template DNA strands trigger replication fork collapse and PARP inhibitor sensitivity.

DNA single-strand breaks (SSBs) disrupt DNA replication and induce chromosome breakage. However, whether SSBs induce chromosome breakage when present behind replication forks or ahead of replication forks is unclear. To address this question, we exploited an exquisite sensitivity of SSB repair-defective human cells lacking PARP activity or XRCC1 to the thymidine analogue 5-chloro-2'-deoxyuridine (CldU). We show that incubation with CldU in these cells results in chromosome breakage, sister chromatid exchange, and cytotoxicity by a mechanism that depends on the S phase activity of uracil DNA glycosylase (UNG). Importantly, we show that CldU incorporation in one cell cycle is cytotoxic only during the following cell cycle, when it is present in template DNA. In agreement with this, while UNG induces SSBs both in nascent strands behind replication forks and in template strands ahead of replication forks, only the latter trigger fork collapse and chromosome breakage. Finally, we show that BRCA-defective cells are hypersensitive to CldU, either alone and/or in combination with PARP inhibitor, suggesting that CldU may have clinical utility.

The Impact of Human DNA Glycosylases on the Activity of DNA Polymerase β toward Various Base Excision Repair Intermediates.

Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligase. Coordination of BER is implemented by multiple protein-protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polβ's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polβ efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5'-dRP-mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polβ's activity toward the model DNA intermediates.

Abasic site-peptide cross-links are blocking lesions repaired by AP endonucleases.

Apurinic/apyrimidinic (AP) sites are abundant DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and as base excision repair (BER) intermediates. AP sites and their derivatives readily trap DNA-bound proteins, resulting in DNA-protein cross-links. Those are subject to proteolysis but the fate of the resulting AP-peptide cross-links (APPXLs) is unclear. Here, we report two in vitro models of APPXLs synthesized by cross-linking of DNA glycosylases Fpg and OGG1 to DNA followed by trypsinolysis. The reaction with Fpg produces a 10-mer peptide cross-linked through its N-terminus, while OGG1 yields a 23-mer peptide attached through an internal lysine. Both adducts strongly blocked Klenow fragment, phage RB69 polymerase, Saccharolobus solfataricus Dpo4, and African swine fever virus PolX. In the residual lesion bypass, mostly dAMP and dGMP were incorporated by Klenow and RB69 polymerases, while Dpo4 and PolX used primer/template misalignment. Of AP endonucleases involved in BER, Escherichia coli endonuclease IV and its yeast homolog Apn1p efficiently hydrolyzed both adducts. In contrast, E. coli exonuclease III and human APE1 showed little activity on APPXL substrates. Our data suggest that APPXLs produced by proteolysis of AP site-trapped proteins may be removed by the BER pathway, at least in bacterial and yeast cells.

Visualizing the coordination of apurinic/apyrimidinic endonuclease (APE1) and DNA polymerase β during base excision repair

Base Excision Repair (BER) is carried out by a series of proteins that function in a step-by-step process to identify, remove, and replace DNA damage. During BER, the DNA transitions through various intermediate states as it is processed by each DNA repair enzyme. Left unrepaired, these BER intermediates can transition into double-stranded DNA breaks and promote genome instability. Previous studies have proposed a short-lived complex consisting of the BER intermediate, the incoming enzyme, and the outgoing enzyme at each step of the BER pathway to protect the BER intermediate. The transfer of BER intermediates between enzymes, known as BER coordination or substrate channeling, remains poorly understood. Here, we utilize single-molecule total internal reflection fluorescence (TIRF) microscopy to investigate the mechanism of BER coordination between apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (Pol β). When preformed complexes of APE1 and the incised abasic site product (APE1 product and Pol β substrate) were subsequently bound by Pol β, the Pol β enzyme dissociated shortly after binding in most of the observations. In the events where Pol β binding was followed by APE1 dissociation during substrate channeling, Pol β remained bound for a longer period of time to allow disassociation of APE1. Our results indicate that transfer of the BER intermediate from APE1 to Pol β during BER is dependent on the dissociation kinetics of APE1 and the duration of the ternary complex on the incised abasic site.

Chemical Tools for the Study of DNA Repair.

ConspectusDNA repair enzymes continuously provide surveillance throughout our cells, protecting the enclosed DNA from the damage that is constantly arising from oxidation, alkylating species, and radiation. Members of this enzyme class are intimately linked to pathways controlling cancer and inflammation and are promising targets for diagnostics and future therapies. Their study is benefiting widely from the development of new tools and methods aimed at measuring their activities. Here, we provide an Account of our laboratory's work on developing chemical tools to study DNA repair processes in vitro, as well as in cells and tissues, and what we have learned by applying them.We first outline early work probing how DNA repair enzymes recognize specific forms of damage by use of chemical analogs of the damage with altered shapes and H-bonding abilities. One outcome of this was the development of an unnatural DNA base that is incorporated selectively by polymerase enzymes opposite sites of missing bases (abasic sites) in DNA, a very common form of damage.We then describe strategies for design of fluorescent probes targeted to base excision repair (BER) enzymes; these were built from small synthetic DNAs incorporating fluorescent moieties to engender light-up signals as the enzymatic reaction proceeds. Examples of targets for these DNA probes include UDG, SMUG1, Fpg, OGG1, MutYH, ALKBH2, ALKBH3, MTH1, and NTH1. Several such strategies were successful and were applied both in vitro and in cellular settings; moreover, some were used to discover small-molecule modulators of specific repair enzymes. One of these is the compound SU0268, a potent OGG1 inhibitor that is under investigation in animal models for inhibiting hyperinflammatory responses.To investigate cellular nucleotide sanitation pathways, we designed a series of "two-headed" nucleotides containing a damaged DNA nucleotide at one end and ATP at the other; these were applied to studying the three human sanitation enzymes MTH1, dUTPase, and dITPase, some of which are therapeutic targets. The MTH1 probe (ARGO) was used in collaboration with oncologists to measure the enzyme in tumors as a disease marker and also to develop the first small-molecule activators of the enzyme.We proceed to discuss the development of a "universal" probe of base excision repair processes (UBER), which reacts covalently with abasic site intermediates of base excision repair. UBER probes light up in real time as the reaction occurs, enabling the observation of base excision repair as it occurs in live cells and tissues. UBER probes can also be used in efficient and simple methods for fluorescent labeling of DNA. Finally, we suggest interesting directions for the future of this field in biomedicine and human health.

Human TREX1 Repairs 3'-End DNA Lesions in Vitro.

Human three-prime repair exonuclease 1 (TREX1) is the major 3' to 5' exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3'-phospho-α,β-unsaturated aldehyde and 3'-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.

FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2’-deoxycytidine and 5-hydroxymethyl-2’-deoxyuridine

Fanconi anemia (FA) is a rare hereditary disorder caused by mutations in any one of the FANC genes. FA cells are mainly characterized by extreme hypersensitivity to interstrand crosslink (ICL) agents. Additionally, the FA proteins play a crucial role in concert with homologous recombination (HR) factors to protect stalled replication forks. Here, we report that the 5-methyl-2’-deoxycytidine (5mdC) demethylation (pathway) intermediate 5-hydroxymethyl-2’-deoxycytidine (5hmdC) and its deamination product 5-hydroxymethyl-2’-deoxyuridine (5hmdU) elicit a DNA damage response, chromosome aberrations, replication fork impairment and cell viability loss in the absence of FANCD2. Interestingly, replication fork instability by 5hmdC or 5hmdU was associated to the presence of Poly(ADP-ribose) polymerase 1 (PARP1) on chromatin, being both phenotypes exacerbated by olaparib treatment. Remarkably, Parp1−/− cells did not show any replication fork defects or sensitivity to 5hmdC or 5hmdU, suggesting that retained PARP1 at base excision repair (BER) intermediates accounts for the observed replication fork defects upon 5hmdC or 5hmdU incorporation in the absence of FANCD2. We therefore conclude that 5hmdC is deaminated in vivo to 5hmdU, whose fixation by PARP1 during BER, hinders replication fork progression and contributes to genomic instability in FA cells.

Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses.

Mitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

Fluorescence Imaging of Mitochondrial DNA Base Excision Repair Reveals Dynamics of Oxidative Stress Responses

AbstractMitochondrial function in cells declines with aging and with neurodegeneration, due in large part to accumulated mutations in mitochondrial DNA (mtDNA) that arise from deficient DNA repair. However, measuring this repair activity is challenging. We employ a molecular approach for visualizing mitochondrial base excision repair (BER) activity in situ by use of a fluorescent probe (UBER) that reacts rapidly with AP sites resulting from BER activity. Administering the probe to cultured cells revealed signals that were localized to mitochondria, enabling selective observation of mtDNA BER intermediates. The probe showed elevated DNA repair activity under oxidative stress, and responded to suppression of glycosylase activity. Furthermore, the probe illuminated the time lag between the initiation of oxidative stress and the initial step of BER. Absence of MTH1 in cells resulted in elevated demand for BER activity upon extended oxidative stress, while the absence of OGG1 activity limited glycosylation capacity.

DNA polymerase θ promotes CAG•CTG repeat expansions in Huntington’s disease via insertion sequences of its catalytic domain

Huntington's disease (HD), a neurodegenerative disease characterized by progressive dementia, psychiatric problems, and chorea, is known to be caused by CAG repeat expansions in the HD gene HTT. However, the mechanism of this pathology is not fully understood. The translesion DNA polymerase θ (Polθ) carries a large insertion sequence in its catalytic domain, which has been shown to allow DNA loop-outs in the primer strand. As a result of high levels of oxidative DNA damage in neural cells and Polθ's subsequent involvement in base excision repair of oxidative DNA damage, we hypothesized that Polθ contributes to CAG repeat expansion while repairing oxidative damage within HTT. Here, we performed Polθ-catalyzed in vitro DNA synthesis using various CAG•CTG repeat DNA substrates that are similar to base excision repair intermediates. We show that Polθ efficiently extends (CAG)n•(CTG)n hairpin primers, resulting in hairpin retention and repeat expansion. Polθ also triggers repeat expansions to pass the threshold for HD when the DNA template contains 35 repeats upward. Strikingly, Polθ depleted of the catalytic insertion fails to induce repeat expansions regardless of primers and templates used, indicating that the insertion sequence is responsible for Polθ's error-causing activity. In addition, the level of chromatin-bound Polθ in HD cells is significantly higher than in non-HD cells and exactly correlates with the degree of CAG repeat expansion, implying Polθ's involvement in triplet repeat instability. Therefore, we have identified Polθ as a potent factor that promotes CAG•CTG repeat expansions in HD and other neurodegenerative disorders.

DNA glycosylase deficiency leads to decreased severity of lupus in the Polb-Y265C mouse model

The Polb gene encodes DNA polymerase beta (Pol β), a DNA polymerase that functions in base excision repair (BER) and microhomology-mediated end-joining. The Pol β-Y265C protein exhibits low catalytic activity and fidelity, and is also deficient in microhomology-mediated end-joining. We have previously shown that the PolbY265C/+ and PolbY265C/C mice develop lupus. These mice exhibit high levels of antinuclear antibodies and severe glomerulonephritis. We also demonstrated that the low catalytic activity of the Pol β-Y265C protein resulted in accumulation of BER intermediates that lead to cell death. Debris released from dying cells in our mice could drive development of lupus. We hypothesized that deletion of the Neil1 and Ogg1 DNA glycosylases that act upstream of Pol β during BER would result in accumulation of fewer BER intermediates, resulting in less severe lupus. We found that high levels of antinuclear antibodies are present in the sera of PolbY265C/+ mice deleted of Ogg1 and Neil1 DNA glycosylases. However, these mice develop significantly less severe renal disease, most likely due to high levels of IgM in their sera.

XRCC1 prevents toxic PARP1 trapping during DNA base excision repair

SummaryMammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1−/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity.

The coordination between DNA polymerase β and ligase governs the formation of mutagenic repair intermediates as an important determinant of faithful base excision repair

Base excision repair (BER) is a critical process for preventing the mutagenic and lethal consequences of DNA damage that arises from endogenous and environmental agents and underlies disease and aging. BER pathway involves a series of sequential enzymatic steps that are tightly coordinated in a process known as the substrate-product channeling between repair proteins so that release of cytotoxic BER intermediates is minimized. DNA ligase I and III catalyze the ultimate ligation step following DNA polymerase (pol) β nucleotide insertion at downstream steps of BER pathway. DNA ligases and DNA polymerases that play roles in DNA replication and repair transactions have long been prime candidates for key regulators of genome stability. However, the ligation efficiency of pol β-promoted mutagenesis products with 3′-DNA mismatches or 8-oxo-2'-deoxyguanosine (8-oxodG) at the downstream steps of BER pathway remains undefined. We show that pol β 8-oxo-2'-deoxyribonucleoside 5'-triphosphate (8-oxodGTP) insertion confounds DNA ligases leading to the formation of ligation failure products with a 5'-adenylate (AMP) block. However, pol β Watson-Crick-like dGTP mismatch insertion opposite T can be efficiently ligated by the BER DNA ligases in vitro. Moreover, we report the mutagenic ligation of pol β 8-oxodGTP insertion products by the LIG1 MgHiFimutant with a perturbed fidelity and enhanced ligation failure by the variants of LIG1 deficiency disease that exhibits the symptoms of developmental abnormalities, immunodeficiency, lymphoma, and predisposition to cancer. Our results also demonstrate that the BER DNA ligases are compromised by subtle changes in all 12 possible noncanonical base pairs at the 3′-end of the nicked repair intermediate. Finally, we found that Aprataxin and Flap Endonuclease 1, as compensatory DNA-end processing enzymes, can remove the 5'-AMP block from the abortive ligation products harboring 3′-8-oxodG or all possible mispairs. These findings contribute to understanding of how the identity of the damaged or mismatch affects the substrate channeling of the repair pathway, importance of mechanism underlying the interplay between pol β and DNA ligases for faithful BER, and how a multi-protein complex can coordinate to maintain the repair efficiency.

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice

N-Nitrosodimethylamine (NDMA) is a DNA-methylating agent that has been discovered to contaminate water, food, and drugs. The alkyladenine DNA glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we study Aag-knockout (Aag-/-) and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine [3MeA]) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switches from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrate a suite of molecular, cellular, and physiological analyses. We find that unrepaired 3MeA is somewhat toxic, but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tip the balance between blocks and breaks and thus dictate the disease consequences of DNA damage.

DNA polymerase β outperforms DNA polymerase γ in key mitochondrial base excision repair activities

DNA polymerase beta (POLβ), well known for its role in nuclear DNA base excision repair (BER), has been shown to be present in the mitochondria of several different cell types. Here we present a side-by-side comparison of BER activities of POLβ and POLγ, the mitochondrial replicative polymerase, previously thought to be the only mitochondrial polymerase. We find that POLβ is significantly more proficient at single-nucleotide gap filling, both in substrates with ends that require polymerase processing, and those that do not. We also show that POLβ has a helicase-independent functional interaction with the mitochondrial helicase, TWINKLE. This interaction stimulates strand-displacement synthesis, but not single-nucleotide gap filling. Importantly, we find that purified mitochondrial extracts from cells lacking POLβ are severely deficient in processing BER intermediates, suggesting that mitochondrially localized DNA POLβ may be critical for cells with high energetic demands that produce greater levels of oxidative stress and therefore depend upon efficient BER for mitochondrial health.

Dynamic Processing of a Common Oxidative DNA Lesion by the First Two Enzymes of the Base Excision Repair Pathway

Base excision repair (BER) is the primary pathway by which eukaryotic cells resolve single base damage. One common example of single base damage is 8-oxo-7,8-dihydro-2ʹ-deoxoguanine (8-oxoG). High incidence and mutagenic potential of 8-oxoG necessitate rapid and efficient DNA repair. How BER enzymes coordinate their activities to resolve 8-oxoG damage while limiting cytotoxic BER intermediates from propagating genomic instability remains unclear. Here we use single-molecule Förster resonance energy transfer (smFRET) and ensemble-level techniques to characterize the activities and interactions of consecutive BER enzymes important for repair of 8-oxoG. In addition to characterizing the damage searching and processing mechanisms of human 8-oxoguanine glycosylase 1 (hOGG1), our data support the existence of a ternary complex between hOGG1, the damaged DNA substrate, and human AP endonuclease 1 (APE1). Our results indicate that hOGG1 is actively displaced from its abasic site containing product by protein–protein interactions with APE1 to ensure timely repair of damaged DNA.

Significance of base excision repair to human health.

Oxidative and alkylating DNA damage occurs under normal physiological conditions and exogenous exposure to DNA damaging agents. To counteract DNA base damage, cells have evolved several defense mechanisms that act at different levels to prevent or repair DNA base damage. Cells combat genomic lesions like these including base modifications, abasic sites, as well as single-strand breaks, via the base excision repair (BER) pathway. In general, the core BER process involves well-coordinated five-step reactions to correct DNA base damage. In this review, we will uncover the current understanding of BER mechanisms to maintain genomic stability and the biological consequences of its failure due to repair gene mutations. The malfunction of BER can often lead to BER intermediate accumulation, which is genotoxic and can lead to different types of human disease. Finally, we will address the use of BER intermediates for targeted cancer therapy.

XRCC1 Prevents Toxic PARP1 Trapping During DNA Base Excision Repair

Mammalian DNA base excision repair (BER) is an essential pathway comprised of damaged base excision by a DNA glycosylase, incision by AP endonuclease-1 (APE1), end processing and gap filling by DNA polymerase β (POLβ), and DNA ligation by DNA ligase I (LIG1) or DNA ligase III (LIG3). In mammals, BER additionally employs PARP1 and/or PARP2 and the scaffold protein XRCC1 to accelerate and coordinate the overall process. Whereas PARP1 and PARP2 are sensor proteins that detect unrepaired DNA single-strand breaks, the essential role of XRCC1 during BER is unknown. Here, we have identified this role. We show that the DNA repair protein complexes that are assembled by XRCC1 compete with and prevent excessive PARP1 engagement during BER, which otherwise leads to PARP1 ‘trapping’ on BER intermediates in a manner reminiscent of that induced by clinical PARP inhibitors. We demonstrate that this elevated engagement and trapping of PARP1 collectively renders BER intermediates inaccessible to POLβ and impedes their repair. Consequently, PARP1 deletion rescues both the accessibility and repair of BER intermediates in XRCC1-/- cells, and also their cellular resistance to DNA base damage. These data demonstrate that PARP1 trapping is an endogenous threat to genome integrity, and identify XRCC1 as an “anti-trapper” that prevents the toxic binding of PARP1 to BER intermediates to ensure their efficient repair.

Excision of Mutagenic Replication-Blocking Lesions Suppresses Cancer But Promotes Cytotoxicity and Lethality in Nitrosamine-Exposed Mice

N-nitrosodimethylamine (NDMA) is a DNA methylating agent that has been discovered to contaminate water, food and drugs. The alkyladenine glycosylase (AAG) removes methylated bases to initiate the base excision repair (BER) pathway. To understand how gene-environment interactions impact disease susceptibility, we studied Aag-/- and Aag-overexpressing mice that harbor increased levels of either replication-blocking lesions (3-methyladenine, or 3MeA) or strand breaks (BER intermediates), respectively. Remarkably, the disease outcome switched from cancer to lethality simply by changing AAG levels. To understand the underlying basis for this observation, we integrated a suite of molecular, cellular and physiological analyses. We found that unrepaired 3MeA is somewhat toxic but highly mutagenic (promoting cancer), whereas excess strand breaks are poorly mutagenic and highly toxic (suppressing cancer and promoting lethality). We demonstrate that the levels of a single DNA repair protein tips the balance between blocks and breaks, and thus dictates the disease consequences of DNA damage.

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.