Model DNA for investigation of mechanism of nucleotide excision repair

The living cell DNA is under permanent attack of a variety of exogenous and endogenous damaging factors. Nucleotide excision repair (NER) is main pathway which removes a wide variety of bulky DNA adducts formed by UV light, electrophilic environmental mutagens, and chemotherapeutic agents. NER process in mammalian cells consistently leads to the very specific excision of damaged DNA fragments 24–32 nucleotides in length. The following DNA repair synthesis and DNA ligation restore intact DNA helix. The main set of the genes inactivated in NER-deficient higher eukaryotic cells was identified; about 30 proteins are involved in the specific multi-subunit complexes responsible for NER process. The specific NER feature is wide substrate specificity and great difference of damages elimination efficiencies. A key limiting step in NER is damage recognition and verification. One of the advanced and upcoming approaches to NER process investigation is based on the application of model DNAs – artificial DNA structures, which are analogs of substrate or intermediates of the repair process. This article reviews our current knowledge concerning the model DNA design, synthesis and application as a tool for the NER process comprehensive study

Role of poly(ADP-ribose)polymerase 2 in DNA repair

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs), which use NAD+ as a substrate, synthesize polymer of (ADP)-ribose (PAR) covalently attached to nuclear proteins including PARP themselves. PARPs constitute a large family of proteins, in which PARP1 is the most abundant and best-characterized member. In spite of growing body of PARPs’ role in cellular processes, PARP2, the closest homolog of PARP1, still remains poorly characterized at the level of its contribution to different pathways of DNA repair. An overview summarizes in vivo and in vitro data on PARP2 implication in specialized DNA repair processes, base excision repair and double strand break repair

Interaction of nucleotide excision repair factors RPA and XPA with DNA containing bulky photoreactive groups imitating damages

Interaction of nucleotide excision repair factors-replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)-with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5′-end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into DNA. The mechanism of damage recognition in the process of nucleotide excision repair is discussed.</p

Interaction of nucleotide excision repair factors RPA and XPA with DNA containing bulky photoreactive groups imitating damages

Interaction of nucleotide excision repair factors-replication protein A (RPA) and Xeroderma pigmentosum complementing group A protein (XPA)-with DNA structures containing nucleotides with bulky photoreactive groups imitating damaged nucleotides was investigated. Efficiency of photoaffinity modification of two proteins by photoreactive DNAs varied depending on DNA structure and type of photoreactive group. The secondary structure of DNA and, first of all, the presence of extended single-stranded parts plays a key role in recognition by RPA. However, it was shown that RPA efficiently interacts with DNA duplex containing a bulky substituent at the 5′-end of a nick. XPA was shown to prefer the nicked DNA; however, this protein was cross-linked with approximately equal efficiency by single-stranded and double-stranded DNA containing a bulky substituent inside the strand. XPA seems to be sensitive not only to the structure of DNA double helix, but also to a bulky group incorporated into DNA. The mechanism of damage recognition in the process of nucleotide excision repair is discussed.</p

Interaction of PARP2 with DNA structures mimicking DNA repair intermediates

Poly(ADP-ribosyl)ation is a posttranslational protein modification significant for the genomic stability and cell survival in response to DNA damage. Poly(ADP-ribosyl)ation is catalyzed by poly(ADP-ribose)polymerases (PARPs). Whereas the role of PARP1 in response to DNA damage has been widely illustrated, the contribution of another DNA-dependent PARP, PARP2, has not been studied so far. Aim. To find out specific DNA targets of PARP2. Methods. The EMSA and the PARP activity tests were used. Results. We evaluated Kd values of PARP2-DNA complexes for several DNA structures mimicking intermediates of different DNA metabolizing processes and tested these DNA as «activators» of PARP1 and PARP2 in poly(ADP-ribose) synthesis. Conclusions. Like PARP1, PARP2 does not show correlation between the activation efficiency and Kd values for DNA. PARP2 was activated most effectively in the presence of over5DNA

Interplay Between Mitophagy and Apoptosis Defines a Cell Fate Upon Co-treatment of Breast Cancer Cells With a Recombinant Fragment of Human κ-Casein and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand

A recombinant fragment of human k-Casein, termed RL2, induces cell death of breast cancer cells; however,molecularmechanisms of RL2-mediated cell death have remained largely unknown. In the current study, we have decoded the molecular mechanism of the RL2-mediated cell death and found that RL2 acts via the induction of mitophagy. This was monitored by the loss of adenosine triphosphate production, LC3B-II generation, and upregulation of BNIP3 and BNIP3L/NIX, as well as phosphatase and tensin homolog-induced kinase 1. Moreover, we have analyzed the cross talk of this pathway with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis upon combinatorial treatment with RL2 and TRAIL. Strikingly, we found two opposite effects of this co-treatment. RL2 had inhibitory effects on TRAIL-induced cell death upon short-term co-stimulation. In particular, RL2 treatment blocked TRAIL-mediated caspase activation, cell viability loss, and apoptosis, which was mediated via the downregulation of the core proapoptotic regulators. Contrary to short-termco-treatment, upon long-term co-stimulation, RL2 sensitized the cells toward TRAIL-induced cell death; the latter observation provides the basis for the development of therapeutic approaches in breast cancer cells. Collectively, our findings have important implications for cancer therapy and reveal the molecular switches of the cross talk between RL2-induced mitophagy and TRAIL-mediated apoptosis.DFG-Publikationsfonds 202

Glycyrrhetinic acid and its derivatives as inhibitors of poly(ADP-ribose)polymerases 1 and 2, apurinic/apyrimidinic endonuclease 1 and DNA polymerase β

Aim. For strengthening the efficiency of monofunctional alkylating antineoplastic drugs it is important to lower the capacity of base excision repair (BER) system which corrects the majority of DNA damages caused by these reagents. The objective was to create inhibitors of the key BER enzymes (PARP1, PARP2, DNA polymerase β, and APE1) by the directed modification of glycyrrhetinic acid (GA). Methods. Amides of GA were produced from the GA acetate by formation of the corresponding acyl chloride, amidation with the appropriate amine and subsequent deacylation. Small library of 2-cyano substituted derivatives of GA methyl esters was obtained by the structural modification of GA framework and carboxylic acid group. The inhibitory capacity of the compounds was estimated by comparison of the enzyme activities in specific tests in the presence of compounds versus their absence. Results. None of tested compounds inhibits PARP1 significantly. Unmodified GA and its morpholinic derivative were shown to be weak inhibitors of PARP2. The derivatives of GA containing keto-group in 11 triterpene framework were shown to be moderate inhibitors of pol β. Compound 3, containing 12-oxo-9(11)-en moiety in the ring C, was shown to be a single inhibitor of APE1 among all compounds studied. Conclusions. The class of GA derivatives, selective pol β inhibitors, was found out. The selective inhibitor of APE1 and weak selective inhibitor of PARP2 were also revealed

Evidence for direct contact between the RPA3 subunit of the human replication protein A and single-stranded DNA

Replication Protein A is a single-stranded (ss) DNA-binding protein that is highly conserved in eukaryotes and plays essential roles in many aspects of nucleic acid metabolism, including replication, recombination, DNA repair and telomere maintenance. It is a heterotrimeric complex consisting of three subunits: RPA1, RPA2 and RPA3. It possesses four DNA-binding domains (DBD), DBD-A, DBD-B and DBD-C in RPA1 and DBD-D in RPA2, and it binds ssDNA via a multistep pathway. Unlike the RPA1 and RPA2 subunits, no ssDNA-RPA3 interaction has as yet been observed although RPA3 contains a structural motif found in the other DBDs. We show here using 4-thiothymine residues as photoaffinity probe that RPA3 interacts directly with ssDNA on the 3′-side on a 31 nt ssDNA

Role of apoptosis genes in aggression revealed using combined analysis of ANDSystem gene networks, expression and genomic data in grey rats with aggressive behavior

Aggressive behavior in animals plays an important role in protecting the territory, offspring, establishing social hierarchical relations, etc. Increased aggression is observed in a number of diseases ( schizophrenia, bipolar disorder, brain degenerative disorders). Neuronal apoptosis is crucial in the maintenance of developmental processes during neurogenesis. Alterations in neuronal apoptosis are observed in aging and neuropathologies accompanied by changes in psycho­emo­ tional state (epilepsy, Alzheimer’s disease, neurotrauma). The expression of key neuronal apoptosis genes (Casp3, Bax and Bcl-xl) in the brain of highly aggressive rats is significantly altered. The aim of this work was to analyze associative networks that describe genetic interactions between genes/proteins involved in neuronal apoptosis, differentially expressed genes and genes with polymorphisms in grey rats with aggressive behavior. Analysis revealed 819 differentially expressed genes in the hypothalamus, ventral tegmental region and periaqueductus Sylvii grey matter in grey rats with aggressive and tame behavior. The Stx1a, Mbp and Th genes have the highest index of betweenness centrality in the associative network of differentially expressed genes. Genome analysis revealed 137 polymorphic genes. Three of them (Lig4, Parp1 and Pigt) were involved in neuronal apoptosis. It was shown that polymorphic and differentially expressed genes were statistically significantly overrepresented among ge nes interacting with neuronal apoptosis genes (p value &lt; 0.01). Three molecular­genetic chains describing connections between polymorphic and neuronal apoptosis genes mediated by differentially expressed genes were reconstructed. Chains included the polymorphic genes Tsc1, Adamts4 and Lgals3, differentially expressed genes Ezr, Acan, Th and 19 neuronal apoptosis genes. It was shown that neuronal apoptosis is closely related to aggressive behavior in animals

ANALYSIS OF THE INTERACTIONS OF NEURONAL APOPTOSIS GENES IN THE ASSOCIATIVE GENE NETWORK OF PARKINSON’S DISEASE

Parkinson’s disease (PD) affects an estimated 7–10 million people worldwide and 210000 people in Russia. PD is accompanied by degeneration of dopaminergic neurons and because of that neuronal apoptosis is an important factor in this disease. Analysis of gene networks is one of the key approaches in systems biology. We previously developed the ANDSystem tool, designed to automatically extract knowledge from scientific publications and reconstruct on this basis associative gene networks describing the molecular genetic mechanisms of biological processes. The aim of this work was prioritization of neuronal apoptosis genes by their involvement in PD pathogenesis, taking into account the structure of the PD associative gene network using ANDSystem. Analysis of the centrality of neuronal apoptosis genes, associated with PD, revealed that mean values of degree, closeness and betweenness centralities statistically significantly exceed such values of all nodes of the PD network. The APOE, CASP3 and GAPDH genes involved in neuronal apoptosis were among the most central genes. Prioritization of neuronal apoptosis genes for which there was no data in ANDSystem on their associations with PD was performed using standard methods (Endeavor and ToppGene) and the criteria of centrality and specificity of genes interactions with the PD gene network. Analysis revealed that genes involved in such processes as positive and negative regulation of neu ronal apoptosis, MAPK and ephrin receptor signaling pathways, are mainly represented among candidate genes with the highest priority (top 50, 70, 100 genes were considered). In particular, TP53, JUN, BCL2, PIK3CA and APP were among candidate genes with the highest priority