An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes.

Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5'-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.

Characterization of Aspergillus niger endo-1,4-ß-glucanase ENG1 secreted from Saccharomyces cerevisiae using different expression vectors.

Heterologous expression of Aspergillus niger endo-1,4-ß-glucanase (ENG1) in Saccharomyces cerevisiae was tested both with an episomal plasmid vector (YEGAp/eng1) and a yeast vector capable of integration into the HO locus of the S. cerevisiae chromosome (pHO-GAPDH-eng1-KanMX4-HO). In both cases, eng1 gene expression in yeast, with its native signal sequence for secretion, was under the control of the strong glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter. We aimed to verify how each expression system affects protein expression, posttranslational modification, and biochemical properties. Expression of eng1 from the episomal plasmid vector YEGAp/eng1 significantly slowed the growth of a yeast cell culture. However, expression of eng1 from the vector integrated into the HO locus of the chromosome did not cause growth suppression, and the enzyme activity in a culture supernatant was maintained throughout the incubation time. ENG1 has optimum catalytic activity at pH 6.0, and is stable in the pH range 5.0-9.0. The enzyme's optimum temperature for catalytic activity at pH 6.0 is 70°C; importantly, more than 95% of the enzyme's initial activity remained after a 2-h incubation at 60°C. The biochemical characterization of ENG1 confirmed the correct expression of the protein and showed that ENG1 expressed by the pHO-GAPDH-eng1-KanMX4-HO vector, in addition to its N-linked sites, is overglycosylated at its O-glycosylation sites compared with ENG1 expressed by the YEGAp/eng1 vector. It is likely that the O-glycosylated form of the A. niger ENG1 retains more stable activity during continuous cultivation of recombinant yeasts than the form that is only N-glycosylated.

Kinetic mechanism of the interaction of Saccharomyces cerevisiae AP-endonuclease 1 with DNA substrates.

The apurinic/apyrimidinic endonuclease from Saccharomyces cerevisiae Apn1 is one of the key enzymes involved in base excision repair of DNA lesions. A major function of the enzyme is to cleave the upstream phosphodiester bond of an apurinic/apyrimidinic site (AP-site), leading to the formation of a single-strand break with 3'-hydroxyl (OH) and 5'-deoxyribose phosphate (dRP) termini. In this study, the pre-steady-state kinetics and conformational dynamics of DNA substrates during their interaction with Apn1 were investigated. A stopped-flow method with detection of the fluorescence intensity of 2-aminopurine and pyrrolocytosine located adjacent or opposite to the damage was used. It was found that upon interaction with Apn1, both DNA strands undergo a number of rapid changes. The location of fluorescent analogs of heterocyclic bases in DNA does not influence the catalytic step of the reaction. Comparison of data obtained for yeast Apn1 and reported data (Kanazhevskaya, L. Yu., Koval, V. V., Vorobjev, Yu. N., and Fedorova, O. S. (2012) Biochemistry, 51, 1306-1321) for human Ape1 revealed some differences in their interaction with DNA substrates.

Initiation of 8-oxoguanine base excision repair within trinucleotide tandem repeats.

Trinucleotide repeat expansion provides a molecular basis for several devastating neurodegenerative diseases. In particular, expansion of a CAG run in the human HTT gene causes Huntington's disease. One of the main reasons for triplet repeat expansion in somatic cells is base excision repair (BER), involving damaged base excision and repair DNA synthesis that may be accompanied by expansion of the repaired strand due to formation of noncanonical DNA structures. We have analyzed the kinetics of excision of a ubiquitously found oxidized purine base, 8-oxoguanine (oxoG), by DNA glycosylase OGG1 from the substrates containing a CAG run flanked by AT-rich sequences. The values of k(2) rate constant for the removal of oxoG from triplets in the middle of the run were higher than for oxoG at the flanks of the run. The value of k(3) rate constant dropped starting from the third CAG-triplet in the run and remained stable until the 3'-terminal triplet, where it decreased even more. In nuclear extracts, the profile of oxoG removal rate along the run resembled the profile of k(2) constant, suggesting that the reaction rate in the extracts is limited by base excision. The fully reconstituted BER was efficient with all substrates unless oxoG was near the 3'-flank of the run, interfering with the initiation of the repair. DNA polymerase ß was able to perform a strand-displacement DNA synthesis, which may be important for CAG run expansion initiated by BER.

Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair.

Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.

Conformational dynamics of human AP endonuclease in base excision and nucleotide incision repair pathways.

APE1 is a multifunctional enzyme that plays a central role in base excision repair (BER) of DNA. APE1 is also involved in the alternative nucleotide incision repair (NIR) pathway. We present an analysis of conformational dynamics and kinetic mechanisms of the full-length APE1 and truncated NDelta61-APE1 lacking the N-terminal 61 amino acids (REF1 domain) in BER and NIR pathways. The action of both enzyme forms were described by identical kinetic schemes, containing four stages corresponding to formation of the initial enzyme-substrate complex and isomerization of this complex; when a damaged substrate was present, these stages were followed by an irreversible catalytic stage resulting in the formation of the enzyme-product complex and the equilibrium stage of product release. For the first time we showed, that upon binding AP-containing DNA, the APE1 structure underwent conformational changes before the chemical cleavage step. Under BER conditions, the REF1 domain of APE1 influenced the stability of both the enzyme-substrate and enzyme-product complexes, as well as the isomerization rate, but did not affect the rates of initial complex formation or catalysis. Under NIR conditions, the REF1 domain affected both the rate of formation and the stability of the initial complex. In comparison with the full-length protein, NDelta61-APE1 did not display a decrease in NIR activity with a dihydrouracil-containing substrate. BER conditions decrease the rate of catalysis and strongly inhibit the rate of isomerization step for the NIR substrates. Under NIR conditions AP-endonuclease activity is still very efficient.

Dimerization of plasmid DNA accelerates selection for antibiotic resistance.

Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell. It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts. However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria. Dimers facilitate segregation of mutants from numerous copies of the parental plasmid. Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria. In contrast, resolution of dimers by site-specific recombination decreases, 10(3)-10(5)-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.

The chemical mutagen dimethyl sulphate induces homologous recombination of plasmid DNA by increasing the binding of RecA protein to duplex DNA.

The role of different DNA damages in the stimulation of homologous recombination was studied by using an in vivo plasmid recombination assay. Dimethyl sulphate (DMS) treatment of plasmid DNA induced a 20-50-fold increase in the frequency of recombinational events. DMS treatment also stimulated RecA protein binding to double-stranded DNA. In contrast, plasmid DNA containing uracil, which, like DMS, is also subject to repair, was less effective in stimulation of recombination. The ability of purified RecA protein to bind DMS-treated or uracil-containing DNA was tested by measuring its ATPase activity. The result indicates that DMS treatment, but not uracil incorporation, stimulates RecA protein binding to DNA. We conclude, that the main reason (or the first step) for stimulation of recombination by mutagens is activation of RecA binding to damaged DNA.

[Effectiveness of chemical mutagenesis in multiple damage to one or both strands of plasmid DNA]. / Effektivnost' khimicheskogo mutageneza pri mnozhestvennykh povrezhdeniiakh odnoi i dvukh nitei plazmidnoi DNK.

Methods for site-directed multiple modification of DNA have been developed and used for modification of either one or two strands of plasmid DNA. Plasmid DNAs modified in the region of the tet gene were transformed into Escherichia coli cells and Tet colonies were screened. It was shown that multiple lesions in one DNA strand performed using either N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or sodium bisulfite were effectively repaired in the cell by error-free mechanism. In contrast, modification of two DNA strands led to induction of mutations. The efficiency of mutagenesis in the case of modification of a local region of one DNA strand with sodium bisulfite and modification of the other strand with MNNG was 1.1-7.9%. Mutations were analysed by restriction mapping and sequencing. All of them were G----A transitions.

Site-directed insertion of long single-stranded DNA fragments into plasmid DNA.

A new site-directed method for inserting long single-stranded DNA fragments into any region of a duplex vector is described. Its major advantage is independence of the location of the restriction sites. The method involves the assembly of single-stranded DNA fragments by ligation to both ends of the inserted fragments of two cohesive flanks that are complementary to the target region. Short oligonucleotide templates are used to direct the ligation. The resulting fragments, designated further as omega fragments with cohesive flanks, are hybridized with a gapped DNA vector. The heteroduplexes are transformed into Escherichia coli cells without enzymatic filling and sealing of gapped DNA. As a consequence of intracellular repair and heteroduplex resolution, insertion mutants are recovered. To demonstrate the method's efficiency, we inserted a 51-nucleotide synthetic DNA fragment containing a modified glucocorticoid receptor binding site into the region of pBR322, near the transcription starting point of the tet gene. The method we developed makes possible site-directed insertion of synthetic and genome-derived DNA fragments at least 200 nucleotides long.

[Introduction of new DNA sequences into previously selected regions of a plasmid genome by means of the formation of heteroduplexes]. / Vvedenie novykh posledovatel'nostei DNK v zaranee izbrannye uchatki genoma plazmidy putem obrazovaniia geterodupleksov.

A new method for obtaining the recombinant DNA based on heteroduplex-initiated site-directed insertion of alien nucleotide sequences is proposed. To generate a single-stranded region, plasmid DNA was nicked with restriction endonuclease in the presence of ethidium bromide with subsequent exonuclease III controlled digestion. The inserted DNA sequences flanked by nucleotide sequences complementary to single-stranded region were annealed with plasmid DNA and E. coli cells were transformed by the resulting heteroduplex molecules. The presented data show the possibility to insert as many as 200 nucleotides. The yield of recombinant DNA varied from 16 to 0.7% as the number of nucleotides inserted correspondingly varied from 15 to 200. The site of insertion does not depend crucially on the localization of the restriction site used.