ABSTRACT
Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´'- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´'-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.
Subject(s)
Cysteine/chemistry , Deoxyribonuclease I/chemistry , Endonucleases/chemistry , Serine/chemistry , Bacillus/enzymology , Binding Sites , Crystallography, X-Ray/methods , DNA/chemistry , DNA/metabolism , Deoxyribonuclease I/genetics , Deoxyribonuclease I/metabolism , Dimerization , Endonucleases/genetics , Endonucleases/metabolism , Hydrogen Bonding , Hydrolysis , Molecular Structure , Mutagenesis, Site-Directed , Protein Subunits/chemistryABSTRACT
The review focuses on the main factors involved in the formation of nonspecific products in isothermal nucleic acid amplification, such as mispriming, ab initio DNA synthesis, and additional activities of DNA polymerases, and discusses approaches to prevent formation of such nonspecific products in LAMP, RPA, NASBA, RCA, SDA, LSDA, NDA, and EXPAR.
Subject(s)
Nucleic Acid Amplification Techniques/methods , Temperature , DNA-Directed DNA Polymerase/metabolismABSTRACT
Communicated by Ramaswamy H. Sarma.
Subject(s)
DNA, Bacterial/chemistry , DNA, Bacterial/metabolism , DNA-Directed DNA Polymerase/metabolism , Deoxyribonucleases, Type II Site-Specific/metabolism , Geobacillus stearothermophilus/enzymology , Computational Biology , Models, Molecular , Nucleic Acid ConformationABSTRACT
PURPOSE: The objective of the study was to estimate the presence of large mtDNA deletions in brain and spleen tissues of mice four months after exposure to 2 and 5 Gy. MATERIALS AND METHODS: The male BALB/c mice underwent X-ray total-body acute radiation. Four months after irradiation, the mice were decapitated, and the samples of spleen and brain tissues were examined. A long-distance PCR was used to detect mtDNA deletions and their levels in samples of brain and spleen tissues. RESULTS: Four months after irradiation the levels of mtDNA deletions in the brain and spleen tissues were higher in animals exposed to 5 Gy than in animals at an irradiation dose of 2 Gy and in control mice. The levels of deletions in the mice brain tissues were higher 4 months than 1 month after X-ray exposure to both doses (2 and 5 Gy). In spleen tissues, a higher level of deletions was observed only at an irradiation dose of 5 Gy. CONCLUSIONS: Our data have shown that ionizing radiation induces an increase of mtDNA copies with deletions in tissues of mice four months after the post-irradiation period. The level of deletions depends on the animal age, type of tissue, irradiation dose and length of the post-irradiation period.
Subject(s)
DNA, Mitochondrial/genetics , Gene Deletion , Radiation Exposure/adverse effects , Animals , Dose-Response Relationship, Radiation , Male , Mice , Mice, Inbred BALB C , Time FactorsABSTRACT
A new format of a very rapid, low-cost and high-productive analysis based on the acid precipitation of radiolabeled DNA was developed. By contrast to the conventional processing of a large number of GF/C discs, the method employs one GF/C strip containing samples on individual teeth. The strip assay was validated by comparison with the glass fiber disk technique; the efficiency was demonstrated by screening E. coli superproducers and fractions obtained at the steps of Bst DNA polymerase, Large Fragment purification by the protocol we developed. The principle proposed allows simultaneous assaying many samples for the activity of different polymerases.
Subject(s)
Bacterial Proteins/chemistry , DNA, Bacterial/chemistry , DNA-Directed DNA Polymerase/chemistry , Escherichia coli/chemistry , Geobacillus stearothermophilus/enzymologyABSTRACT
In the absence of added DNA, thermophilic DNA polymerases synthesize double-stranded DNA from free dNTPs, which consist of numerous repetitive units (ab initio DNA synthesis). The addition of thermophilic restriction endonuclease (REase), or nicking endonuclease (NEase), effectively stimulates ab initio DNA synthesis and determines the nucleotide sequence of reaction products. We have found that NEases Nt.AlwI, Nb.BbvCI, and Nb.BsmI with non-palindromic recognition sites stimulate the synthesis of sequences organized mainly as palindromes. Moreover, the nucleotide sequence of the palindromes appeared to be dependent on NEase recognition/cleavage modes. Thus, the heterodimeric Nb.BbvCI stimulated the synthesis of palindromes composed of two recognition sites of this NEase, which were separated by AT-reach sequences or (A)n (T)m spacers. Palindromic DNA sequences obtained in the ab initio DNA synthesis with the monomeric NEases Nb.BsmI and Nt.AlwI contained, along with the sites of these NEases, randomly synthesized sequences consisted of blocks of short repeats. These findings could help investigation of the potential abilities of highly productive ab initio DNA synthesis for the creation of DNA molecules with desirable sequence.
