The role of cysteine residues in the Interaction of nicking endonuclease BspD6I with DNA

Abrosimova, L.A.; Samsonova, A.R.; Perevyazova, T.A.; Yunusova, A.K.; Artyukh, R.I.; Romanova, E.A.; Zheleznaya, L.A.; Oretskaya, T.S.; Kubareva, E.A.

Авторы: Abrosimova L.A., Samsonova A.R., Perevyazova T.A., Yunusova A.K., Artyukh R.I., Romanova E.A., Zheleznaya L.A., Oretskaya T.S., Kubareva E.A.
Журнал: Molecular Biology
Том: 54
Номер: 4
Год издания: 2020
Издательство: Maik Nauka/Interperiodica Publishing
Местоположение издательства: Russian Federation
Первая страница: 599
Последняя страница: 610
Оригинальные версии: Роль остатков цистеина во взаимодействии никующей эндонуклеазы BspD6I с ДНК
DOI: 10.1134/S0026893320040020
Аннотация: Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. Theenzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in thecomplex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NEBspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.
Добавил в систему: Никифорова Людмила Алексеевна

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The role of cysteine residues in the Interaction of nicking endonuclease BspD6I with DNAстатья Исследовательская статья

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