2019  1,023
2018  0,932
2017  0,977
2016  0,799
2015  0,662
2014  0,740
2013  0,739
2012  0,637
2011  0,658
2010  0,654
2009  0,570
2008  0,849
2007  0,805
2006  0,330
2005  0,435
2004  0,623
2003  0,567
2002  0,641
2001  0,490
2000  0,477
1999  0,762
1998  0,785
1997  0,507
1996  0,518
1995  0,502
Vol 54(2020) N 4 p. 599-610; DOI 10.1134/S0026893320040020 Full Text

L.A. Abrosimova1*, A.R. Samsonova2, T.A. Perevyazova3, A.K. Yunusova3, R.I. Artyukh3, E.A. Romanova4, L.A. Zheleznaya3+, T.S. Oretskaya4, E.A. Kubareva4

The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA

1Chemistry Department, Moscow State University, Moscow, 119991 Russia
2SABNP, University of Evry, INSERM U1204, Universite Paris-Saclay, Evry 91025 France
3Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia
4Belozersky Institute of Physico-Сhemical Biology, Moscow State University, Moscow, 119991 Russia

Received - 2020-03-05; Revised - 2020-03-05; Accepted - 2020-03-16

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes 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 the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(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.

nicking endonuclease, BspD6I, mutant forms, DNA duplex, 2-pyridyldithio group, crosslinking, Bacillus species D6I