Mechanisms of Catalytic Action and Chemical Modifications of Endonucleases WEN1 and WEN2 from Wheat Seedlings

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L. I. Fedoreyeva^1,2 and B. F. Vanyushin^1,2*

¹All-Russian Research Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, ul. Timiryazevskaya 42, 127550 Moscow, Russia

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; E-mail: vanyush@belozersky.msu.ru

^* To whom correspondence should be addressed.

Received March 26, 2012; Revision received July 18, 2012

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Hydrolysis of DNA catalyzed by wheat endonucleases WEN1 and WEN2 is pronouncedly processive. A correlation has been revealed between appearance of new products of DNA hydrolysis with different length and conformational changes in the enzymes. The first conformational conversion of the endonucleases is associated with appearance of large fragments of DNA hydrolysis with length longer than 500 bp, and the second conversion is associated with formation of oligonucleotides with length of 120-140 bp, and the third conversion is associated with formation of short oligonucleotides and mononucleotides. Formation of the DNA—enzyme complex is accompanied by appearance of fluorescence at λ = 410-440 nm. The intensity, positions, and numbers of maximums of the fluorescence spectra of DNA—WEN1 and DNA—WEN2 complexes are different and depend on the methylation status of the DNA and on the presence of Mg²⁺. The endonucleases hydrolyze DNA by two mechanisms: one is metal-independent, and the other depends on one or two Mg²⁺ ions. One Mg²⁺ ion is located inside the catalytic center of WEN1, whereas the WEN2 center contains two Mg²⁺ ions. The first (site-specific) stage of DNA hydrolysis does not depend on Mg²⁺. Mg²⁺ ions evoke changes in the site specificity of the endonuclease action (WEN1) and abolish their ability to recognize the methylation status of DNA. Products of DNA hydrolysis by endonucleases WEN1 and WEN2 in the presence of Mg²⁺ are similar in length (120-140 bp). The endonucleases have at least two centers (domains) – catalytic and substrate-binding. Two histidine and apparently two lysine plus two dicarboxylic amino acid residues are present inside the catalytic center of WEN1. The catalytic center of WEN2 contains at least one histidine residue and apparently two residues of aspartic or glutamic acid, which are involved in coordination of the metal ions. The catalytic centers of WEN1 and WEN2 seem to be formed, respectively, by HD/E(D/EK)KH and HD/ED/E amino acid residues. The site-specificity of the endonuclease action is due to the DNA-binding domain. This domain contains dicarboxylic amino acid residues, which seem to be responsible for sensitivity of the enzymes to the methylation status of DNA. The hydroxyl groups of tyrosine residues in the enzymes also seem to contribute to recognizing methylated bases in DNA.

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KEY WORDS: endonucleases, DNA hydrolysis kinetics, catalytic mechanism, DNA methylation, wheat, plants

DOI: 10.1134/S0006297913010057

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