Processing character of the action of wheat endonucleases WEN1 and WEN2. Kinetic parameters |
| |
Authors: | L I Fedoreyeva B F Vanyushin |
| |
Institution: | (1) Institute of Molecular Science, Shanxi University, 030006 Taiyuan, Shanxi Province, China;(2) Department of Chemistry and Biochemistry, University of Massachusetts, Dartmouth, MA 02747, USA;(3) Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Molecular Science, Shanxi University, 030006 Taiyuan, Shanxi Province, China;(4) Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA |
| |
Abstract: | The wheat seedling endonucleases WEN1 and WEN2 dependent on Mg2+, Ca2+, and S-adenosyl-L-methionine (SAM) and sensitive to the substrate DNA methylation status have an expressed processing action. The enzymes hydrolyze
DNA at a few subsequent stages: first, they split λ phage DNA specifically at CNG-sites (WEN1) with liberation of large fragments;
second, they hydrolyze these fragments to 120–140 bp oligonucleotides that finally are hydrolyzed to very short fragments
and mononucleotides. Initial stages of DNA hydrolysis may proceed in the absence of Mg2+, but subsequent hydrolysis stages are very strongly stimulated by Mg2+. It cannot be ruled out that modulation of enzymatic activity with Mg2+ and probably with DNA fragments formed is associated with reorganization of the structure of eukaryotic (wheat) endonucleases
with respective changes in their catalytic properties and site specificity of action. Michaelis constant value for WEN1 endonuclease
on hydrolysis of methylated λ phage DNA containing Cm5CWGG and Gm6ATC sites is four-fold lower compared with that observed on hydrolysis of unmethylated λ phage DNA. This may indicate that
affinity of WEN1 enzyme to methylated DNA is higher than that to unmethylated DNA. In the presence of SAM, the Michaelis constant
for WEN2 on the DNA hydrolysis stage characterized by formation of 120–140 bp fragments is decreased, but for WEN1 it is increased
by 1.5–2.0-fold. This means that SAM inhibits WEN1 but stimulates WEN2. Thus, wheat endonucleases WEN1 and WEN2 differ significantly
in affinities to substrate DNAs with different methylation status, in velocities of DNA hydrolysis, and time of production
of DNA fragments of similar length. It seems that the investigated plant endonucleases can hydrolyze DNA in the nucleus as
well to both large and very short fragments including mononucleotides, that is, in particular, essential for utilization of
cell nucleic acid material during apoptosis. |
| |
Keywords: | |
本文献已被 PubMed SpringerLink 等数据库收录! |
|