Wheat coleoptile endonuclease Wen2 is dependent on S-adenosyl-L-methionine and sensitive to DNA methylation status.
Endonuclease WEN2 with an apparent molecular mass 21.5 kD was isolated from subcellular vesicular fraction obtained from aging apoptotic coleoptiles of 8-day-old etiolated wheat seedlings and partially characterized. Similar to wheat endonuclease WEN1 of the same origin described earlier, the WEN2 enzyme is a neutral Ca2+,Mg2+,Mn2+-dependent endonuclease. Both enzymatic activities were found also in nuclei from the same cells. Mn2+ activates WEN2 more efficiently than Mg2+ or Ca2+. High ionic strength, Zn2+, and EDTA inhibit the enzyme completely. In the presence of Mg2+, elevated WEN2 activity was observed at pH between 5.5 and 7.7 and at 37 degrees C, and without Mg2+ added it was observed in narrower pH range (from pH 6.8 to pH 7.7). The enzyme is active even at high temperature (65 degrees C). WEN2 splits preferentially unmethylated, but WEN1 - methylated lambda phage DNA. Double-stranded DNA is a preferential substrate to be hydrolyzed with WEN2. S-Adenosyl-L-methionine (SAM) significantly activates endonuclease WEN2 (the optimal SAM concentration is 0.3 mM). Contrary to strong stimulating action on WEN1, the competitive inhibitors of the DNA methylation reaction (SAM analogs S-adenosyl-L-homocysteine and S-isobutyladenosine) at concentration 0.3 mM increase WEN2 activity slightly. It is suggested that WEN2 may take part in apoptotic DNA degradation. Thus, in plants there are endonucleases that recognize methylation status of substrate DNAs and are modulated by the methyl group donor, SAM, in different fashions. Therefore, all this may indicate the presence of a restriction-modification (R-M) system in higher plants.
Fedoreyeva
L I
LI
Institute of Agricultural Biotechnology, Russian Academy of Agricultural Sciences, Moscow, 127550, Russia.
Sobolev
D E
DE
Vanyushin
B F
BF
D017209:Apoptosis; D018548:Cotyledon / Q000201:enzymology*; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D004720:Endonucleases / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*; D036226:Seedlings / Q000378:metabolism; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000502:physiology
D010940:Plant Proteins; D012436:S-Adenosylmethionine; D004247:DNA; D012435:S-Adenosylhomocysteine; D004720:Endonucleases