A M.BssECI DNA Methyltransferase Forms 5'-m4CCNNGG-3'. Sensitivity of Restriction Endonucleases to the New Methylation

Автор: V.S. Dedkov, N.A. Mikhnenkova, Z.K. Djanobilova, and M.V. Tarasova

Страница: 32-42

A M.BssECI DNA Methyltransferase Forms 5'-m4CCNNGG-3'. Sensitivity of Restriction Endonucleases to the New Methylation

Biotekhnologiya, 2012, N 2, P. 32-42

UDC 577.152

Section:  “Producers, Biology, Selection, and Gene Engineering”

V.S. Dedkov ^*,  N.A. Mikhnenkova,  Z.K. Djanobilova,  and  M.V. Tarasova

SibEnzyme Ltd,  630117, Novosibirsk Russia

e-mail:  dedkov@sibenzyme.ru

A novel DNA methyltransferase, M.BssECI, has been isolated and purified from Bacillus stearothermophilus EC cells. The enzyme methylates a surface cytosine residue in a 5'-CCNNGG-3' sequence with the formation of N4-methylcytosine, 5'-m4CCNNGG-3'. The approaches to enzyme isolation, purification (gel-filtration on a Biogel A-0,5m with the following chromatography on benzyl-DEAE-cellulose and heparin-sepharose) and identification of the methylated DNA sequence were developed. The optimum conditions and the activity of the novel methyltransferase were determined using phage λ DNA on the basis of the BssECI restriction endonuclease blockage. A base that was methylated within the recognized sequence was detected using the [³H]-labeling of the oligonucleotide duplex. The specificity of M.BssECI was investigated using self-methylated Adeno-2 DNA taking into account the sensitivity of the BssECI, MvaI and MspI restriction endonucleases to methylated DNA; computer modeling was also employed. The isolated enzyme can be used in studying of character of DNA methylation; in particular, it permitted to distinguish the m4C-methylated cytosine from m5C. DNAs of phages λ and Т7 methylated by M.BssECI were applied to the investigation of the sensitivity of some restriction endonucleases to the methylation of their recognition sites.

Key words:  computer modeling,  DNA methyltransferase,  study on specificity.

The full English version of the article was published in “Biotechnology in Russia”, 2012, Issue 2, pp. 32-42 as V.S. Dedkov, N.A. Mikhnenkova, Z.K. Djanobilova, M.V. Tarasova “A M. BssECI DNA Methyltransferase Forms 5'-m4CCNNGG-3'. Sensitivity of Restriction Endonucleases to New Methylation”.

It is contained at the Russian Scientific Electron Library website:  http://elibrary.ru/item.asp?id=21092707

15.06.2015, 1651 просмотр .