Recognition and cleavage of double-stranded DNA by yeast VMA1-derived endonuclease.
DNA endonuclease derived from the yeast VMA1-gene product recognizes and cleaves 31 base-pairs of double-stranded DNA (dsDNA). Mixtures of the endonuclease (VDE) with a full DNA substrate consisting of 34 base-pairs, with nicked substrates each having a nick in either DNA chain, and with cleaved substrates each having a cleaved-off chain are prepared. Molecular weights (MWs) of eluted peaks from gel filtration columns were estimated from elution profiles in the presence of Mg2+ ions. Each mixture exhibited an elute peak at about 63k MW, larger than the MW of VDE unbound to dsDNA. This indicates that VDE and dsDNA substrates form stable complexes. The mixture of VDE either with the full substrate or with the nicked substrate having a nick in the anti-sense chain eluted an additional 25k-MW peak, which presumably corresponds to a cleaved product. The complex of VDE with the full substrate was eluted at 62k-MW location in the absence of Mg2+ ions and yielded a single crystal. Stable complexes of VDE either with the dsDNA substrates or with the cleaved products are obtainable.
Miyamoto
S
S
Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan.
Mizutani
R
R
Satow
Y
Y
Kawasaki
M
M
Ohya
Y
Y
Anraku
Y
Y
D001483:Base Sequence; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D006180:Proton-Translocating ATPases; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae; D008274:Magnesium