Isolation and characterization of biochemical properties of DNA methyltransferase FauIA modifying the second cytosine in the nonpalindromic sequence 5'-CCCGC-3'.
A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.
Chernukhin
V A
VA
NPO SibEnzyme, ul. Timakova 9, 630117 Novosibirsk, Russia. Walera@sibenzyme.ru
Kashirina
Yu G
YG
Sukhanova
K S
KS
Abdurashitov
M A
MA
Gonchar
D A
DA
Degtyarev
S Kh
SKh
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular; D003596:Cytosine / Q000737:chemistry*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry / Q000302:isolation & purification*; D015254:DNA Modification Methylases / Q000737:chemistry* / Q000302:isolation & purification*; D004269:DNA, Bacterial; D005417:Flavobacterium; D005798:Genes, Bacterial; D007700:Kinetics; D008745:Methylation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D003596:Cytosine; D015254:DNA Modification Methylases; C113510:DNA modification methylase protein, Flavobacterium aquatile; D004248:DNA (Cytosine-5-)-Methyltransferases