The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding.
All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.
Wyszynski
M W
MW
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Gabbara
S
S
Kubareva
E A
EA
Romanova
E A
EA
Oretskaya
T S
TS
Gromova
E S
ES
Shabarova
Z A
ZA
Bhagwat
A S
AS
D001483:Base Sequence; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005437:Flucytosine / Q000378:metabolism; D008745:Methylation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D013379:Substrate Specificity
D004247:DNA; D005437:Flucytosine; D015257:DNA-Cytosine Methylases; C046545:CCWGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D003545:Cysteine