Functional mapping of the EcoRV DNA methyltransferase by random mutagenesis and screening for catalytically inactive mutants.
M.EcoRV is an alpha-adenine DNA methyltransferase. According to structure predictions, the enzyme consists of a catalytic domain, which has a structure similar to all other DNA-methyltransferases, and a smaller DNA-recognition domain. We have investigated this enzyme by random mutagenesis, using error-prone PCR, followed by selection for catalytically inactive mutants. 20 single mutants were identified that are completely inactive in vivo as His6- and GST-fusion proteins. 13 of them could be overexpressed and purified. All of these mutants are also inactive in vitro. 5 of the mutations are located near the putative binding site for a flipped adenine residue (C192R, D193G, E212G, W231R, N239H). All of these variants bind to DNA, demonstrating the importance of this region of the protein in catalysis. Only the W231R mutant could be purified with high yields. It binds to DNA and AdoMet and, thus, behaves like a bona fide active site mutant. According to the structure prediction Trp231 corresponds to Val121 in M.HhaI, which forms a hydrophobic contact to the flipped target cytosine. 4 of the remaining purified variants are located within a small region of the putative DNA-recognition domain (F115S, F117L, S121P, C122Y). F117L, S121P and C122Y are unable to bind to DNA, suggesting a critical role of this region in DNA binding. Taken together, these results are in good agreement with the structural model of M.EcoRV.
Friedrich
T
T
Institut für Biochemie, Fachbereich Biologie, Justus-Liebig-Universität, Giessen, Germany.
Roth
M
M
Helm-Kruse
S
S
Jeltsch
A
A
D002384:Catalysis; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism*; D013329:Structure-Activity Relationship
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; C056127:DNA modification methylase EcoRV; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)