[Purification and site-directed mutagenesis of DNA methyltransferase SssI].
Prokaryotic DNA methyltransferase SssI (M.SssI) methylates C5 position of cytosine residue in CpG sequences. To obtain functionally active M.SssI and its mutants as His6-tagged proteins, bacterial strains have been produced. To test a possible role of Ser300 in recognition of CpG site by this enzyme, M.SssI mutants containing Ser300 replacements with Gly or Pro have been obtained. These replacements have practically no effect on DNA binding and methylation by M.SssI except small disturbance of DNA binding affinity in the case of S300P mutant. It indicates that there are no interactions of both the side chain and, probably, the main chain of Ser300 with DNA. A replacement of highly conserved Va1188 residue with Ala has been performed. Vall88 may participate in the stabilization of the flipped target cytosine during reaction. The replacement results in a 5-fold decrease of dissociation constant of the enzyme-substrate complex and a 2-fold decrease of initial velocity of DNA methylation. Though there are no noticeable differences in the functioning of the mutant in comparison with the wild-type enzyme, the formation of contact between Val 188 and cytosine could not be excluded. In the case of V 188A mutant the contact may be probably formed between Ala and cytosine residue.
Darií
M V
MV
Kirsanova
O V
OV
Drutsa
V L
VL
Kochetkov
S N
SN
Gromova
E S
ES
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D019175:DNA Methylation; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity / Q000235:genetics
D001426:Bacterial Proteins; C081573:DNA modification methylase SssI; D015257:DNA-Cytosine Methylases