Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation.
In vitro compartmentalisation (IVC), a technique for selecting genes encoding enzymes based on compartmentalising gene translation and enzymatic reactions in emulsions, was used to investigate the interaction of the DNA cytosine-5 methyltransferase M.HhaI with its target DNA (5'-GCGC-3'). Crystallography shows that the active site loop from the large domain of M.HhaI interacts with a flipped-out cytosine (the target for methylation) and two target recognition loops (loops I and II) from the small domain make almost all the other base-specific interactions. A library of M.HhaI genes was created by randomising all the loop II residues thought to make base-specific interactions and directly determine target specificity. The library was selected for 5'-GCGC-3' methylation. Interestingly, in 11 selected active clones, 10 different sequences were found and none were wild-type. At two of the positions mutated (Ser252 and Tyr254) a number of different amino acids could be tolerated. At the third position, however, all active mutants had a glycine, as in wild-type M.HhaI, suggesting that Gly257 is crucial for DNA recognition and enzyme activity. Our results suggest that recognition of base pairs 3 and 4 of the target site either relies entirely on main chain interactions or that different residues from those identified in the crystal structure contribute to DNA recognition.
Lee
Yin-Fai
YF
The MRC Laboratory of Molecular Biology and. Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK.
Tawfik
Dan S
DS
Griffiths
Andrew D
AD
D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015257:DNA-Cytosine Methylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004655:Emulsions; D015723:Gene Library; D008957:Models, Genetic; D008958:Models, Molecular; D009154:Mutation; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014158:Transcription, Genetic
D000596:Amino Acids; D003062:Codon; D004655:Emulsions; D004247:DNA; C037664:DNA modification methylase HhaI; D015257:DNA-Cytosine Methylases