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Nucleic Acids Research, 2002, Vol. 30, No. 22 4937-4944
© 2002 Oxford University Press

Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation

Yin-Fai Lee, Dan S. Tawfik1,2 and Andrew D. Griffiths*

The MRC Laboratory of Molecular Biology and 1 Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK and 2 Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot 76 100, Israel

*To whom correspondence should be addressed. Tel: +44 1223 402113; Fax: +44 1223 402140; Email: griff{at}mrc-lmb.cam.ac.uk
Present address: Yin-Fai Lee, Institute of Cancer Research, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, UK

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'). Crystallog raphy 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.


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