ArticleNucleic acids research2002
Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation.
Article in Nucleic acids research, 2002. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed, 59 citations in OpenAlex.
- Ultrahigh-Throughput Enzyme Engineering and Discovery inChemical reviews · 2023Review
- Droplet Microfluidics and Directed Evolution of Enzymes: An Intertwined Journey.Angewandte Chemie (International ed. in English) · 2021Review
- Split & mix assembly of DNA libraries for ultrahigh throughput on-bead screening of functional proteins.Nucleic acids research · 2020Article
- A high-throughput screen for antibiotic drug discovery.Biotechnology and bioengineering · 2014 · on this mapArticle
- Metadynamics simulation study on the conformational transformation of HhaI methyltransferase: an induced-fit base-flipping hypothesis.BioMed research international · 2014Article
- Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution.Nucleic acids research · 2013Article
- Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage.Nucleic acids research · 2012Article
- Microfluidic compartmentalized directed evolution.Chemistry & biology · 2010Article
- Molecular and enzymatic profiles of mammalian DNA methyltransferases: structures and targets for drugs.Current medicinal chemistry · 2010Review
- Microfluidic systems for biosensing.Sensors (Basel, Switzerland) · 2010Review
- A directed evolution design of a GCG-specific DNA hemimethylase.Nucleic acids research · 2009Article
- Novel method for high-throughput colony PCR screening in nanoliter-reactors.Nucleic acids research · 2009Article
- In vivo DNA protection by relaxed-specificity SinI DNA methyltransferase variants.Journal of bacteriology · 2008Article
- The BsaHI restriction-modification system: cloning, sequencing and analysis of conserved motifs.BMC molecular biology · 2008Article
- Nano-enabled synthetic biology.Molecular systems biology · 2007Article
- Selection of ribozymes that catalyse multiple-turnover Diels-Alder cycloadditions by using in vitro compartmentalization.Proceedings of the National Academy of Sciences of the United States of America · 2005Article
- In vitro selection of restriction endonucleases by in vitro compartmentalization.Nucleic acids research · 2004Article
- DNA display for in vitro selection of diverse peptide libraries.Nucleic acids research · 2003Article
- Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization.The EMBO journal · 2003Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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