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Engineering and Directed Evolution of DNA Methyltransferases

  • Paola Laurino
  • Liat Rockah-Shmuel
  • Dan S. Tawfik
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 945)

Abstract

DNA methyltransferases (MTases) constitute an attractive target for protein engineering, thus opening the road to new ways of manipulating DNA in a unique and selective manner. Here, we review various aspects of MTase engineering, both methodological and conceptual, and also discuss future directions and challenges. Bacterial MTases that are part of restriction/modification (R/M) systems offer a convenient way for the selection of large gene libraries, both in vivo and in vitro. We review these selection methods, their strengths and weaknesses, and also the prospects for new selection approaches that will enable the directed evolution of mammalian DNA methyltransferases (Dnmts). We explore various properties of MTases that may be subject to engineering. These include engineering for higher stability and soluble expression (MTases, including bacterial ones, are prone to misfolding), engineering of the DNA target specificity, and engineering for the usage of S-adenosyl-L-methionine (AdoMet) analogs. Directed evolution of bacterial MTases also offers insights into how these enzymes readily evolve in nature, thus yielding MTases with a huge spectrum of DNA target specificities. Engineering for alternative cofactors, on the other hand, enables modification of DNA with various groups other than methyl and thus can be employed to map and redirect DNA epigenetic modifications.

Keywords

Directed Evolution Gene Library Methylation Site Enzyme Variant Consensus Amino Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

MTase

DNA methyltransferase

Dnmt

Mammalian DNA methyltransferase

R/M

Restriction/modification

IPTG

Isopropyl β-D-1-thiogalactopyranoside

IVC

In vitro compartmentalization

PCR

Polymerase chain reaction

MeDIP

Methylated DNA immunoprecipitation

CpG

5’-C-phosphate-G-3’

NMR

Nuclear magnetic resonance

ELISA

Enzyme-linked immunosorbent assay

DIG

Digoxigenin

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

References

  1. Augustyniak W, Brzezinska AA, Pijning T, Wienk H, Boelens R, Dijkstra BW, Reetz MT. Biophysical characterization of mutants of Bacillus subtilis lipase evolved for thermostability: factors contributing to increased activity retention. Protein Sci. 2012;21(4):487–97. doi: 10.1002/pro.2031.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bennett BD, Kimball EH, Gao M, Osterhout R, Van Dien SJ, Rabinowitz JD. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat Chem Biol. 2009;5(8):593–9. doi: 10.1038/nchembio.186.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bernath K, Magdassi S, Tawfik DS. In vitro compartmentalization (IVC): a high-throughput screening technology using emulsions and FACS. Discov Med. 2004;4(20):49–53.PubMedGoogle Scholar
  4. Bernath K, Magdassi S, Tawfik DS. Directed evolution of protein inhibitors of DNA-nucleases by in vitro compartmentalization (IVC) and nano-droplet delivery. J Mol Biol. 2005;345(5):1015–26. doi: 10.1016/j.jmb.2004.11.017.CrossRefPubMedGoogle Scholar
  5. Bloom JD, Labthavikul ST, Otey CR, Arnold FH. Protein stability promotes evolvability. Proc Natl Acad Sci U S A. 2006;103(15):5869–74. doi: 10.1073/pnas.0510098103.CrossRefPubMedPubMedCentralGoogle Scholar
  6. Borgel J, Guibert S, Weber M. Methylated DNA immunoprecipitation (MeDIP) from low amounts of cells. Methods Mol Biol. 2012;925:149–58. doi: 10.1007/978-1-62703-011-3_9.CrossRefPubMedGoogle Scholar
  7. Bothwell IR, Islam K, Chen Y, Zheng W, Blum G, Deng H, Luo M. Se-adenosyl-L-selenomethionine cofactor analogue as a reporter of protein methylation. J Am Chem Soc. 2012;134(36):14905–12. doi: 10.1021/ja304782r.CrossRefPubMedPubMedCentralGoogle Scholar
  8. Brebi-Mieville P, Ili-Gangas C, Leal-Rojas P, Noordhuis MG, Soudry E, Perez J, Roa JC, Sidransky D, Guerrero-Preston R. Clinical and public health research using methylated DNA immunoprecipitation (MeDIP): a comparison of commercially available kits to examine differential DNA methylation across the genome. Epigenetics. 2012;7(1):106–12. doi: 10.4161/epi.7.1.18647.CrossRefPubMedPubMedCentralGoogle Scholar
  9. Chahar S, Elsawy H, Ragozin S, Jeltsch A. Changing the DNA recognition specificity of the EcoDam DNA-(adenine-N6)-methyltransferase by directed evolution. J Mol Biol. 2010;395(1):79–88. doi: 10.1016/j.jmb.2009.09.027.CrossRefPubMedGoogle Scholar
  10. Chaikind B, Ostermeier M. Directed evolution of improved zinc finger methyltransferases. PLoS One. 2014;9(5):e96931. doi: 10.1371/journal.pone.0096931.CrossRefPubMedPubMedCentralGoogle Scholar
  11. Cohen HM, Griffiths AD, Tawfik DS, Loakes D. Determinants of cofactor binding to DNA methyltransferases: insights from a systematic series of structural variants of S-adenosylhomocysteine. Org Biomol Chem. 2005;3(1):152–61. doi: 10.1039/b415446k.CrossRefPubMedGoogle Scholar
  12. Cohen HM, Tawfik DS, Griffiths AD. Promiscuous methylation of non-canonical DNA sites by HaeIII methyltransferase. Nucleic Acids Res. 2002;30(17):3880–5.CrossRefPubMedPubMedCentralGoogle Scholar
  13. Cohen HM, Tawfik DS, Griffiths AD. Altering the sequence specificity of HaeIII methyltransferase by directed evolution using in vitro compartmentalization. Protein Eng Des Sel. 2004;17(1):3–11. doi: 10.1093/protein/gzh001.CrossRefPubMedGoogle Scholar
  14. Comstock LR, Rajski SR. Conversion of DNA methyltransferases into azidonucleosidyl transferases via synthetic cofactors. Nucleic Acids Res. 2005a;33(5):1644–52. doi: 10.1093/nar/gki306.CrossRefPubMedPubMedCentralGoogle Scholar
  15. Comstock LR, Rajski SR. Methyltransferase-directed DNA strand scission. J Am Chem Soc. 2005b;127(41):14136–7. doi: 10.1021/ja054128y.CrossRefPubMedGoogle Scholar
  16. Dalby PA. Strategy and success for the directed evolution of enzymes. Curr Opin Struct Biol. 2011;21(4):473–80. doi: 10.1016/j.sbi.2011.05.003.CrossRefPubMedGoogle Scholar
  17. Dalhoff C, Lukinavicius G, Klimasauskas S, Weinhold E. Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases. Nat Chem Biol. 2006;2(1):31–2. doi: 10.1038/nchembio754.CrossRefPubMedGoogle Scholar
  18. Daujotyte D, Vilkaitis G, Manelyte L, Skalicky J, Szyperski T, Klimasauskas S. Solubility engineering of the HhaI methyltransferase. Protein Eng. 2003;16(4):295–301.CrossRefPubMedGoogle Scholar
  19. Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R. Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet. 2013;9(6):e1003602. doi: 10.1371/journal.pgen.1003602.CrossRefPubMedPubMedCentralGoogle Scholar
  20. Friedrich T, Roth M, Helm-Kruse S, Jeltsch A. Functional mapping of the EcoRV DNA methyltransferase by random mutagenesis and screening for catalytically inactive mutants. Biol Chem. 1998;379(4–5):475–80.CrossRefPubMedGoogle Scholar
  21. Gerasimaite R, Vilkaitis G, Klimasauskas S. A directed evolution design of a GCG-specific DNA hemimethylase. Nucleic Acids Res. 2009;37(21):7332–41. doi: 10.1093/nar/gkp772.CrossRefPubMedPubMedCentralGoogle Scholar
  22. Goldsmith M, Tawfik DS. Enzyme engineering by targeted libraries. Methods Enzymol. 2013;523:257–83. doi: 10.1016/B978-0-12-394292-0.00012-6.CrossRefPubMedGoogle Scholar
  23. Gowher H, Jeltsch A. Molecular enzymology of the catalytic domains of the Dnmt3a and Dnmt3b DNA methyltransferases. J Biol Chem. 2002;277(23):20409–14. doi: 10.1074/jbc.M202148200.CrossRefPubMedGoogle Scholar
  24. Gowher H, Liebert K, Hermann A, Xu G, Jeltsch A. Mechanism of stimulation of catalytic activity of Dnmt3A and Dnmt3B DNA-(cytosine-C5)-methyltransferases by Dnmt3L. J Biol Chem. 2005;280(14):13341–8. doi: 10.1074/jbc.M413412200.CrossRefPubMedGoogle Scholar
  25. Griffiths AD, Tawfik DS. Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization. EMBO J. 2003;22(1):24–35. doi: 10.1093/emboj/cdg014.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Hart DJ, Waldo GS. Library methods for structural biology of challenging proteins and their complexes. Curr Opin Struct Biol. 2013;23(3):403–8. doi: 10.1016/j.sbi.2013.03.004.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Hughes JA, Brown LR, Ferro AJ. Expression of the cloned coliphage T3 S-adenosylmethionine hydrolase gene inhibits DNA methylation and polyamine biosynthesis in Escherichia coli. J Bacteriol. 1987;169(8):3625–32.CrossRefPubMedPubMedCentralGoogle Scholar
  28. Jeltsch A, Sobotta T, Pingoud A. Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants. Protein Eng. 1996;9(5):413–23.CrossRefPubMedGoogle Scholar
  29. Jin SG, Kadam S, Pfeifer GP. Examination of the specificity of DNA methylation profiling techniques towards 5-methylcytosine and 5-hydroxymethylcytosine. Nucleic Acids Res. 2010;38(11):e125. doi: 10.1093/nar/gkq223.CrossRefPubMedPubMedCentralGoogle Scholar
  30. Jochens H, Bornscheuer UT. Natural diversity to guide focused directed evolution. Chembiochem. 2010;11(13):1861–6. doi: 10.1002/cbic.201000284.CrossRefPubMedGoogle Scholar
  31. Jurkowska RZ, Siddique AN, Jurkowski TP, Jeltsch A. Approaches to enzyme and substrate design of the murine Dnmt3a DNA methyltransferase. Chembiochem. 2011;12(10):1589–94. doi: 10.1002/cbic.201000673.CrossRefPubMedGoogle Scholar
  32. Jurkowski TP, Anspach N, Kulishova L, Nellen W, Jeltsch A. The M.EcoRV DNA-(adenine N6)-methyltransferase uses DNA bending for recognition of an expanded EcoDam recognition site. J Biol Chem. 2007;282(51):36942–52. doi: 10.1074/jbc.M706933200.CrossRefPubMedGoogle Scholar
  33. Kobayashi I. Behavior of restriction-modification systems as selfish mobile elements and their impact on genome evolution. Nucleic Acids Res. 2001;29(18):3742–56.CrossRefPubMedPubMedCentralGoogle Scholar
  34. LaMonte BL, Hughes JA. In vivo hydrolysis of S-adenosylmethionine induces the met regulon of Escherichia coli. Microbiology. 2006;152(Pt 5):1451–9. doi: 10.1099/mic.0.28489-0.CrossRefPubMedGoogle Scholar
  35. Laurino P, Toth-Petroczy A, Meana-Pañeda R, Lin W, Truhlar DG, Tawfik DS. An ancient fingerprint indicates the common ancestry of Rossmann fold enzymes utilizing different ribose based cofactors. PloS Biol. 2016;14(3):e1002396.Google Scholar
  36. Lee YF, Tawfik DS, Griffiths AD. Investigating the target recognition of DNA cytosine-5 methyltransferase HhaI by library selection using in vitro compartmentalisation. Nucleic Acids Res. 2002;30(22):4937–44.CrossRefPubMedPubMedCentralGoogle Scholar
  37. Lin Q, Jiang F, Schultz PG, Gray NS. Design of allele-specific protein methyltransferase inhibitors. J Am Chem Soc. 2001;123(47):11608–13.CrossRefPubMedGoogle Scholar
  38. Lu ZJ, Markham GD. Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii. J Biol Chem. 2002;277(19):16624–31. doi: 10.1074/jbc.M110456200.CrossRefPubMedGoogle Scholar
  39. Lukinavicius G, Lapinaite A, Urbanaviciute G, Gerasimaite R, Klimasauskas S. Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA. Nucleic Acids Res. 2012;40(22):11594–602. doi: 10.1093/nar/gks914.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Lukinavicius G, Tomkuviene M, Masevicius V, Klimasauskas S. Enhanced chemical stability of adomet analogues for improved methyltransferase-directed labeling of DNA. ACS Chem Biol. 2013;8(6):1134–9. doi: 10.1021/cb300669x.CrossRefPubMedGoogle Scholar
  41. Miller OJ, Bernath K, Agresti JJ, Amitai G, Kelly BT, Mastrobattista E, Taly V, Magdassi S, Tawfik DS, Griffiths AD. Directed evolution by in vitro compartmentalization. Nat Methods. 2006;3(7):561–70. doi: 10.1038/nmeth897.CrossRefPubMedGoogle Scholar
  42. Miyazaki K. Creating random mutagenesis libraries by megaprimer PCR of whole plasmid (MEGAWHOP). Methods Mol Biol. 2003;231:23–8. doi: 10.1385/1-59259-395-X:23.PubMedGoogle Scholar
  43. Mohn F, Weber M, Schubeler D, Roloff TC. Methylated DNA immunoprecipitation (MeDIP). Methods Mol Biol. 2009;507:55–64. doi: 10.1007/978-1-59745-522-0_5.CrossRefPubMedGoogle Scholar
  44. Morley KL, Kazlauskas RJ. Improving enzyme properties: when are closer mutations better? Trends Biotechnol. 2005;23(5):231–7. doi: 10.1016/j.tibtech.2005.03.005.CrossRefPubMedGoogle Scholar
  45. Mruk I, Blumenthal RM. Real-time kinetics of restriction-modification gene expression after entry into a new host cell. Nucleic Acids Res. 2008;36(8):2581–93. doi: 10.1093/nar/gkn097.CrossRefPubMedPubMedCentralGoogle Scholar
  46. Nomura W, Barbas 3rd CF. In vivo site-specific DNA methylation with a designed sequence-enabled DNA methylase. J Am Chem Soc. 2007;129(28):8676–7. doi: 10.1021/ja0705588.CrossRefPubMedGoogle Scholar
  47. Pignot M, Siethoff C, Linscheid M, Weinhold E. Coupling of a Nucleoside with DNA by a Methyltransferase. Angew Chem Int Ed Engl. 1998;37(20):3.CrossRefGoogle Scholar
  48. Pljevaljcic G, Pignot M, Weinhold E. Design of a new fluorescent cofactor for DNA methyltransferases and sequence-specific labeling of DNA. J Am Chem Soc. 2003;125(12):3486–92. doi: 10.1021/ja021106s.CrossRefPubMedGoogle Scholar
  49. Raleigh EA, Wilson G. Escherichia coli K-12 restricts DNA containing 5-methylcytosine. Proc Natl Acad Sci U S A. 1986;83(23):9070–4.CrossRefPubMedPubMedCentralGoogle Scholar
  50. Rockah-Shmuel L, Tawfik DS. Evolutionary transitions to new DNA methyltransferases through target site expansion and shrinkage. Nucleic Acids Res. 2012;40(22):11627–37. doi: 10.1093/nar/gks944.CrossRefPubMedPubMedCentralGoogle Scholar
  51. Rockah-Shmuel L, Tawfik DS, Goldsmith M. Generating targeted libraries by the combinatorial incorporation of synthetic oligonucleotides during gene shuffling (ISOR). Methods Mol Biol. 2014;1179:129–37. doi: 10.1007/978-1-4939-1053-3_8.CrossRefPubMedGoogle Scholar
  52. Rockah-Shmuel L, Toth-Petroczy A, Sela A, Wurtzel O, Sorek R, Tawfik DS. Correlated occurrence and bypass of frame-shifting insertion-deletions (InDels) to give functional proteins. PLoS Genet. 2013;9(10):e1003882. doi: 10.1371/journal.pgen.1003882.CrossRefPubMedPubMedCentralGoogle Scholar
  53. Rockah-Shmuel L, Toth-Petroczy A, Tawfik DS. Systematic mapping of protein mutational space by prolonged drift reveals the deleterious effects of seemingly neutral mutations. PLoS Comput Biol. 2015;11(8):e1004421. doi: 10.1371/journal.pcbi.1004421.CrossRefPubMedPubMedCentralGoogle Scholar
  54. Samuelson JC, Morgan RD, Benner JS, Claus TE, Packard SL, Xu SY. Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants. Nucleic Acids Res. 2006;34(3):796–805. doi: 10.1093/nar/gkj483.CrossRefPubMedPubMedCentralGoogle Scholar
  55. Sapienza PJ, Dela Torre CA, McCoy WH, Jana SV, Jen-Jacobson L. Thermodynamic and kinetic basis for the relaxed DNA sequence specificity of “promiscuous” mutant EcoRI endonucleases. J Mol Biol. 2005;348(2):307–24. doi: 10.1016/j.jmb.2005.02.051.CrossRefPubMedGoogle Scholar
  56. Siddique AN, Nunna S, Rajavelu A, Zhang Y, Jurkowska RZ, Reinhardt R, Rots MG, Ragozin S, Jurkowski TP, Jeltsch A. Targeted methylation and gene silencing of VEGF-A in human cells by using a designed Dnmt3a-Dnmt3L single-chain fusion protein with increased DNA methylation activity. J Mol Biol. 2013;425(3):479–91. doi: 10.1016/j.jmb.2012.11.038.CrossRefPubMedGoogle Scholar
  57. Stemmer WP. Rapid evolution of a protein in vitro by DNA shuffling. Nature. 1994;370(6488):389–91. doi: 10.1038/370389a0.CrossRefPubMedGoogle Scholar
  58. Szomolanyi E, Kiss A, Venetianer P. Cloning the modification methylase gene of Bacillus sphaericus R in Escherichia coli. Gene. 1980;10(3):219–25.CrossRefPubMedGoogle Scholar
  59. Tawfik DS. Accuracy-rate tradeoffs: how do enzymes meet demands of selectivity and catalytic efficiency? Curr Opin Chem Biol. 2014;21:73–80. doi: 10.1016/j.cbpa.2014.05.008.CrossRefPubMedGoogle Scholar
  60. Tawfik DS, Griffiths AD. Man-made cell-like compartments for molecular evolution. Nat Biotechnol. 1998;16(7):652–6. doi: 10.1038/nbt0798-652.CrossRefPubMedGoogle Scholar
  61. Timar E, Groma G, Kiss A, Venetianer P. Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution. Nucleic Acids Res. 2004;32(13):3898–903. doi: 10.1093/nar/gkh724.CrossRefPubMedPubMedCentralGoogle Scholar
  62. Tokuriki N, Tawfik DS. Chaperonin overexpression promotes genetic variation and enzyme evolution. Nature. 2009a;459(7247):668–73. doi: 10.1038/nature08009.CrossRefPubMedGoogle Scholar
  63. Tokuriki N, Tawfik DS. Stability effects of mutations and protein evolvability. Curr Opin Struct Biol. 2009b;19(5):596–604. doi: 10.1016/j.sbi.2009.08.003.CrossRefPubMedGoogle Scholar
  64. Ulrich A, Andersen KR, Schwartz TU. Exponential megapriming PCR (EMP) cloning--seamless DNA insertion into any target plasmid without sequence constraints. PLoS One. 2012;7(12):e53360. doi: 10.1371/journal.pone.0053360.CrossRefPubMedPubMedCentralGoogle Scholar
  65. Vranken C, Deen J, Dirix L, Stakenborg T, Dehaen W, Leen V, Hofkens J, Neely RK. Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry. Nucleic Acids Res. 2014;42(7):e50. doi: 10.1093/nar/gkt1406.CrossRefPubMedPubMedCentralGoogle Scholar
  66. Wang R, Zheng W, Luo M. A sensitive mass spectrum assay to characterize engineered methionine adenosyltransferases with S-alkyl methionine analogues as substrates. Anal Biochem. 2014;450:11–9. doi: 10.1016/j.ab.2013.12.026.CrossRefPubMedGoogle Scholar
  67. Wijma HJ, Floor RJ, Jekel PA, Baker D, Marrink SJ, Janssen DB. Computationally designed libraries for rapid enzyme stabilization. Protein Eng Des Sel. 2014;27(2):49–58. doi: 10.1093/protein/gzt061.CrossRefPubMedPubMedCentralGoogle Scholar
  68. Williams R, Peisajovich SG, Miller OJ, Magdassi S, Tawfik DS, Griffiths AD. Amplification of complex gene libraries by emulsion PCR. Nat Methods. 2006;3(7):545–50. doi: 10.1038/nmeth896.CrossRefPubMedGoogle Scholar
  69. Zhao MT, Whyte JJ, Hopkins GM, Kirk MD, Prather RS. Methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) using low amounts of genomic DNA. Cell Reprogram. 2014;16(3):175–84. doi: 10.1089/cell.2014.0002.CrossRefPubMedGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2016

Authors and Affiliations

  • Paola Laurino
    • 1
  • Liat Rockah-Shmuel
    • 1
  • Dan S. Tawfik
    • 1
  1. 1.Department of Biological ChemistryWeizmann Institute of ScienceRehovotIsrael

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