Centre of Excellence
Founded in 1975 as the all Union Research Institute of Applied Enzimology, currently, the Institute of Biotechnology is mainly involved in research and training in the fields of biotechnology and molecular biology, including research and development of recombinant biomedical proteins, genetic and molecular studies of restriction modification phenomenon, developing of viruses diagnostics, epigenetic study of small RNA, drug design and synthesis, bioinformatics.
Laboratory of Biological DNA Modification
Chief Scientist and Head
Prof. Saulius Klimašauskas, Ph.D., Dr.Habil.
phone: 370 5 2602114
fax: 370 5 2602116
e-mail: klimasau@ibt.lt
Employees
Giedrius Vilkaitis, Ph.D.
Edita Kriukienė, Ph.D.
Rūta Gerasimaitė, M.Sc.
Zdislav Staševskij, M.Sc.
Giedrė Urbanavičiūtė, M.Sc.
Alexandra Plotnikova, B.Sc.
Ala Žilionienė
Postdoctoral trainees
Viktoras Masevičius, Ph.D.
PhD students
Miglė Gudeliauskaitė, M.Sc.
Zita Liutkevičiūtė, M.Sc.
Simona Jachimovičiūtė, M.Sc.
Tomas Radzvilavičius, M.Sc.
Research Overview
AdoMet-dependent methyltransferases (MTases), which represent more than 3% of the proteins in the cell, catalyze the transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules.
In DNA, enzymatic methylation of nucleobases serves to expand the information content of the genome in organisms ranging from bacteria to mammals. Postreplicative methylation is accomplished by DNA methyltransferases yielding 5-methylcytosine, N4-methylcytosine or N6-methyladenine. Genomic DNA methylation is a key epigenetic regulatory mechanism in high eukaryotes. DNA methylation profiles (occurrence of 5-methylcytosine) are highly variable across different genetic loci, cells and organisms, and are dependent on tissue, age, sex, diet, and other factors. Aberrant DNA methylation correlates with a number of pediatric syndromes and cancer, or predisposes individuals to various other human diseases. However, research into the epigenetic misregulation and its diagnostics is hampered by the limitations of available analytical techniques. We aim to develop new approaches to genome-wide profiling of DNA methylation for epigenome studies and improved diagnostics.
Besides their diverse biological roles, DNA MTases are attractive models for studying structural aspects of DNA-protein interaction. Bacterial enzymes recognize an impressive variety (over 200) of short sequences in DNA. As shown first for the HhaI MTase, access to the target base, which is buried within the stacked double helix, is gained in a remarkably elegant manner: by rotating the nucleotide completely out of the DNA helix and into a concave catalytic pocket in the enzyme (Klimašauskas, S. et al., Cell 1994, 76: 357-369). This general mechanistic feature named "base-flipping" is shared by numerous other DNA repair and DNA modifying enzymes. Our laboratory has a long standing interest in studies of mechanistic and structural aspects of DNA methylation with partucular focus on the HhaI DNA cytosine-5 methyltransferase (M.HhaI) from the bacterium Haemophilus haemolyticus as the paradigm model system.
Although the methylation of biopolymers generally occurs in a highly specific manner, the naturally transferred methyl group has limited utility for practical applications. On the other hand, the ability of most MTases to catalyze highly specific covalent modifications of biopolymers makes them attractive molecular tools, provided that the transfer of larger chemical entities can be achieved. Our goal is to redesign the methyltransferase reactions for targeted covalent deposition of desired functional or reporter groups onto biopolymer molecules such as DNA and RNA.
Kinetic and molecular mechanisms of DNA methylation
Enzymatic DNA cytosine-5 methylation is a complex reaction that proceeds via multiple steps such as binding of cofactor AdoMet and substrate DNA, flipping of the target cytosine, conformational rearrangement of the mobile catalytic loop, activation of the target cytosine via formation of a transient covalent bond, the methyl transfer. We use mutagenesis, biochemical analysis, steady-state and transient kinetic analysis, fluorescence spectroscopy and x-ray diffraction to delineate the elementary steps on the reaction pathway of HhaI MTase (Klimašauskas S. et al., EMBO J. 1998, 17: 317-324; Serva S., et al., Nucleic Acids Res. 1998, 26: 3473-3479; Vilkaitis G. et al., J. Biol. Chem. 2001, 276: 20924-20934; Merkiene and Klimašauskas, 2005) and related enzymes (Vilkaitis et al., 2005; Subach et al., 2007) in collaboration with groups of Prof. Shoji Tajima, Institute for Protein Research, Osaka University, Japan and Prof. Elizaveta Gromova, Moscow State University, Russia.
Rotation of a nucleotide out of the DNA helix (base flipping) is a mechanistic feature used by numerous modification and repair enzymes to gain access to their target bases buried in double-helical DNA (Klimašauskas and Liutkevičiūtė, 2009). Using mutagenesis, fluorescence spectroscopy and enzyme kinetics we demonstrated that M.HhaI uses a protruding Gln237 residue for active opening the target C:G pair in DNA by a "push-and-bind" mechanism (Daujotytė, D. et al., Structure, 2004, 12: 1047-1055). Further insights into dynamics of base flipping in the HhaI MTase system are sought by NMR structural studis in collaboration with Prof. Thomas Szyperski, SUNY Buffalo, NY, USA. To this end, functional variants of the enzyme that show a significantly increased solubility and long term stability have been engineered (Daujotytė, D. et al., Protein Engin. 2003, 16: 295-301).
DNA base flipping is known to be employed by a wide variety of DNA enzymes, and therefore simple and reliable laboratory techniques for such analyses are desired. Fluorescence methods had been emplyoed for the determination and spectroscopic studies of base flipping in solution, and 2-aminopurine is often used as a fluorescent nucleobase probe (Holz B. et al., Nucleic Acids Res. 1998, 26: 1076-1083; Neely et al., 2005). We have recently demonstrated the first application of chloracetaldehyde to detect individual extrahelical cytosines in the model M.HhaI-DNA complex, and validated it in unexplored systems including other DNA cytosine methyltransferases and restriction endonucleases (Daujotyte et al., 2008).
Targeted covalent labeling of biopolymers
Our goal is to convert MTases into alkyltransferases for sequence-specific covalent modification of DNA and other biopolymers. Our strategy is based on designing novel synthetic analogues of the natural cofactor AdoMet. In collaboration with the group of Prof. Elmar Weinhold, Institute of Organic Chemistry, RWTH Aachen, Germany, we have synthesized a series of model AdoMet analogs with sulfonium-bound extended side chains replacing the methyl group. We demonstrated that allylic and propargylic side chains can be efficiently transferred by DNA MTases with high sequence- and base-specificity (Dalhoff et al., 2006), which provides a novel enabling technique for sequence-specific covalent derivatization of DNA (reviewed in: Breindl, A. BioWorld Today 2005 16:1; Borman, S. Chem. Eng. News 2005, 83:12; Mercer, A.C. & Burkart, M.D. Nature Chem. Biol. 2006, 2: 8-11). These cofactors are termed double-activated AdoMet analogs because the reactive carbon located between the sulfonium center and the unsaturated bond is activated for transfer by both adjacent groups (Klimašauskas and Weinhold, 2007); see further links: pubs.acs.org/cen/news/83/i49/8349notw7.html; www.hhmi.org/news/klimasauskas20051127.html
This novel approach named mTAG (methyltransferase-directed Transfer of Activated Groups) is a convenient and robust technique that is suitable for routine laboratory use. Using model DNA MTases along with their novel cofactors, we demonstrate that: i) the mTAG approach can be used for efficient sequence-specific amino-functionalization of plasmid or bacteriophage DNA; ii) subsequent amine-specific chemoligations (with corresponding NHS esters) lead to covalent labeling of the natural DNA with reporter groups (biotin, fluorophores (Lukinavičius et al., 2007). These findings envision numerous applications of the new labeling technique in functional studies, DNA-based nanotechnologies and medical diagnostics (Klimašauskas and Weinhold, 2007). 800 DNA MTases that recognize over 200 different DNA sequences spanning 2-8 base pairs are currently known, offering an unprecedented experimental control over sequence-specific manipulation of DNA with many potential applications ranging from probes for genetic screening technologies to molecular building blocks in DNA-based nanobiotechnology. Moreover, the newly developed cofactors should in principle be suitable for sequence-specific transfer of functional groups or other chemical entities to RNA and proteins using appropriate MTases as catalysts.
Novel approaches to epigenome profiling
Genomic DNA methylation is a key epigenetic regulatory mechanism in high eukaryotes, however, research into the epigenetic regulation is hampered by limitations of current analytical techniques. We therefore aim to develop new experimental approaches to genome-wide profiling of DNA methylation for epigenome studies and improved diagnostics. Our approach is based on selective mTAG labeling and enrichment of unmethylated CpG sites (Gerasimaitė et al., 2009) in the genome (note that premethylated target sites cannot be labeled) followed by analysis of the enriched fractions on tiling microarrays (in collaboration with Prof. Art Petronis, CAMH, Toronto, Canada).
Functional analysis of the miRNA methyltransferase HEN1
MicroRNAs and siRNAs are small non-coding double-stranded RNA molecules that control gene activity in a homology-dependent manner - a process named RNA interference. Since their discovery in 1993, numerous microRNAs have been identified and recognized as important regulators of gene expression in both plants and animals. Many microRNAs have well-defined developmental and tissue-specific expression pattern, but a great number of microRNAs and their roles are still unknown.
The biogenesis of miRNAs and siRNAs in plants differs from that in animals as it involves an additional methylation step catalyzed by the HEN1 methyltransferase. HEN1 from Arabidopsis catalyzes the transfer methyl groups from AdoMet onto the 2'OH group of the 3'-terminal nucleotide of small RNAs, like miRNA/miRNA* and siRNA/siRNA*. The methylation is imperative in the biogenesis of microRNA in Arabidopsis since microRNAs in hen1 mutants are reduced in abundance or are totally absent. A number of biochemical approaches have been developed in our laboratory along with the group of Prof. Xuemei Chen at UC Riverside, USA to examining the unique methyltransferase HEN1 (Yang et al., 2007). To determine the structural organization of this 942 residue protein, a series of truncated variants are being constructed and analyzed.
Recent publications
N. Miropolskaya, I. Artsimovitch, S. Klimašauskas, V. Nikiforov, and A. Kulbachinskiy
Allosteric control of catalysis by the F loop of RNA polymerase.
Proc. Natl. Acad. Sci. USA, 2009, 106, 18942-18947.
A directed evolution design of a GCG-specific DNA hemimethylase.
Nucleic Acids Res. 2009, 37, 7332-7341.
Z. Liutkevičiūtė, G. Lukinavičius, V. Masevičius, D. Daujotytė, and S. Klimašauskas
Cytosine-5 methyltransferases add aldehydes to DNA.
Nature Chem. Biol., 2009, 5, 400-402.
S. Klimašauskas and Z. Liutkevičiūtė
Experimental approaches to study DNA base flipping.
DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution.
ed. H. Grosjean, (2009), Landes Bioscience, pp.19-32.
E. Weinhold and S. Klimašauskas
S-Adenosyl-L-methionine and analogs.
DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution.
ed. H. Grosjean, (2009), Landes Bioscience, pp. 639-642.
D. Daujotytė, Z. Liutkevičiūtė, G. Tamulaitis and S. Klimašauskas
Chemical mapping of cytosines enzymatically flipped out of the DNA helix.
Nucleic Acids Res., 2008, 36, e57.
S. Klimašauskas and G. Lukinavičius
Chemistry of AdoMet-dependent methyltransferases.
Wiley Encyclopedia of Chemical Biology, (2008), wecb335
G. Lukinavičius, V. Lapienė, Z. Staševskij, C. Dalhoff, E. Weinhold and S. Klimašauskas
Targeted labeling of DNA by methyltransferase-directed Transfer of Activated Groups (mTAG).
J. Amer. Chem. Soc., 2007, 129, 2758-2759.
S. Klimašauskas and E. Weinhold
A new tool for biotechnology: AdoMet-dependent methyltransferases.
Trends Biotechnol., 2007, 25, 99-104.
Z. Yang, G. Vilkaitis, B. Yu, S. Klimašauskas and X. Chen
Approaches for studying microRNA and small interfering RNA methylation in vitro and in vivo.
Methods Enzymol., 2007, 247, 139-154.
A. Sevostyanova, A. Feklistov, N. Barinova, E. Heyduk, I. Bass, S. Klimašauskas, T. Heyduk, and A. Kulbachinskiy
Specific recognition of the -10 promoter element by the free RNA polymerase sigma subunit.
J. Biol. Chem., 2007, 282, 22033-22039.
O.M. Subach, D.V. Maltseva, A. Shastry, A. Kolbanovskiy, S. Klimašauskas, N.E. Geacintov, and E.S. Gromova
The stereochemistry of benzo[a]pyrene-2'-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases.
FEBS J., 2007, 274, 2121-2134.
C. Dalhoff, G. Lukinavičius, S. Klimašauskas and E. Weinhold
Synthesis of S-adenosyl-L-methionine analogs and their use for sequence-specific transalkylation of DNA by methyltransferases.
Nature Protocols, 2006, 1, 1879-1886.
A. Feklistov, N. Barinova, A. Sevostyanova, E. Heyduk, I. Bass, I. Vvedenskaya, K. Kuznedelov, E. Merkiene, E. Stavrovskaya, S. Klimašauskas, V. Nikiforov, T. Heyduk, K. Severinov and A. Kulbachinskiy
A basal promoter element recognized by free RNA polymerase sigma subunit determines promoter recognition by RNA polymerase holoenzyme.
Mol. Cell, 2006, 23, 97-107.
C. Dalhoff, G. Lukinavičius, S. Klimašauskas and E. Weinhold
Direct transfer of extended groups from synthetic cofactors by DNA methyltransferases.
Nature Chem. Biol., 2006, 2, 31-32.
E. Merkiene and S. Klimašauskas
Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase.
Nucleic Acids Res., 2005, 33, 307-315.
R.K. Neely, D. Daujotyte, S. Gražulis, S.W. Magennis, D.T.F. Dryden, S. Klimašauskas and A.C. Jones
Time-Resolved Fluorescence of 2-Aminopurine as a Probe of Base Flipping in M.HhaI-DNA Complexes.
Nucleic Acids Res., 2005, 33, 6953-6960.
G. Vilkaitis, I. Suetake, S. Klimašauskas and S. Tajima
Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase.
J. Biol. Chem., 2005, 280, 64-72.