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.
Department 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: saulius.klimasauskas (at) bti.vu.lt
Employees
Giedrius Vilkaitis, Ph.D.
Edita Kriukienė, Ph.D.
Rasa Rakauskaitė, Ph.D.
Viktoras Masevičius, Ph.D.
Zita Liutkevičiūtė, Ph.D.
Zdislav Staševskij, M.Sc.
Giedrė Urbanavičiūtė, M.Sc.
Miglė Tomkuvienė, M.Sc.
Simona Baranauskė, M.Sc.
Aleksandr Osipenko, M.Sc.
Stasė Butkytė, M.Sc.
Robertas Juškėnas, 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. 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).
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). 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 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). Most recently, we have described a direct observation, in a chemically unperturbed system, of methyltransferase-induced flipping of the target cytosine residue out of the DNA helix and its subsequent covalent activation in the catalytic centre of the enzyme. The proposed approach is based on monitoring small hyperchromicity changes in DNA and is likely to be applicable for the study of other systems involving base flipping. Combined with analysis of tryptophan-engineered variants of the HhaI methyltransferase, the temporal order and kinetics of the individual steps in the catalytic cycle of M.HhaI can be established with unprecedented thoroughness (Gerasimaitė et al., 2011).
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, 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). 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).
This novel approach named mTAG (methyltransferase-directed Transfer of Activated Groups) is a convenient and robust technique that is suitable for routine laboratory use (Lukinavičius et al., 2007, 2012 and 2013). 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 ( Neely et al., 2010; see commentary in Highlights Chem. Sci.) 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 (Tomkuvienė et al., 2012) 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).
In the absence of the S-adenosylmethionine cofactor, bacterial cytosine-5 MTases can catalyze catalyze reversible covalent addition of exogenous aliphatic aldehydes to their target residues in DNA, thus yielding corresponding 5-hydroxyalkylcytosines (Liutkevičiūtė et al., 2009). Moreover, our most recent studies demonstrate the ability of the MTases to direct condensation of aliphatic thiols and selenols with 5-hydroxymethylcytosine in DNA to yield 5-alkylchalcogenomethyl derivatives (Liutkevičiūtė et al., 2011 ). These atypical reactions demonstrate a surprizing catalytic versatility of these enzymes and pave new ways for the sequence-specific derivatization and analysis of 5-hydroxymethylcytosine, a recently discovered nucleobase in mammalian DNA (Kriukienė et al., 2012) .
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 have been constructed and analyzed (Vilkaitis et al., 2010).
Recent publications
A. Plotnikova, S. Baranauskė, A. Osipenko, S. Klimašauskas and G. Vilkaitis
Mechanistic insights into small RNA recognition and modification by the HEN1 methyltransferase.
Biochem J., 2013 Apr 29 [Epub ahead of print].
G. Lukinavičius, M. Tomkuvienė, V. Masevičius and S. Klimašauskas
Enhanced chemical stability of AdoMet analogues for improved methyltransferase-directed labeling of DNA.
ACS Chem. Biol., 2013, Article ASAP
G. Lukinavičius, A. Lapinaitė, G. Urbanavičiūtė, R. Gerasimaitė and S. Klimašauskas
Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.
Nucleic Acids Res., 2012, 40, (22) 11594–11602.
T. Khare, S. Pai, K. Koncevičius, M. Pal, E. Kriukienė, Z. Liutkevičiūtė, M. Irimia, P. Jia, C. Ptak, M. Xia, R. Tice, M. Tochigi, S. Moréra, A. Nazarians, D. Belsham, A.H.C. Wong, B.J. Blencowe, S.C. Wang, P. Kapranov, R. Kustra, V. Labrie, S. Klimašauskas and A. Petronis
5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary.
Nature Struct. Mol. Biol., 2012, 19, (10) 1037–1043.
E. Kriukienė, Z. Liutkevičiūtė and S. Klimašauskas
5-Hydroxymethylcytosine – the elusive epigenetic mark in mammalian DNA.
Chem. Soc. Rev., 2012, 41, (21) 6916–6930.
M. Tomkuvienė, B. Clouet-d'Orval, I. Černiauskas, E. Weinhold and S. Klimašauskas
Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases.
Nucleic Acids Res., 2012, 40, (14) 6765-6773.
R. Sakaguchi, A. Giessing, Q. Dai, G. Lahoud, Z. Liutkevičiūtė, S. Klimašauskas, J. Piccirilli, F. Kirpekar and Y.-M. Hou
Recognition of guanosine by dissimilar tRNA methyltransferases.
RNA , 2012, 18, 1687–1701.
Z. Liutkevičiūtė, E. Kriukienė, I. Grigaitytė, V. Masevičius and S. Klimašauskas
Methyltransferase-directed derivatization of 5-hydroxymethylcytosine in DNA.
Angew. Chem. Int. Ed., 2011, 50, 2090-2093; Very Important Paper.
R. Gerasimaitė, E. Merkienė and S. Klimašauskas
Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase.
Nucleic Acids Res., 2011, 39, 3771-3780; Featured Article.
R.K. Neely, P. Dedecker, J. Hotta, G. Urbanavičiūtė, S. Klimašauskas and J. Hofkens
DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution.
Chem. Sci., 2010, 1, 453-460; Edge Article, commentary in Highlights Chem. Sci.
G. Vilkaitis, A. Plotnikova and S. Klimašauskas
Kinetic and functional analysis of the small RNA methyltransferase HEN1:
The catalytic domain is essential for preferential modification of duplex RNA.
RNA , 2010, 16, 1935-1942.
N. Miropolskaya, V. Nikiforov, S. Klimašauskas, I. Artsimovitch, and A. Kulbachinskiy
Modulation of RNA polymerase activity through trigger loop folding.
Transcription, 2010, 1, 89-94.
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. F1000 evaluation
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. F1000 evaluation
Chemical mapping of cytosines enzymatically flipped out of the DNA helix.
Nucleic Acids Res., 2008, 36, e57.
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.
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. F1000 evaluation
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. Daujotytė, 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. F1000 evaluation
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.
Reviews and Book chapters
E. Kriukienė, Z. Liutkevičiūtė and S. Klimašauskas
5-Hydroxymethylcytosine – the elusive epigenetic mark in mammalian DNA.
Chem. Soc. Rev., 2012, 41, 6916–6930.
Experimental approaches to study DNA base flipping.
DNA and RNA Modification Enzymes: Structure, Mechanism, Function and Evolution.
ed. H. Grosjean (2009), Landes Bioscience, p. 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, p. 639-642.
S. Klimašauskas, Z. Liutkevičiūtė and D. Daujotytė
Biophysical approaches to study DNA base flipping.
Biophysics and the Challenges of Emerging Threats. ed. J.D. Puglisi (2009), Springer Science, p. 50-64.
S. Klimašauskas S. and G. Lukinavičius
Chemistry of AdoMet-dependent methyltransferases.
Wiley Encyclopedia of Chemical Biology. (2008), ed. T.P. Begley, Wiley-Blackwell, ch.335, p. 1–10.
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.
Project