Founded in 1975 as an independent research institution, now part of Vilnius University, the Institute of Biotechnology strives to maintain the high standards of excellence in scientific endeavour, research training and technological advance with its main focus in a broadly defined field of molecular biotechnology including nucleic acid and protein technologies, bioinformatics, molecular diagnostics, drug design, next generation epigenomic and gene editing technologies. The Institute provides an interface between advanced education, basic research and technological development for the economic and social benefit of Lithuania.
Chief Scientist and Head
phone: +370 5 2234350
fax: +370 5 2234367
e-mail: saulius.klimasauskas (at) bti.vu.lt
Employees PhD students
Giedrius Vilkaitis, Ph.D. Stasė Butkytė, M.Sc.
Edita Kriukienė, Ph.D. Janina Ličytė, M.Sc.
Rasa Rakauskaitė, Ph.D. Milda Mickutė, M.Sc.
Viktoras Masevičius, Ph.D. Povilas Gibas, M.Sc.
Juozas Gordevičius, Ph.D. Milda Rudytė, M.Sc.
Miglė Tomkuvienė, Ph.D.
Vaidotas Stankevičius, Ph.D.
Zdislav Staševskij, M.Sc.
Giedrė Urbanavičiūtė, M.Sc.
Audronė Rukšėnaitė, M.Sc.
Aleksandr Osipenko, M.Sc.
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 to study the 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 (Daujotytė 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. We have synthesized a series of model AdoMet analogs with sulfonium-bound extended side chains replacing the methyl group (Dalhoff et al., 2006; Klimašauskas and Weinhold, 2007). This novel enabling technology named mTAG (methyltransferase-directed Transfer of Activated Groups) is a convenient and robust technique that is suitable for routine laboratory use. In particular, we demonstrated that propargylic side chains can be efficiently transferred by DNA MTases with high sequence- and base-specificity (Lukinavičius et al., 2007, 2012 and 2013; Masevičius et al., 2016) offering many potential applications for genomic (Neely et al., 2010; see commentary in Highlights Chem Sci) and epigenomic (see below) studies. Moreover, the newly developed cofactors are suitable for sequence-specific transfer of functional groups or other chemical entities to RNA (Tomkuvienė et al., 2012; Plotnikova et al., 2014) 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; Kriukienė et al. 2013; Labrie et al., 2016) in the genome 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) or decarboxylation of 5-carboxylcytosines (Liutkevičiūtė et al., 2014) in DNA. 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).
Methylation of small non-coding RNA
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 for 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; Plotnikova et al., 2013 and 2014; Baranauskė et al., 2015).
Z. Staševskij, P. Gibas, J. Gordevičius, E. Kriukienė, and S. Klimašauskas
Tethered Oligonucleotide-Primed sequencing, TOP-seq: a high resolution economical approach for DNA epigenome profiling.
Mol. Cell, 2017, 65(3): 554–564.
M. Tomkuvienė, E. Kriukienė, and S. Klimašauskas
DNA labeling using DNA methyltransferases.
Adv. Exp. Med. Biol., 2016, 945: 511-535.
V. Labrie, O. J. Buske, E. Oh, R. Jeremian, C. Ptak, G. Gasiūnas, A. Maleckas, R. Petereit, A. Žvirbliene, K. Adamonis, E. Kriukienė, K. Koncevičius, J. Gordevičius, A. Nair, A. Zhang, S. Ebrahimi, G. Oh, V. Šikšnys, L. Kupčinskas, M. Brudno, and A. Petronis
Lactase nonpersistence is directed by DNA-variation-dependent epigenetic aging.
Nature Struct. Mol. Biol. 2016, 23(6): 566-573.
V. Myrianthopoulos, P. F. Cartron, Z. Liutkevičiūtė, S. Klimašauskas, D. Matulis, C. Bronner, N. Martinet, and E. Mikros
Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation.
Eur. J. Med. Chem., 2016, 114: 390–396.
V. Masevičius, M. Nainytė, and S. Klimašauskas
Synthesis of S-adenosyl-L-methionine analogs with extended transferable groups for methyltransferase-directed labeling of DNA and RNA.
Curr. Protoc. Nucleic Acid Chem., 2016, 64: 1.36.1-1.36.13.
D. Esyunina, M. Turtola, D. Pupov, I. Bass, S. Klimašauskas, G. Belogurov, and A. Kulbachinskiy
Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases.
Nucleic Acids Res., 2016, 44(3): 1298–1308.
R. Rakauskaitė, G. Urbanavičiūtė, A. Rukšėnaitė, Z. Liutkevičiūtė, R. Juškėnas, V. Masevičius, and S. Klimašauskas
Biosynthetic selenoproteins with geneticallyencoded photocaged selenocysteines.
Chem. Commun., 2015, 51(39): 8245-8248.
S. Baranauskė, M. Mickutė, A. Plotnikova, A. Finke, Č. Venclovas, S. Klimašauskas, and G. Vilkaitis
Functional mapping of the plant small RNAmethyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.
Nucleic Acids Res., 2015, 43(5): 2802-2812.
A. Plotnikova, A. Osipenko, V. Masevičius, G. Vilkaitis, and S. Klimašauskas
Selective covalent labeling of miRNA and siRNA duplexes using HEN1 methyltransferase.
J. Am. Chem. Soc., 2014, 136(39): 13550–13553.
Z. Liutkevičiūtė, E. Kriukienė, J. Ličytė, M. Rudytė, G. Urbanavičiūtė, and S. Klimašauskas
Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases.
J. Am. Chem. Soc., 2014, 136(16): 5884−5887.
N. Miropolskaya, D. Esyunina, S. Klimašauskas, V. Nikiforov, I. Artsimovitch, and A. Kulbachinskiy
Interplay between the trigger loop and the F loop during RNA polymerase catalysis.
Nucleic Acids Res., 2014, 42(1): 544–552.
E. Kriukienė, V. Labrie, T. Khare, G. Urbanavičiūtė, A. Lapinaitė, K. Koncevičius, D. Li, T. Wang, S. Pai, C. Ptak, J. Gordevičius, S.C. Wang, A. Petronis, and S. Klimašauskas
DNA unmethylome profiling by covalent capture of CpG sites.
Nature Commun., 2013, 4: 2190.
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, 453: 281-290.
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, 8: 1134-1139.
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.
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.
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.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
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. Am. 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.
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.
M. Tomkuvienė, E. Kriukienė, and S. Klimašauskas
DNA labeling using DNA methyltransferases.
DNA Methyltransferases - Role and Function.
ed. A. Jeltsch and R.Z. Jurkowska (2016) Springer International Publishing, p. 511-535.
E. Kriukienė, Z. Liutkevičiūtė, and S. Klimašauskas
5-Hydroxymethylcytosine – the elusive epigenetic mark in mammalian DNA.
Chem. Soc. Rev., 2012, 41, 6916–6930.