Purification, crystallization, and preliminary x-ray diffraction analysis of an M.HhaI-AdoMet complex

Kumar, Sanjay; Cheng, Xiaodong; Pflugrath, J.W.; Roberts, Richard J.

doi:10.1021/bi00151a035

Cited by 64 publications

(69 citation statements)

References 34 publications

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“…Deionized Milli-Q water was used for all of the studies. M.HhaI was prepared according to a method described previously (20). The following oligonucleotides were synthesized (New England Biolabs) and used as substrates in the methylation reactions: 5Ј-GACTGGTACAGTATCAGGCGCTGACCCA-CAACATCCG-3Ј and 5Ј-TCGGATGTTGTGGGTCAGCGCCTGATACT-GTACCAGT-3Ј.…”

Section: Methodsmentioning

confidence: 99%

“…The resultant plasmids containing the mutations were sequenced on both strands to confirm that no undesired codon changes had occurred. Mutant M.HhaI genes were subcloned into the overexpression vector pUHE25, and the mutant methyltransferases were purified according to the protocol followed for the wild type enzyme (20). Both of the mutant methyltransferases, W41I and W41Y, were as active as the wild type M.HhaI with no significant changes in their kinetic parameters (data not shown).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Swaminathan¹,

Sankpal²,

Rao³

et al. 2002

Journal of Biological Chemistry

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Methyltransferase-catalyzed modification of substrate DNA, RNA, or protein serves as a key signal in the regulation and maintenance of diverse biological processes in a range of organisms from prokaryotes to eukaryotes (1, 2). Subsequent to its isolation from Hemophilus haemolyticus nearly a quarter century ago, HhaI DNA methyltransferase (M.HhaI) 1 has been subjected to incisive investigations yielding detailed insights into the reaction chemistry (3-7). M.HhaI is an S-adenosyl-Lmethionine (AdoMet) dependent DNA methyltransferase that catalyzes the covalent attachment of a methyl group at the C-5 position of the aromatic ring of the first cytosine in the specific sequence 5Ј-GCGC-3Ј. Its ability to perform the methyl transfer within the B-DNA helix was rationalized by a localized "DNA backbone rotation" by nearly 180°(base flipping) without significantly bending or kinking of the rest of the duplex (8 -11). Base flipping serves to deliver the base into a concave catalytic site in the enzyme (8). The Gln 287 side chain fills up the gap generated concomitantly in the DNA bound with M.HhaI, leading to a stable and specific interruption of the aromatic -stacked base pairs, a feature recently utilized to evaluate long range electron migration through DNA (12). M.HhaI methylates all of the cytosines in poly(dG-dC) DNA (3), with hemimethylated DNA being the preferred substrate (13). Both the reaction product S-adenosyl-L-homocysteine (AdoHcy) (14) and, by implication, the cofactor AdoMet (7), appear to share the ability to drive M.HhaI to trap the target cytosine in the catalytic site to form a productive complex. These results portray AdoMet and AdoHcy as important modulators of the multiple binding modes of the DNA-bound enzyme.Despite a wealth of information on the structural, dynamic, and kinetic aspects of the M.HhaI mechanism, little attention has been paid to the energetics of AdoMet and AdoHcy recognition reactions. No molecule other than ATP serves as a cofactor in more diverse reactions than does AdoMet (15). It was, therefore, of interest to understand the thermodynamic basis of recognition of the ubiquitous cofactor AdoMet by methyltransferases, a feature shared among structurally related enzymes that catalyze reactions with diverse substrate specificities (16 -18). For example, a snapshot of a step that follows methyl transfer has been elucidated by crystallographic analyses of the DNA intermediate covalently bound to M.HhaI (8) and M.HaeIII (19). Despite its absence in the original crystallization mix of the ternary complex, it was AdoHcy that was found, fortuitously, to reside within the cofactor binding pocket (8) shared by AdoMet (16). This illustrates that the transfer of the methyl group from AdoMet to the target cytosine results in the formation not only of the modified (i.e. methylated) DNA but also the transformation of AdoMet into AdoHcy. On the other * This work was supported by grants from the Department of Biotechnology, Government of India (to D. N. R. and A. S.) and the Department of Scien...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Swaminathan¹,

Sankpal²,

Rao³

et al. 2002

Journal of Biological Chemistry

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“…The cofactor SAM is often strongly associated with methyltransferases and may be retained in preparations even after several steps of chromatographic purification (Kumar et al, 1992). Substantial amounts of bound SAM (over 10 to 20 mol%) may interfere with certain biochemical and kinetic experiments.…”

Section: Methyltransferase Assays As Monitored By β-Eliminationmentioning

confidence: 99%

“…Several rounds of dialysis against a suitable buffer are usually sufficient to remove most of the bound cofactor. The detection of endogenous SAM is based on in vitro enzymatic reactions, in which increasing amounts of a methyltransferase are incubated with a fixed amount of its substrate without added SAM (Kumar et al, 1992). For example, methylation reactions can be performed with 0.2 μM miR173/miR173★ duplex and several concentrations of GST-HEN1 in the range 0.2 to 3 μM.…”

Section: Methyltransferase Assays As Monitored By β-Eliminationmentioning

confidence: 99%

Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo

Yang¹,

Vilkaitis²,

Yu³

et al. 2007

Methods in Enzymology

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The biogenesis of microRNAs (miRNAs) in plants is similar to that in animals, however, the processing of plant miRNAs consists of an additional step, the methylation of the miRNAs on the 3′ terminal nucleotides. The enzyme that methylates Arabidopsis miRNAs is encoded by a gene named HEN1, which has been shown genetically to be required for miRNA biogenesis in vivo. Small interfering RNAs (siRNAs) are also methylated in vivo in a HEN1-dependent manner. Our biochemical studies demonstrated that HEN1 is a methyltransferase acting on both miRNAs and siRNAs in vitro. HEN1 recognizes 21 to 24 nt small RNA duplexes, which are the products of Dicer-like enzymes, and transfers a methyl group from S-adenosylmethionine (SAM) to the 2′ OH of the last nucleotides of the small RNA duplexes. Here we describe methods to characterize the biochemical activities of the HEN1 protein both in vitro and in vivo, and methods to analyze the methylation status of small RNAs in vivo.

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“…For the purification of M⅐HhaI and M⅐HhaI-dod, E. coli RRI cells containing pSP72.HhaI or pSP72.HhaI-dod were cultured in LuriaBertani medium with 50 g͞ml ampicillin, and the enzyme was purified essentially as described in ref. 18. For the purification of M⅐HhaI-dod that purification was modified to use High-S fast protein liquid chromatography (FPLC) (Bio-Rad), with final purification obtained with a His-Trap (Pharmacia) affinity column (19).…”

Section: Molecular Cloning Of the M⅐hhai-dod Fusion Proteinmentioning

confidence: 99%

Nucleoprotein-based nanoscale assembly

Smith

Niu²,

Baker³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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A system for addressing in the construction of macromolecular assemblies can be based on the biospecificity of DNA (cytosine-5) methyltransferases and the capacity of these enzymes to form abortive covalent complexes at targeted 5-f luorocytosine residues in DNA. Using this system, macromolecular assemblies have been created using two representative methyltransferases: M⅐HhaI and M⅐MspI. When 5-f luorocytosine (F) is placed at the targeted cytosine in each recognition sequence in a synthetic oligodeoxynucleotide (GFGC for M⅐HhaI or FCGG for M⅐MspI), we show that the first recognition sequence becomes an address for M⅐HhaI, while the second sequence becomes an address for M⅐MspI. A chimeric enzyme containing a dodecapeptide antigen linked to the C terminus of M⅐HhaI retained its recognition specificity. That specificity served to address the linked peptide to the GFGC recognition site in DNA. With this assembly system components can be placed in a preselected order on the DNA helix. Axial spacing for adjacent addresses can be guided by the observed kinetic footprint of each methyltransferase. Axial rotation of the addressable protein can be guided by the screw axis of the DNA helix. The system has significant potential in the general construction of macromolecular assemblies. We anticipate that these assemblies will be useful in the construction of regular protein arrays for structural analysis, in the construction of protein-DNA systems as models of chromatin and the synaptonemal complex, and in the construction of macromolecular devices.Macromolecular assembly is easily approached with DNA. Branching through the formation of Watson-Crick paired duplexes in the shape of a Y or an X is now well known (1-4), and the feasibility of assembling 2-dimensional quadrilaterals and 3-dimensional cubes on which more extended structures can be based has been demonstrated (5, 6). However, the stable, site-directed attachment of labile enzymes and proteins to a DNA scaffold presents a formidable challenge in macromolecular fabrication. Candidate procedures in which the Watson-Crick base-pairing homology or triple-helix basepairing homology of an oligodeoxynucleotide is used to direct a tethered moiety to a preselected site in DNA (3, 4, 7-9) involve extremes of pH or temperature that can destroy the native structure of these proteins. Attachment systems based on antibodies directed against DNA are likely to lack specificity. On the other hand, antibodies to a hapten could be used to decorate a matrix depending on the pattern of haptens laid down during synthesis. The disadvantage here is that all hapten moieties are equivalent, and thus selective addressing would not be possible unless a series of haptens and antibodies directed to them could be developed. While a system of distinct haptens and antibodies is possible (3), it would be necessary to develop a set of hapten-phosphoramidites and the corresponding series of bifunctional antibodies to utilize this approach. Moreover, the use of noncovalent linkages sacrifice...

show abstract

Purification, crystallization, and preliminary x-ray diffraction analysis of an M.HhaI-AdoMet complex

Cited by 64 publications

References 34 publications

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Approaches for Studying MicroRNA and Small Interfering RNA Methylation In Vitro and In Vivo

Nucleoprotein-based nanoscale assembly

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