Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase

Chen, Lin; MacMillan, Andrew M.; Chang, W.; Ezaz-Nikpay, Khosro; Lane, William S.; Verdine, Gregory L.

doi:10.1021/bi00110a002

Cited by 245 publications

(172 citation statements)

References 20 publications

Supporting

Mentioning

166

Contrasting

Unclassified

Order By: Relevance

“…The ESI-MS approach used in this study to identify amino acid residues involved in nucleophilic attack on a DNA substrate may be used to analyze other DNA glycosylases and DNA-protein complexes and may, in some cases, replace more time-consuming methods of identifying the peptide portions of these complexes (28). A stable covalent bond, linking protein to DNA, is required for ESI-MS analysis; two new ionization techniques, electrospray and matrix-assisted laser desorption, provide accurate mass assignments for large complexes of this type.…”

Section: Discussionmentioning

confidence: 99%

NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

Zharkov

Rieger

Iden

et al. 1997

Journal of Biological Chemistry

175

163

View full text Add to dashboard Cite

Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli. The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), ␤-elimination (AP lyase), and ␦-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA. Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A. P. (1995) J. Biol. Chem. 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride. By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA. This experimental approach may be applicable to the analysis of other protein-DNA complexes.Fpg 1 protein, a DNA base excision repair enzyme with Nglycosylase and AP lyase activities (1-3), efficiently removes 8-oxoguanine (8-oxoGua) and formamidopyrimidines from oxidatively damaged DNA (4 -7). We have shown previously (8) that this reaction involves an imino-enzyme-substrate (Schiff base) intermediate and that the amino group involved is located within a 72-amino acid fragment of Fpg protein containing the N terminus. The four-cysteine zinc finger motif located near the C terminus (9, 10) is utilized in binding oxidatively damaged DNA (10, 11). The 8-oxo moiety is a critical structural determinant by which Fpg protein recognizes duplex DNA substrates containing 8-oxoguanine (5).It has been proposed that Schiff bases participate in the catalytic action of AP endonucleases (12) and DNA glycosylases that possess AP lyase activity (13-16). Sodium borohydride and cyanoborohydride have been used to trap Schiff base intermediates as covalently linked DNA-protein complexes in reactions catalyzed by Fpg protein (8, 16) and by bacteriophage T4 endonuclease V, Micrococcus luteus UV endonuclease and Escherichia coli endonuclease III (15, 16). A mechanism involving nucleophilic attack on C1Ј of the modified deoxynucleoside targeted for excision (8,(15)(16)(17) has been proposed to explain the catalytic action of these enzymes. Nucleophilic attack, facilitated by protonation of the base, effects cleavage of the N-glycosidic bond. The AP lyase activity of Fpg protein is a concerted reaction involving ␤-and ␦-elimination reactions (2, 18, 19), producing a gap in one strand of the duplex demarcated by 3Ј-and 5Ј-phosphate termini (2, 18).Several nucleophiles capable of forming Schiff base intermediates (20) are located within the 72-residue N-terminal fragment of Fpg protein. Basic nitrogen functional groups are found in the N-terminal proline, the free amino group of Lys-56, and eight arginine residues. Following enzymatic digestion of the covalently linked complex, we used elect...

show abstract

Section: Discussionmentioning

confidence: 99%

NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

Zharkov

Rieger

Iden

et al. 1997

Journal of Biological Chemistry

175

163

View full text Add to dashboard Cite

show abstract

“…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)(4)(5)(6)(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.…”

mentioning

confidence: 99%

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Swaminathan¹,

Sankpal²,

Rao³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

“…2 A was 82.2 hr Ϫ1 , while that for 5-fluorocytosine in the same oligodeoxynucleotide background was 0.8 hr Ϫ1 . This reduction in reaction rate (Ͼ100-fold) occurs under conditions where complex formation can be as high as 95% of input enzyme (13,21). In the experiments reported here, where the complexes were incubated for 2.5 hr before separation, they are expected to be 100% covalent at C6 but only about 38% irreversible due to the slow rate of methyltransfer at C5 (see Fig.…”

Section: Resultsmentioning

confidence: 87%

“…They have well characterized DNA sequence specificities (11) and they form abortive covalent complexes (Fig. 1) between an active-site cysteine and 5-fluorocytosine in their DNA recognition sequences (12)(13)(14)(15). In this report, we demonstrate this approach to macromolecular assembly using two representative methyltransferases: M⅐HhaI and M⅐MspI.…”

mentioning

confidence: 78%

Nucleoprotein-based nanoscale assembly

Smith

Niu²,

Baker³

et al. 1997

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

View full text Add to dashboard Cite

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

Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase

Cited by 245 publications

References 20 publications

NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

NH2-terminal Proline Acts as a Nucleophile in the Glycosylase/AP-Lyase Reaction Catalyzed by Escherichia coli Formamidopyrimidine-DNA Glycosylase (Fpg) Protein

Water-assisted Dual Mode Cofactor Recognition by HhaI DNA Methyltransferase

Nucleoprotein-based nanoscale assembly

Contact Info

Product

Resources

About