DNA Methyltransferase HhaI DNMT3, DNMT1, DNA restriction modification protein
Peptides and Backbones
Ben Douglass '98, Aaron Downs'00, David Marcey and Aris Kaksis 2014 Riga Stradins University
1MHT0Marz , 1MHTMarz+DNA , 1HMYMarz
July 2011 Molecule of the Month by David Goodsell Protein Data Bank publication 2011 Contents:
I.Background
 II. Reaction
III. HhaI DNA Methyltransferase Monomer Structure
IV. Binding Mechanism
V. HhaI DNA Methyltransferase Complexed with Cofactor: AdoMet binding
VI. HhaI DNA Methyltransferase Complexed with DNA and AdoMet
VII. Future Studies
I. Background
     There are three classes of methyltransferases. Two of the classes methylate exocyclic nitrogens to convert adenine to N6-methyladenine and cytosine to N4-methylcytosine. The third class methylates the fifth cytosine carbon to convert it to 5-methylcytosine; this class is referred to as m5c-methyltransferases. All family members of m5c-methyltransferases are built upon a common architecture of ten conserved motifs (conserved blocks of amino acids). The majority of these conserved, structural motifs are located on the surface of the DNA binding cleft of the molecule . 
     Methyltransferases recognize specific DNA sequences and transfer a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to nucleic acxid nitrogenous bases. Methyltransferases are employed in restriction, modification and mismatch repair systems in prokaryotes, and have been implicated in many molecular processes in eukaryotes including regulation of gene expression, genomic imprinting, DNA repair, mutagenesis, and chromatin organization as histone methylation.

II. HhaI DNA methyltransferase
III. DNMT1 DNA Methyltransferase Monomer Structure:

     HhaI DNA methyltransferase 1MHTMarz,
5MHTMarz,6MHTMarz is a m5c-methyltransferase
that recognizes the. 5'-GCGC-3' sequence in double stranded DNA and flip out DNA C cytosine
DNA binding Zn finger motifs pyrimidine, purine bases G,C,G,C blink purple and flip out DNA C427 cytosine on chain. Backbone thin off .
     Between double strands of DNA
and methylates the C427 cytosine of the recognition sequence.
     DNMT1 DNA methyltransferase 3PT6Marz is a m5c-methyltransferase that recognizes the. 5'-GCGG-3' sequence in double stranded DNA
DNA binding Zn finger motifs pyrimidine, purine bases G,C,G,G blink purple. Backbone thin off
1MHTMarz is a m5c-methyltransferase that recognizes the. 5'-GCGC-3' sequence in double stranded DNA. Backbone thin off .
DNA binding Zn finger motifs pyrimidine, purine bases G,C,G,C , Reaction (Eqution)
     The reaction after Glu119 residue hydrogen bond interaction with target cytosine. Mechanism of m5c-methyltransferases begins with a nucleophilic attack by a cysteine residue CPK color (Cys81 ) on the sixth cytosine carbon C6 of the targeted cytosine base and C5 for methylation -CH3 block blinking.
     This attack creates a covalent intermediate that serves to activate the fifth cytosine carbon C5 as a carbanion that is stabilized by resonance react6MHT. The carbanion attacks the methyl group of AdoMet nucleophilically, which causes the addition of the methyl group to the fifth carbon. AdoMet is altered to AdoHcy after the methyl group is removed by the carbanion. There is a concerted disengagement of the cysteine residue from the sixth cytosine carbon, addition of a hydrogen ion H+ to the cysteine residue Cys81, and the formation of a double bond between the fifth and sixth cytosine carbons >C=C<. 
     Methyltransferases recognize specific DNA sequences and transfer a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to nucleic acxid nitrogenous bases. Methyltransferases are employed in restriction, modification and mismatch repair systems in prokaryotes, and have been implicated in many molecular processes in eukaryotes including regulation of gene expression, genomic imprinting, DNA repair, mutagenesis, and chromatin organization as histone methylation.
     The molecule is comprised of 327 amino acids and
has dimensions of 40 Å x 50 Å x 60 Å. HhaI has three domains, the large domain and small domain, connected by a hinge region < >. Note the pronounced cleft between the large domain and small domain.
This cleft is capable of binding double stranded B-DNA
at 5'-GCGC-3'
    3'-CGCG-5'.
     The large domain < > contains a core beta sheet with six strands (shown in ribbons)< >. Five of the strands are parallel to one another, and the remaining one strand form a hairpin beta sheet turn at one side of the five parallel strands. The helices core beta sheet complex is sandwiched between two alpha helices and two alpha helices plus one beta strand < >. There is also an alpha helix that lies in front of the beta sheet core < > . The large domain contains all or parts of ten of the conserved motifs found in m5c methyltransferases.
     The small domain contains five beta strands . Five beta strands are configured in an antiparallel formation and are arranged in a circular formation that resembles a pinwheel (spirālveida rotoram vējā). There are two strands above and below the antiparallel group, orientated in opposite directions.
     The hinge region < > is composed of an alpha-loop-alpha structure that connects the large and small domain. The hinge region is comprised of parts of conserved motifs IX and X. The first half of motif IX is in the small domain, and provides much of the structural backbone for the small domain. The second half of motif X is located in the large domain.
     The amino acid sequences between motifs VIII and IX are not conserved in all members of m5c-methyltransferases and are labeled the variable region < > . This region has the greatest heterogeneity in size, sequence, and composition among family members. The variable region spans the entire length of the HhaI molecule and folds to form the majority of the small domain. There is a small sub region < > within the variable region that is responsible for sequence specific recognition and target base selection. The target recognition domains < > are located on the side of the cleft in the small domain and directly interact with the major groove of the DNA molecule. 

IV. Binding Mechanism

The HhaI DNA methyltransferase molecule has two seperate binding pockets for DNA and AdoMet. The pockets are in close proximity, and allow the three molecules to interact. HhaI binds DNA, bringing about structural changes that enables the binding of AdoMet to form a tertiary complex. 

V. HhaI DNA Methyltransferase Complexed with Cofactor: AdoMet binding

Click here to see HhaI DNA Methyltransferase structure 1hmy when complexed with AdoMet. ,
Backbone thin off
1MHT0Marz , 1MHTMarz+DNA , 1HMYMarz
     The binding of AdoMet by HhaI methyltransferase is unique. The AdoMet binding site is located in the large domain .
     The site is a hydrophobic pocket Phe18,Ala19,Gly20,Leu21,Gly22,
Gly23,Phe24,Ala27,Trp41,Pro57,Gly59,Ile61,Ile74,Cys76,Ala77,Gly78,
Phe79,Pro80,Gly98,Leu100,Phe101,Phe102,Val121,Val115,Val306,Val307
bounded by < > the single alpha helix in front of the beta sheet < > which is both side double-double helices beta sheet core< > sandvich and by five residues in four conserved motifs : < > motif I Phe18, < > motif III Asp6, < > motif IV Pro80,Gln82, and < > motif X Asn304. Other residues: cyan Gly20,Glu40,Leu100 and Trp41 green surrounding the AdoMet binding pocket interact with the cofactor to tightly bind it to the protein < >. These residues (partially conserved or barely conserved) use a variety of hydrogen bonds and side chain interactions to accomplish the binding of the cofactor. AdoMet is inserted into the pocket so that the methionine projects into the cleft. There is a glycine rich loop Gly20,Leu21,Gly22 redorange inside the binding pocket that serves to position the AdoMet molecule so that the methionine region is in close proximity with the DNA < >.
     DNMT3 adds methyl groups to Cytosine bases in DNA during development of an organism. These methyl groups are important for epigenetic controlling genes in differentiation development of cell types. 
Genetics and Epigenetics.
     The information in DNA does not end at the simple genetic sequence of bases. Cells layer additional forms of control on top of the genetic code, creating "epigenetic" information that modifies the use of baseparing genes in sequences. Nucleosomes control is performed by histone metilation. Bases Cytosine in the DNA are methylated, modifying the read process during protein synthesis.
Is clear how imprinted genes are targeted for methylation. Imprinted genes in mammals are often associated with differentially methylated regions (DMs), which show DNA methylation patterns that depend on the parent of origin. How the imprinting machinery recognizes DMRs is unknown.
     The Dnmt3 family includes three members: two de novo CpG methyltransferases, namely Dnmt3a and Dnmt3b, and an enzymatically inactive paralogue, Dnmt3L, that functions as a regulatory factor in cells.
DNMT3 DNA methyltransferase 2QRVMarz is a m5c-methyltransferase that recognizes the 5'-GCGC-3' sequence in double stranded DNA.
     The complex contains two monomers of Dnmt3a-C and two of Dnmt3L-C, forming a tetramer
(Dnmt3L–Dnmt3a–Dnmt3a–Dnmt3L) with two Dnmt3L–Dnmt3a interfaces (about 906 angstrem square interface area). Hydrophobic bonds of two pairs of phenylalanine residues (F728 and F768 of Dnmt3a, and F261 and F301 of Dnmt3L) with 12 contact points .
     One Dnmt3a–Dnmt3a interface with two pairs of salt bridges formed between R881-D872, D872-H869, H869-E851, E851-R881 (about 944 angstrem square) .
     The Dnmt3L–Dnmt3a interface of Dnmt3L also supports a Dnmt3L homodimer (Fig. 1a, b). Dnmt3a2 might use the same interface to form a Dnmt3a2 homo-oligomer.
     Both interfaces (Dnmt3a–Dnmt3L and
Dnmt3a–Dnmt3a) are essential for catalysis. Dnmt3L might stabilize the conformation of the active-site loop of Dnmt3a (residues 704–725 before helix αD 725-738 , containing the key nucleophile Cys 706).
     TheDnmt3L–Dnmt3a–Dnmt3a–Dnmt3L AdoHcy was found only in Dnmt3a-C.
     Dimerization by means of the Dnmt3a–Dnmt3a interface brings two active sites together and effectively doubles the DNA-binding surface.
     This model indicates that dimeric Dnmt3a could methylate two CpGs separated by one helical turn in one binding event. (Fig. 1a, b) , (Fig. 2a, b)

VI. HhaI DNA Methyltransferase Complexed with DNA and AdoMet
Click here to see HhaI DNA Methyltransferase structure when complexed with DNA and AdoMet.
     HhaI DNA methyltransferase 1MHTMarz is a m5c-methyltransferase that recognizes the. 5'-GCGC-3' sequence in double stranded DNA
DNA binding Zn finger motifs pyrimidine, purine bases G,C,G,C between double strands of DNA .
< > DNA binds to HhaI methyltransferase in the binding cleft formed by the three domains and is situated so that the major groove faces the small domain and the minor groove faces the large domain. There are three sites of protein/DNA interaction: two glycine rich loops  in the small domain and the active site loop in the large domain < >. The HhaI monomer conformation is altered by DNA binding. This change brings the active site loop towards the binding cleft, where it contacts the minor groove of DNA and binds in three places < >. The binding of DNA also brings the small domain closer to the binding cleft so that the glycine rich loops can come into contact with the major groove of the DNA molecule < >.
     In order for the HhaI molecule to methylate the cytosine base, the DNA must undergo structural distortions. Commonly, structural distortions caused by DNA binding proteins result in DNA bends. These proteins usually contact the surface of bases in the major or minor grooves and interact with the external, phosphodiester backbone. In the case of HhaI, however, the buried atoms of cytosine bases of DNA must be accessible for methylation chemistry, requiring drastic distortions to make the bases accessible to the methyltransferase. The phosphodiester backbone of DNA is distorted in such a manner that the phosphates on either side of the target cytosine are shifted outward, increasing the distance between the phosphates on the two strands of DNA. The shifting of the phosphates allows the target cytosine to flip out of the DNA helix through the minor groove < >. Once out of the helix, the target cytosine is held in place by three conserved motifs surrounding the binding cleft in the large domain using hydrogen bonds and salt bridges < >. A simple removal of  the target cytosine would be enrgetically unfavorable, and two residues located on two separate loops move into the helix to occupy the position vacated by the target cytosine, thus restoring stacking interactions < >. These residues are unique to HhaI methyltransferase. The conformational changes bring the target cytosine, the catalytic nucleophile of the active site loop (cysteine81), and the methyl donor (AdoMet) into close proximity < >.

VII. Fresh replicate DNA methylase DNMT1 PDB 3PT6

     The DNA methyltransferase DNMT1, shown here from PDB entry 3PT6, as DNA is being replicated, adds the proper methyl groups to the new DNA strands C.
     Methyl groups are almost always added to Cytosine bases with the sequence:
---CG---
---GC--- that both strands have a Cytosine, so in a methylated region of DNA, both strands will have a methyl group. When the DNA is replicated, each of the new DNA double helices will have one old strand, complete with methyl groups, and one new strand, which is not methylated. So, DNMT1 just needs to look for CG base steps where only one strand has a methyl group and DNMT1 add to second strand C methyl group. De novo methylation ensures that only hemimethylated CpG dinucleotides gain access to the active site.
     Genomic methylation patterns in mammals is required for monoallelic expression of imprinted genes, for the transcriptional silencing of retrotransposon, and for X chromosome inactivation in females.
Structural overview of mDNMT1(646-1602) DNA 19 bp nucleotide oligomer complex with bound AdoHcy. (Fig. 1a, b)
DNMT1(646-1600) DNA methyltransferase 3PT6Marz , 3PTAMarz , 4DA4Marz , 4DKJMarz , 3MHTMarz is a m5c-methyltransferase that recognizes the. 5'-GCGG-3' sequence in double stranded DNA
DNA binding Zn finger motifs pyrimidine, purine bases G,C,G,G . (Fig. 2a, b)
The conserved Zn finger 3PT6Zn651-699
Zn 2 Coordinative Bond between residues
Cys656,Cys659,Cys662,Cys694
Zn 4 Coordinative Bond between residues
Cys667,Cys670,Cys673,Cys689 is indicated as yellow stick.
The conserved Zn finger 3PTAZn651-699
Zn 2 Coordinative Bond between residues
Cys653Cys656Cys659Cys691
Zn 5 Coordinative Bond between residues
Cys664,Cys667,Cys670,Cys686 is indicated as yellow stick.
The conserved Zn finger 4DA4Zn1477-1506
The conserved Zn finger4DA4Zn794-902
Zn 1702 Coordinative Bond between residues
CYS1479,CYS1481,CYS1487,HIS1504
Zn 1703 Coordinative Bond between residues
HIS796,CYS823,CYS897,CYS900 is indicated as yellow stick.

References
1. Cheng, X., Kumar, S., Posfai,P.,Pflugrath, J., Roberts, R.(1993). Crystal Structure of the HhaI DNA Methyltransferase Complexed with S-Adenosyl-L-Methionine. Cell. 74:299-307. PDB:1MHT
2. Klimasauskas, S., Kumar, S., Roberts, R., Cheng, X.(1994). HhaI Methyltransferase Flips Its Target Base Out of the DNA Helix. Cell. 76: 357-369.PDB:1MHT
3 . Nature. 2007 September 13; 449(7159): 248–251. DNA DNMT3 Methylase PDB: 2QRV
4 . Science 25 February 2011: Vol. 331 no. 6020 pp. 1036-1040 DNA DNMT1 Methylase PDB:3PT6,3PTA
5 . July 2011 Molecule of the Month by David Goodsell PDB DNA Methyltransferases HhaI Methylase PDB: 2QRV,3PT6 1MHT=DNA=DNMT3,DNMT1
6 . PNAS 2011: vol. 108 no. 22 mouse3PT6+DNA+3PTAhuman; 3AV4; Hha1 5mht;1MHT;AdoHcy 3PT9 DMT1
7. Science 10 February 2012:  Vol. 335 no. 6069 pp. 709-712 PDB: 4DA4,3PT6,3PTA,3MHT,4DKJ