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Oligoduplex Structure and Dimethylated DNA Base-Flipping Activity of the N-terminal Domain of E. coli Endonuclease McrB

Maggie Koenecke '15 and Eric Engelbrecht '14


I. Introduction

The McrBC is a modified cytosine restriction enzyme. The protein is an Escheria coli endonuclease with high affinity for DNA containing 5-methylcytosine (5mC). The N-terminal domain responsible for DNA binding shows a unique DNA-binding fold specifically adapted to recognize 5mC.

In complex with DNA, McrB N-terminal domain (McrB-N) flips out the cytosine bases in dimethylated, hemimethylated, and nonmethylated DNA. The protein makes similar contacts with these different types of DNA; consequently, only the interactions with dimethylated DNA are shown.

II. General Structure

The N-terminal domain of McrB is a composed of a polypeptide chain 161 amino acids in length. The McrB N-terminal recognition domain folds into three α-helices and an anti-parallel beta sheet composed of five β-strands in the order: α1, β1, β2, β3, β4, α2, β5, and α3. The N-terminal α1 packs on the convex side of the beta-sheet with the C-terminal α3.   The concave side of the β-sheet is oriented towards the DNA. Three loops from the beta-sheet grip the minor groove of DNA, which is made significantly wide compared to B-DNA because the is bent 29° toward the major groove.

Two McrB-N monomers bind to the 5’-AmC sites on each strand of the dimethylated DNA. These monomers do not contact each other (in crystal). Both 5mC residues are flipped out of the DNA and positioned in the proteins’ binding pockets.

III. Protein-DNA Interactions

Two McrB-N monomers make identical contacts with dimethylated DNA. McrB-N approaches the minor groove and amino acids from loops α1β1, β1β2, and β2β3 interact with the DNA. Direct and water-mediated interactions with the 3'- and 5'-phosphates of the extrahelical 5mC base are made by Ser 38, Thr45, Ser46, Trp49, Glu58, Ala59, and Ser60. Gln21, Ser22, Thr23, and Lys 24 (from α1β1 loop) contact the 3'- and 5'-phosphates of C11 on the opposite strand. Only Tyr41 and Asn 43 contact the DNA bases directly. occupies the space left by the flipped out 5mC and accepts a hydrogen bond from the orphaned guanine at its backbone oxygen. The backbone nitrogen of is a hydrogen bond donor with adjacent C bases interspaced between the two 5'-RmC sequences.These interactions are crucial for McrB-N base-flipping activity and subsequent complex stability.

McrBC is able to discriminate between purines versus pyrimidines upstream of the 5mC, which is demonstated by the fact that it only cleaves DNA between two 5'-RmC sites. McrB-N affinity to oligoduplexes with 5'-RmC is ~100 times higher than oligoduplexes with 5'-YmC sites. It is possible that McrBC evolved to recognize and cleave 5'-RmC because that particular oligoduplex motif was consistent with bacteriophage DNA modifications.

IV. Affinity for 5mC

is positioned in the protein-binding pocket and adopts an anti-conformation. Residues Trp49, Leu68, Tyr 117, Tyr64, Ala59, Ser60 and backbone atoms of 82-85 form the walls of the binding pocket. A single mediates several interactions between residues in the binding pocket and nearby bases. Tyr117 with the flipped out cytosine while Ile82, Asp84, and Thr85 are involved in hydrogen bonds with the flipped out base.

The substituent 4-amino-group donates a hydrogen bond to .The backbone N of donates to the N3 atom and the O2 atom of 5mC interacts with the backbone N and side chain OG atom of . Most hydrogen bond interactions are made by the backbone of β4α2 loop, so it is unsurprising that Ile82, Asp84, and Thr85 are not conserved in McrB-N homologues. These direct hydrogen bonds in the binding pocket are only compatible with C or 5mC residues, excluding A, T, and G bases.

Discrimination between C and 5mC bases is made possible by Van der Waals interactions between the 5-methyl group and the side chains of . The significance of these interactions is reflected by the conservation of Tyr64 and Leu68 among Mcr-B homologues.  

V. Implications

Endonucleases are involved in several cellular mechanisms, including genetic recombination, DNA damage repair, and even removing arrested RNA polymerase (bacterial Uvr(A)BC). It is thought that in the on-going evolutionary arms race between bacteria and bacteriophages, bacteria evolved the ability to methylate their DNA so unmethylated foreign DNA could be easily recognized and cleaved by an endonuclease. In response, bacteriophages evolved the ability to methylate/hydroxymethylate their DNA during DNA replication. One thought is that bacteria evolved to combat foreign DNA with a methyl/hydroxymethyl-specific endonuclease in the form of Mcr-BC. It is possible that McrBC preferably binds and cleaves 5'-RmC because invasive bacteriophages modified their DNA with this motif.

VI. References

Gast, F., Brinkmann, T., Pieper, U., Kruger, T., NoyerWeidner, M., & Pingoud, A. (1997). The recognition of methylated DNA by the GTP-dependent restriction endonuclease McrBC resides in the N-terminal domain of McrB. Biological Chemistry, 378(9), 975-982.

Pieper, U., Brinkmann, T., Kruger, T., NoyerWeidner, M., & Pingoud, A. (1997). Characterization of the interaction between the restriction endonuclease McrBC from E-coli and its cofactor GTP. Journal of Molecular Biology, 272(2), 190-199.

Pieper, U., Schweitzer, T., Groll, D., & Pingoud, A. (1999). Defining the location and function of domains of McrB by deletion mutagenesis. Biological Chemistry, 380(10), 1225-1230.

Sukackaite, R., Grazulis, S., Tamulaitis, G., & Siksnys, V. (2012). The recognition domain of the methyl-specific endonuclease McrBC flips out 5-methylcytosine. Nucleic Acids Research, 40(15), 7552-7562.

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