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
Contents:
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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