8/8/2019 Recognition of hemi-methylated DNA by UHRF1 (Ronen Zangi, Euskal Herriko Unibersitatea)
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Recognition of hemi-methylated DNA by UHRF1
Ronen Zangi
Department of Organic Chemistry I
University of the Basque Country, UPV/EHU
San Sebastian, Spain
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The Epigenetic Code
Epigenetics: extra layer of instructions that influence gene activity without altering the
DNA sequence.
LeDinhLuong;http://cnx.org/
content/m26565/
DNA Methylation (CpG sequence)
A stable epigenetic mark that regulates gene expression/silencing through
unmethylated/methylated CpG islands in the promoter region of the DNA.
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Inheritance and Maintenance of DNA Methylation
Each type of cell has its own methylation pattern
Patterns of methylated cytosines are propagated with fidelity > 99%
and their stable inheritance for more than 80 cell generations.
T TCGACGAA
T T C G A T C G A5
53
3
T TCGACGAA
T TCGACGAA T TCGACGAA
T T C G A T C G A5
53
3
T TCGACGAA
T T C G A T C G A5
53
3
DNMT1
DNMT1
Replication
T T C G A T C G A5
53
3
5
53
3T T C G A T C G A
original methylation pattern is maintainedDNA immediately after replication
M
M
M
M
M
M
M
M
DNMT1 has to distinguish hemimethylated from either unmethylated or symmetrically
dimethylated DNA strands.
The selectivity is actually performed by UHRF1 (Np95).
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UHRF1DNA Interaction
UHRF1 exhibits strong preferential binding to hemimethylated DNA through its SET
and RING-associated domain.
The methyl-cytosine that is recognized is flipped out of the DNA helix.
The broken WC hydrogen bonds are
replaced by hydrogen bonds with
Asn-Lys-Arg finger of UHRF1 that
intrudes into the DNA.
Nature455,822(2008
)
UHRF1 can also bind to DNMT1 =
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Aims of the Project
1. How can UHRF1 distinguish so accurately hemimethylated
from either unmethylated or fullymethylated CpG sites?
NH
O
NH2
N
NH
O
NH2
N
CH3H
The difference is one methyl group -
no (direct) change of electrostatics
Gtransfer of methane from SDS towater is 4 kJ/mol = K300K 0.2
Half of the atoms surrounding the
methyl group (in the UHRF1-DNA complex) are
hydrophilic with potential of forming hydrogen bonds.
2. Base flipping process:
a. of the recognized methylcytosine
What is intrinsic propensity of methylated cytosine to
flip out of the helix relative to unmethylated cytosine? Does the UHRF1-DNA interaction involve active or
passive base flipping?
b. of the target cytosine to b e methylated
Does the flipped methylcytosine increases the propensity
of flipping the target cytosine on the opposite strand?
3
3 5
5
C
G
T
G
T
A
C
A
G
C
G
A
C
T
DNMT1
UHRF1
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Necessity for Supercomputer
All-atom Molecular Dynamics Simulations of Biological Macromolecules
12-mer DNA: 5GGGCCCGCAGGG3/5CCCTGCGGGCCC3
Neutral system: + 22 Na+ = Particle-Mesh Ewald for electrostatics
Explicit solvent: 8300 water molecules = 6.56.56.5 nm3
SRA domain of UHRF1: 212 residues
Gromacs 4.0.7
0 4 8 12 16 20 24 28 32
# of processors
0.0
2.0
4.0
6.0
8.0
10.0
12.0
14.0
16.0
twall
/ts
imulation
[hour/ns]
Performance on Marenostrum
Prediction based on 100% scaling of 2-cpu job
Scaling Performance
60% efficiency
73% efficiency
Few Long Runs (200 ns):
32 processors, submission
(& resubmission) to the 72 h queue
Many Short Runs (520 ns):
216 processors, submission
(& resubmission) to the 24 (72) h queue
Free energy calculations using the Thermodynamic Integration Technique:
GBA = G(B)G(A) =BA
G
d =BA
H
d
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Results: BI BII transitions in DNA
0 20 40 60 80 100 120 140 160 180 200Time [ns]
0.0
0.2
0.4
0.6
RMSD
[nm
]
unmethylated CpG
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Results: BI BII transitions in DNA
0 20 40 60 80 100 120 140 160 180 200Time [ns]
0.0
0.2
0.4
0.6
RMSD
[nm
]
unmethylated CpG
hemimethylated CpG
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Results: BI BII transitions in DNA
0 20 40 60 80 100 120 140 160 180 200Time [ns]
0.0
0.2
0.4
0.6
RMSD
[nm
]
unmethylated CpG
hemimethylated CpG
fullymethylated CpG
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Results: Stability of Fully/Hemi-Methylated CpG site
Thermodynamic cycles with alchemistry free energy transformation
G
mC G
mC
+G
mC G
mC
Fullymethylated
Formation of
DNA
G (fully)
G1
G2
Hemimethylated
Formation of
DNA
G (hemi)
G
mC G
C
+G
mC G
C
G4G3
G
C G
C
+Formation of
Unmethylated
DNA
G (un)
G
C G
C
forward backward average
G1 -129.7 -129.5 -129.6
G2 -128.3 -127.0 -127.6
G3 -130.7 -130.5 -130.6
G4 -127.1 -127.1 -127.1
Gf(fully)Gf(hemi) = G1G2 = 2.0 kJ/mol
Gf(hemi)Gf(un) = G3G4 = 3.5 kJ/mol
The formation of fullymethylated CpG
site is more favorable by 5.5 kJ/mol
relative to that of unmethylated CpG site.
Methylation of cytosine
stabilizes the DNA double helix
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Acknowledgments
Caterina Bianchi
Funding Agencies:
International Reintegration Grant
Computer Facility: