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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: