Molecular Understanding of Efficient DNA Repair

Machinery of Photolyase

Chuang TanChemical Physics ProgramThe Ohio State University

2012.06.19

Prof. Dongping Zhong LabDepartments of Physics, Chemistry, and BiochemistryPrograms of Biophysics, Chemical Physics, and BiochemistryThe Ohio State University

Human Genetics and Genomics, Third edition. Bruce Korf (2006)

N

O

O P

O

OH

O

N

O

OH

HN

N

N

N

OH

CH3

CH3

O

4

HN NH

CH3 H3C

O

HO

O

O

O

PHO O

OH

N

O

HO

HN

N

O

OH

NH

CH3 CH3

O

P

O

OO

O O

O

HOO

OHO

55

UV light

Thymine dinucleotide(TpT)

Cyclobutane pyrimidine dimer (CPD or T<>T)

(6-4) photoproduct(64PP)

O

O O

O

+

~80% ~20%

UV-induced DNA damage1) Cyclobutane Pyrimidine Dimer2) (6-4) Products

Introduction

Cause genetic mutationsBlock replication and transcription……

Skin Cancer!!

DNA Damage

Introduction DNA Repair

Sancar Chem. Rev. 103, 2203 (2003)

Repair the UV-induced DNA damage using 300-500 nm light as energy source.

Photolyase

Kao et al. Cell Biochem. Biophys. 48, 32 (2007)

The repair process involves a series of light-driven electron transfers and bond breakages.

QuestionHow does photolyase modulate this so complicated repair process?

Important residues at the active site

Three charged/polar residues R232(R226), E283 (E274) and R350 (R342) have hydrophilic interaction with dimer and the flavin ring; N386 (N378) forms H-bond with flavin ring, and the sulfur atom of M353 (M345) may have interaction with the 3’-thymine. The mutation of these residues results in the decrease of the repair efficiency.

E. coli Photolyase WT E274A R226A R342A N378C N378S M345A

Quantum Yield 0.82 0.38 0.53 0.48 0.67 0.62 0.72

Methodology Ultrafast Time Resolved Techniques

Pump-Probe method:

One laser pulse initiates the reaction and sets the time zero. (Pump laser)

Second laser pulse delays in time and probes the signal at each time delay. (Probe laser)

Results

LFET

FETforward kk

k

BETSP

SPbackward kk

k

,

backwardforwardldQuantumYie

WT

Nor

mal

ized

ΔA

Results

Results

  Φ τlifetime <τFET> ΦFET <τSP> <τBET> ΦSP <τER>

WT 0.82 1300 236 0.85 88 2400 0.96 625

M345A 0.72 1169 140 0.89 88 368 0.81 200

N378C 0.67 3374 1181 0.74 88 839 0.91 500

N378S 0.62 1351 675 0.67 88 1242 0.93 462

R226A 0.53 1309 480 0.73 50 126 0.72 1412

R342A 0.48 1600 595 0.73 83 166 0.66 416

E274A 0.38 1044 615 0.62 31 52 0.62 75

Results

How to understand these ET rates?

All times are in unit of picosecond.

  ΔGFET λo, FET λi, FET λo, ER λi, ER

WT -0.440 0.226 0.840 0.395 0.840

M345A -0.510 0.245 0.840 0.455 0.840

N378C -0.310 0.255 0.860 0.490 0.880

N378S -0.365 0.250 0.845 0.475 0.850

R226A -0.383 0.245 0.830 0.400 0.820

R342A -0.380 0.365 0.650 0.690 0.680

E274A -0.379 0.245 0.870 0.640 0.880

Results

All energies are in unit of eV.

Discussion

TTFADHTTFADH

  ΔGFET λo, FET λi, FET λo, ER λi, ER

WT -0.440 0.226 0.840 0.395 0.840

N378C -0.310 0.255 0.860 0.490 0.880

N378S -0.365 0.250 0.845 0.475 0.850

00//)( EG

TTTTFADHFADH

FADHFADH /

  <τFET> <τSP> <τBET> <τER>

WT 236 88 2400 625

N378C 1181 88 839 500

N378S 675 88 1242 462

increases

The mutation of N378 destroys the H-bond with the flavin ring and change the redox potential of flavin, leading to the smaller driving force for the forward ET from FADH− to the dimer and the slower ET rate.

The mutation of this residue makes the active site more flexible, then causes the increase of the solvent reorganization energy.

Discussion

  ΔGFET λo, FET λi, FET λo, ER λi, ER

WT -0.440 0.226 0.840 0.395 0.840

M345A -0.510 0.245 0.840 0.455 0.840

R226A -0.383 0.245 0.830 0.400 0.820

R342A -0.380 0.365 0.650 0.690 0.680

E274A -0.379 0.245 0.870 0.640 0.880

  <τFET> <τSP> <τBET> <τER>

WT 236 88 2400 625M345A 140 88 368 200R226A 480 50 126 1412R342A 595 83 166 416E274A 615 31 52 75

TTFADHTTFADH00//

)( EGTTTTFADHFADH

TTTT /

changes

The mutation of these charged/polar residues makes the active site more flexible, then leads to the increase of the solvent reorganization energy.

The mutation of these residues at the binding site diminishes the stabilization of anionic CPD radical, which results in the faster non-repaired back ET.

The lower quantum yields of mutants result from a combination of two-step competitions: the forward electron transfer competing with lifetime emission and the ring splitting competing with non-repaired back electron transfer.

The mutation of N378 destroys the H-bond with the flavin ring and changes the redox potential of the flavin, leading to the slower forward electron transfer from FADH− to the DNA lesion and the decrease in repair efficiency.

The mutation of the three charged/polar residues at the binding site (E274, R226 and R342) diminishes the stabilization of anionic CPD radical, which results in the slower forward electron transfer and the faster non-repaired back electron transfer. These dynamics changes cause the loss of the quantum yield.

Discussion

Conclusions

With femtosecond-resolved laser spectroscopy, we revealed the ultrafast dynamics of the DNA repair in several photolyase mutants.

As a precision machinery, photolyase controls a series of critical electron transfers and ring splitting of pyrimidine dimer through the modulation of redox potentials and reorganization energies, and the stabilization of the anionic intermediates by the interactions from its active site residues, maintaining the dedicated balance of all the reaction steps and achieving the maximum repair efficiency.

Conclusions

Acknowledgements

Advisor: Prof. Dongping Zhong

Zheyun LiuJiang LiXunmin GuoYa-ting KaoLijuan WangAll group members

$$$:National Institutes of HealthPackard Foundation FellowshipOSU Pelotonia FellowshipAmerican Heart Association Fellowship

Thank You!Thank You!

Repair quantum yields

BindingR342 has direct and indirect interaction with DNA backbone, so it affects the DNA binding and lower the binding constant by more than one order compared to WT.

Repair efficiencyThe charged/polar residues E274, R226 and R342 have hydrophilic interaction with CPD; N378 form H-bond with flavin ring; and M345 has interaction with both CPD and flavin. Thus, the mutation of these residues will result in the decrease of the repair efficiency.

N378SN378CM345AR342AR226AE274A

Substrate Concentration (mM)

WT

0.0 0.2 0.4 0.6 0.8 1.0

Rep

air

Eff

icie

ncy

0.0

0.5

1.0

Visible Light (min)0 50 100 150 200

Abs

orpt

ion

0.0

0.2

0.4

0.6

0.8

E274A

WT

R226A

M345A

N378C

N378S

R342A

B

A

[PL] = 10-6

M

[PL] = 10-6

M

E. coli Photolyase WT E274A R226A R342A N378C N378S M345A

Quantum Yield 0.82 0.38 0.53 0.48 0.67 0.62 0.72

Up-conversion

Sum-frequency generation u=f + p

Transient absorption

S = – [ log(I/I0)pump-on – log( I/I0)pump-off ]

2 2 00

2 00

0

2 0 20

20

20

0 2

( ) ( ) ( )2 2

At transition state: ( ) ( ), set q=q*

2*

( )2* ( *)

2

2

( )*

4

r p

r p

r

a aV q q V q q q G

V q V q

aq G

qaq

aq G

G V qaq

aq

GG

*

B

G

k Tetk e

Marcus ET theory

Diabatic and Adiabatic ET

Diabatic

Adiabatic

Diabatic processRapidly changing conditions prevents the system from adapting its configuration during the process.

Adiabatic processGradually changing conditions allow the systme to adapt its configuration during the process.

When the solvent motion is sufficient slow, the diabatic reaction will become adiabatic, and the rate will be independent on Hrp.

In our case, the solvent relaxation is not slow compared to ET, we treat the ET as diabatic process.

Sumi-Marcus 2D model2

2

22 00

0

00

0

2 20 0

2*

( , ) + 2 2

( )( , ) ( )

2 2At transition state:

( , ) ( , ), set q=q*

*

1 1Given: = , ,

2 2At a given X:

( )( *, ) (0, )

2

r

p

r p

r r

a XV q X q

X XaV q X q q G

V q X V q X

G XXq

aq

o i o X i aq

X Xc oG V q X V X

i

X

0 2

0

( )

2

Gc

2 *2 1 ( )( ) exp

4 Bi B

J G Xk X

k Tk T

Semi-classical Expression:

Reaction-diffusion equation

The electron transfer can be described by the reaction-diffusion equation. The probability distribution of reactant at time t and at the solvent coordinate X is:

L is a Fokker-Planck operator that determines the stochastic motion along the solvent polarization coordinate and has the form:

Where Dp is the diffusion coefficient for the solvent fluctuations, and V(X) is

the potential.

Thus,

Reaction Time and Energy

1. The driving forces and solvent reorganization energies are diverse while the intramolecule vibrational reorganization energies and the energies from the high-frequency vibration modes are almost invariant except R342A.

2. Both the driving force and the solvent reorganization energy influence the ET.

3. In forward ET, the solvent reorganization energy are not variant too much in different mutants, so the driving forces influence ET dominantly.

N3783.33

TTFADHTTFADH

00//)(96485 EG

TTTTFADHFADH

TTTT / does not change

FADHFADH / increases

G

Forward ET in N378 mutants

M345