Mapping Thymine Dimer Splitting in Damaged DNA by Photolyase Zheyun Liu, Chuang Tan, Jiang Li,...
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Transcript of Mapping Thymine Dimer Splitting in Damaged DNA by Photolyase Zheyun Liu, Chuang Tan, Jiang Li,...
Mapping Thymine Dimer Splitting in Damaged DNA by
Photolyase
Zheyun Liu, Chuang Tan, Jiang Li, Xunmin Guo, Lijuan Wang and Dongping Zhong
Department of Physics, Chemistry, and Biochemistry, The Ohio State University, Columbus
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%
http://www.caribvoice.org/health.html
Most photodamage of DNA occurredas cyclobutane pyrimidine dimer.
Overexposure of UVA and UVB light will cause the DNA damage, whichmay induce skin neoplasm.
Introduction: DNA damage
CPD photolyase specifically repairs
cyclobutane pyrimidine dimer
Chem. Rev., 103, 2203 (2003)
N
HN
O
O N
NH
O
O
R
NH
N
NH
N O
O
H3C
H3C
P
N
HN
O
O N
NH
O
O
N
HN
O
O N
NH
O
O
N
HN
O
O N
NH
O
O
N
HN
O
O N
NH
O
O
MTHF *
Light (300~ 500 nm)
Excitation Energy Transfer
1FADH *FADH
.
.
e-
e-
P
P
P
P
Cyclic Electron Transfer
FADH --
MTHF
Substrate Product
Ultrafast time-resolved spectroscopy
•Pump-Probe method:
• One laser pulse initiates the reaction and sets the time zero. (Pump laser)
• Second laser pulse delays in time and takes the signal at each time delay. (Probe laser)
Enzyme Complex
hpu
Ground State
State 1
hpr
kq
State 2
hpr
kdkf
Detecting the weak signals of intermediates
The key of differentiate signal of state 2 from total signal is to determine the signal of state 1 first !
Charge separation (k1) occursin 170 ps.
Charge recombination (k2)occurs in 560 ps.
Flavin retains same redox state during the photocycle.
Proc. Natl. Acad. Sci. USA 102, 16128 (2005)
•How to monitor the evolution of CPD substrate with the injection of one electron?
•How to measure the dynamics of each elementary steps in reaction?
Delay Time (ps)
Dissect the DNA repair reaction into elementary steps
Delay Time (ps)
N
HN
O
O N
NH
O
O
R
NH
N
NH
N O
O
H3C
H3C
P
N
HN
O
O N
NH
O
O
N
HN
O
O N
NH
O
O
N
HN
O
O N
NH
O
O
N
HN
O
O N
NH
O
O
MTHF *
Light (300~ 500 nm)
Excitation Energy Transfer
1FADH *FADH
.
.
e-
e-
P
P
P
P
Cyclic Electron Transfer
FADH --
MTHF
Substrate Product
70 ps
520 ps
Capturing DNA intermediates and
products in UV probe-range
N
NHR
O
O
N
HNR
O
ON
HN
N
NHR R
O
O O
O
3'5'
T-T - T
- T
N
NHR
O
O
The absorption coefficients of intermediates and products
FET: 170 ps
SP1: <10 ps
SP2: 70 ps
BET: >1500 ps
ER: 520 ps
FET: forward electron transfer; SP1, splitting of C5-C5’ bond of CPD dimer; SP2, splitting of C6-C6’ bond of CPD dimer; BET: back electron transfer from unpaired CPD;ER: back electron transfer from repaired thymine monomer.
The photocycle of CPD repair by photolyase
Acknowledgement
•Dr. Dongping Zhong
•Lijuan Wang
•Chuang Tan, Dr. Jiang Li, Dr. Xunmin Guo, and Dr. Ya-Ting Kao
•All other group members
Funding: NIH, PackardFoundation Fellowship
Thank you!