1. Molecular Understanding of Efficient DNA Repair Machinery of Photolyase Chuang Tan Chemical Physics Program The Ohio State University 2012.06.19 Prof. Dongping Zhong Lab Departments of Physics, Chemistry, and Biochemistry Programs of Biophysics, Chemical Physics, and Biochemistry The Ohio State University

2. Human Genetics and Genomics, Third edition. Bruce Korf (2006) UV-induced DNA damage 1)Cyclobutane Pyrimidine Dimer 2)(6-4) Products Introduction Cause genetic mutations Block replication and transcription …… Skin Cancer!! DNA Damage

3. 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. Question How does photolyase modulate this so complicated repair process?

4. 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 PhotolyaseWTE274AR226AR342AN378CN378SM345A Quantum Yield0.820.380.530.480.670.620.72

5. 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)

6. Results, WT Normalized Δ A

7. Results

9. Φτ lifetime Φ FET Φ SP WT0.8213002360.858824000.96625 M345A0.7211691400.89883680.81200 N378C0.67337411810.74888390.91500 N378S0.6213516750.678812420.93462 R226A0.5313094800.73501260.721412 R342A0.4816005950.73831660.66416 E274A0.3810446150.6231520.6275 Results How to understand these ET rates? All times are in unit of picosecond.

10. ΔG FET λ o, FET λ i, FET λ o, ER λ i, ER WT-0.4400.2260.8400.3950.840 M345A-0.5100.2450.8400.4550.840 N378C-0.3100.2550.8600.4900.880 N378S-0.3650.2500.8450.4750.850 R226A-0.3830.2450.8300.4000.820 R342A-0.3800.3650.6500.6900.680 E274A-0.3790.2450.8700.6400.880 Results All energies are in unit of eV.

11. Discussion ΔG FET λ o, FET λ i, FET λ o, ER λ i, ER WT-0.4400.2260.8400.3950.840 N378C-0.3100.2550.8600.4900.880 N378S-0.3650.2500.8450.4750.850 WT236882400625 N378C118188839500 N378S675881242462 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.

12. Discussion ΔG FET λ o, FET λ i, FET λ o, ER λ i, ER WT-0.4400.2260.8400.3950.840 M345A-0.5100.2450.8400.4550.840 R226A-0.3830.2450.8300.4000.820 R342A-0.3800.3650.6500.6900.680 E274A-0.3790.2450.8700.6400.880 WT236882400625 M345A14088368200 R226A480501261412 R342A59583166416 E274A615315275 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.

13.  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

14. 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

15. Acknowledgements Advisor: Prof. Dongping Zhong Zheyun Liu Jiang Li Xunmin Guo Ya-ting Kao Lijuan Wang All group members $$$: National Institutes of Health Packard Foundation Fellowship OSU Pelotonia Fellowship American Heart Association Fellowship

16. Thank You!

17. Repair quantum yields Binding R342 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 efficiency The 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. E. coli PhotolyaseWTE274AR226AR342AN378CN378SM345A Quantum Yield0.820.380.530.480.670.620.72

18. Up-conversion Sum-frequency generation  u =  f +  p

19. Transient absorption S = – [ log(I/I 0 ) pump-on – log( I/I 0 ) pump-off ]

20. Marcus ET theory

21. Diabatic and Adiabatic ET Diabatic Adiabatic Diabatic process Rapidly changing conditions prevents the system from adapting its configuration during the process. Adiabatic process Gradually 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 H rp. In our case, the solvent relaxation is not slow compared to ET, we treat the ET as diabatic process.

22. Sumi-Marcus 2D model Semi-classical Expression:

23. 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 D p is the diffusion coefficient for the solvent fluctuations, and V(X) is the potential. Thus,

25. 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.

26. N378 3.33 does not change increases Forward ET in N378 mutants

27. M345