Dynamics and Mechanism of Efficient DNA Repair Studied by Active-site Mutations Chuang Tan Chemical Physics Program The Ohio State University Prof. Dongping Zhong Lab Departments of Physics, Chemistry, and Biochemistry Programs of Biophysics, Chemical Physics, and Biochemistry The Ohio State University

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

Introduction DNA Repair Sancar Chem. Rev. 103, 2203 (2003) Repair the UV-induced DNA damage using 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?

Introduction Active-site Residues A. nidulans E. coli FADH - - CPD-like DNA lesion E283 N386 R232 R350 E274 N378 R226 R342 E. coliWTE274AR226AR342AN378CN378S Quantum Yield Three charged/polar residues R232(R226), E283 (E274) and R350 (R342) have hydrophilic interaction with dimer and the flavin ring; and N386 (N378) forms H-bond with flavin ring. The mutation of these residues will result in the decrease of the repair efficiency.

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, WT Normalized Δ A

Results Active-site Mutations WTN378CN378S FET (ps) SP (ps)70 BET (ps) ER (ps) N378C N378S WTN378CN378S Ф forward Ф backward Ф total Residue N378 forms a H-bond with the N5 of the flavin ring, the mutation of this residue will destroy the H-bond and change the redox potential of the flavin, leading to the slower FET and the loss of quantum yield. Normalized Δ A

Results Active-site Mutations E274A R226AR342A Normalized Δ A

Results Active-site Mutations Mees et al. Science 306, 1789 (2004) WTE274AR226AR342A FET (ps) SP (ps) BET (ps) ER (ps) WTE274AR226AR342A Ф forward Ф backward Ф total (E274A) (R342A) (R226A)

Conclusions With femtosecond-resolved laser spectroscopy, we revealed the ultrafast dynamics of the DNA repair in several photolyase mutants. The mutation of N378 destroys the H-bond with the flavin ring and change the redox potential of flavin, leading to the slower forward ET from FADH − to the dimer and the decrease in repair efficiency. The mutation of the three charged/polar residues at the binding site (E274, R226, R342) diminishes the stabilization of anionic CPD radical, which results in the slower forward ET and faster non-repaired back ET. These dynamic changes cause the loss of the quantum yield. Conclusions

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

Thank You!

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

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

WTE274AR226AR342AN378CN378S T lifetime

WTE274AR226AR342AN378CN378S T FET β T lifetime Ф forward