American Society for Photobiology

ASP Conference 2016: 21-26 May 2016
Tampa Marriott Waterside Hotel & Marina

Single Session

[Schedule Grid]

11 - Dynamics and Mechanism of DNA Repair by Photolyase: Experiment and Theory - Part 1

Florida 3   10:00 - 12:00

Chair(s): Dongping Zhong
11-1   10:00  Dynamics and mechanisms of UV-damged DNA repair by photolyases Dongping Zhong*, The Ohio State University

Abstract: UV radiation can damage DNA to mainly form cyclobutane pyrimidine dimer or (6-4) photoproduct. Such lesion may eventually lead to skin cancer. Photolyase, a flavin photo enzyme, can revert such damage with high repair efficiency. Here, we combine femtosecond spectroscopy and molecular biology and have completely mapped out the entire repair evolution at the most fundamental level by following the dynamics from the initial reactants, to the fleeting intermediates and to the final repaired products. By resolving more than six elementary steps in the complex enzymatic reaction, we captured three electron-transfer reactions and bond-breaking and -forming processes. These dynamics are in synergy to achieve a maximum repair efficiency. Various mutations were also carried out to identify the critical residues in the active site for function. We carefully examined the Class I, Class II, Class III and ssDNA photolyases and observed a unified electron transfer mechanism and determine the critical role of the unique folded structure of cofactor.

11-2   10:30  Microbial members of the photolyase/cryptochrome family: Common structures, common mechanisms ? LO Essen*, Philipps University

Abstract: Members of the photolyase-cryptochrome family (pc-family) are manifold involved in light-driven DNA repair and blue-light dependent signaling. All members utilize in their C-terminal, alpha-helical domain a U-shaped FAD chromophore for catalysis or light absorption [1,2]. However, unlike other, well studied "canonical" representatives of the pc-family, e. g. plant [3] and animal cryptochromes or class I and (6-4) photolyases, several subfamilies (class II photolyases, proteobacterial cryptochromes) developed novel electron transfer pathways for its photoreduction as well as hitherto unknown antenna cofactors. The advent of bifunctional members of the pc-familiy, e.g. DASH-type and algal cryptochromes, blurred the distinction between cryptochromes and photolyases. For example we found (6-4) photolyase activity for the animal-like cryptochrome of the green algae Chlamydomonas reinhardtii (CraCRY) that otherwise shows blue- and red-light activity in vivo. Its 1.9Å structure reveals the common bilobal architecture, where the N-terminal Rossma-like fold harbors 8-hydroxydeazaflavin as antenna. The co-crystal structure with (6-4) lesion comprising duplex DNA proves that CraCry is indeed a fully-fledged (6-4) photolyase. To investigate photoactivation of CraCRY, the conserved tryptophan triad was mutated and analyzed by UV/Vis spectroscopy. CraCRY forms FADH* upon blue light illumination without any reductive agents. Our data show that Y373 is the endogenous electron donor to the tryptophan triade by forming a long-lived radical state. Other examples of the microbial pc-family will be presented, which indicate a repeated re-usage of DNA-photolyases as signaling proteins.

11-3   11:00  Utilizing Light for Repair of Light-induced DNA Damages: The Clever Mode of Action of DNA Photolyases S Faraji*, University of Southern California, USA ; A Dreuw, Ruprecht-Karls University of Heidelberg, Germany

Abstract: UV radiation triggers various chemical reactions in DNA such as intra-strand cross-linking between adjacent pyrimidines causing genetic mutations. The pyrimidine dimers are supposed to be the major players in the formation of skin cancer. DNA photolyases are enzymes initiating cleavage of mutagenic photolesions by a photo-induced electron transfer from flavin adenine dinucleotide to the lesion. Using state-of-the-art hybrid quantum mechanical/molecular mechanical dynamics, we have carried out a series of simulations to completely map out the entire evolution of functional processes involved in molecular mechanism of this important biological function. We have demonstrated that the electron catalyzing the repair is generated via an intermolecular Coulombic decay (ICD) process [1]. In fact, this is the first example for ICD in a real biological system. We have presented the most energetically feasible electron-induced splitting mechanism in which the initial step is electron transfer coupled to proton transfer from the protonated Histidine to the lesion, which proceeds simultaneously with intramolecular OH transfer along an oxetane-like transition state [2]. In agreement with recent experimental time resolved findings [3], the photolesion can be split and original bases restored. The experimental spectroscopic signature of the detected 6-4PP intermediate is assigned theoretically to a specific molecular structure determining the operating molecular mechanism of the electron-induced restoration of (6-4) photolesions. Thereby, all pieces of the electron-induced (6-4) photolesion repair puzzle are finally put together [4]. 1. P. Harbach , M. Schneider, S. Faraji, A. Dreuw, J. Phys. Chem. Lett. 4, 943 (2013). 2. S. Faraji and A. Dreuw, J. Phys. Chem. Lett. 3, 227 (2012), S. Faraji, G. Groenhof and A. Dreuw, J. Phys. Chem. B. 117, 10071 (2013) 3. J. Li et al, Nature 466, 887 (2010). S. Faraji and A. Dreuw, Ann. Rev. Phys. Chem. 64, (2014). 4. S. Faraji, D. Zhong, and A. Dreuw, Angew. Chem. Int. Ed. Engl, accepted for publication (2016).

11-4   11:30  The blue light-dependent phosphorylation of Arabidopsis CRY2 C. Lin*, UCLA

Abstract: Arabidopsis cryptochrome 2 (CRY2) undergoes blue light-dependent phosphorylation, which regulate the function and protein turnover of CRY2. A study of blue light-dependent CRY2 phosphorylation by mass spectrometry and mutagenesis analyses demonstrate that CRY2 contains two types of phosphorylation in the CCE domains. One type of CRY2 phosphorylation occurs in a serine-cluster of the CCE domain that causes electrophoretic mobility upshift, whereas the other type of phosphorylation occurs outside of the serine cluster that do not cause mobility upshift. We identified four closely related protein kinases, referred to as CIK1 to CIK4 (CRY-Interacting Kinase 1 to 4), and investigated the substrate specificity, phosphorylation sites, and physiological functions of those kinases. Results of our biochemical analyses demonstrate that CIKs phosphorylate the serine-cluster of CRY2 in the substrate-specific and blue light-specific manner. Genetics study demonstrates that CIKs are major protein kinases that phosphorylate CRY2, resulting in blue light-dependent ubiquitination and degradation of the photoreceptor.

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