28 - Dynamics and mechanism of DNA repair by photolyase: experiment and theory - Part 2
Florida 3 10:00 - 12:00
|Chair(s): Dongping Zhong|
10:00 DNA Bases beyond Watson and Crick T Carell*, LMU Munich
Abstract: Epigenetic information is stored in the form of modified cytosine bases in the genome. Setting and erasing of epigenetic imprints controls the complete development process. I will discuss the latest results related to the function and distribution of the epigenetic marker bases 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), 5-carboxycytosine (caC) and 5-hydroxymethyluracil. These nucleobases control epigenetic programming of stem cells and some of these bases are also detected at relatively high levels in brain tissues. I will particularly cover mass spectroscopic approaches to decipher the biological functions of the new bases of which some were described in the past as pure DNA lesions. In particular, results from quantitative mass spectrometry, new covalent-capture proteomics mass spectrometry, and isotope tracing techniques will be reported. The data allow us to unravel the chemistry in stem cells and the protein networks that are controlled by the epigenetic base modifications.
10:30 On The Time Scale Of The Repair Of (6-4) Photoproducts By Their DNA Photolyase J Yamamoto, Osaka University
; K Brettel*, I2BC/CEA Saclay
Abstract: UV irradiation induces two major types of harmful crosslinks between adjacent pyrimidines in DNA: cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). In many organisms, these lesions are repaired by photolyases, flavoproteins that require light for their catalytic action (see comprehensive review by A. Sancar, Chem Rev. 2003, 103, 2203-2237). For CPDs, the repair mechanism involves electron transfer (ET) to the lesion from the photoexcited state of the fully reduced flavin cofactor FADH", splitting of the intra-dimer bonds and return of the excess electron to the semi-reduced flavin FADH°. These reactions take place within ~1 ns and are highly efficient (repair quantum yield of 50-100%). Repair of 6-4PPs by (6-4) photolyase has a much lower quantum yield (about 5-10%), and the repair mechanism is less understood. Li, Liu, Tan, Guo, Wang, Sancar and Zhong (Nature 2010, 466, 887-890) provided evidence for ET from excited FADH" to the 6-4PP and proton transfer from a histidine residue within 500 ps, but electron return did not take place in the accessible time window of 3 ns, indicating that repair of 6-4PPs takes much longer than that of CPDs. From flash sequence experiments on a seconds time scale, we have provided evidence that two successive photoreactions are required for the repair of the 6-4PP, each of them presumably involving ET from excited FADH" to the lesion and electron return to FADH° (Yamamoto et al., Angew. Chem. Int. Ed. 2013, 52, 7432-7436). Here we will present results of recent transient absorption experiments (using a dedicated setup that provides a time resolution of up to 300 ps without excessive signal averaging; Byrdin et al., Rev. Sci. Instrum. 2009, 80, 043102) that aimed at establishing the time scale of 6-4PP repair by monitoring electron return for each of the two photoreactions in the nano- and microsecond range.
11:00 Light-induced difference FTIR spectroscopy of photolyase H Kandori*, Nagoya Institute of Technology
Abstract: Photolyases (PHRs) are DNA repair enzymes by light, which specifically revert UV-induced photoproducts, cyclobutane pyrimidine dimers (CPD) or (6-4) photoproduct, into normal bases. Flavin adenine dinucleotide (FAD) is the chromophore of PHRs, and enzymatically active fully reduced state (FADH^-) is formed by light-induced electron/proton transfers from oxidized and neutral radical states (activation process). Upon illumination of the FADH^- form, the repair of the photoproduct is initiated by the electron transfer from FADH^- to the photoproduct. Molecular mechanism of activation and repair processes of PHRs is intriguing, and light-induced difference Fourier-transform infrared (FTIR) spectroscopy is a powerful tool for this aim. Hydrated film samples have been used for photoreceptive proteins such as rhodopsins, which can monitor even a single water molecule . Nevertheless, for FTIR study of PHRs, we wanted a sample with higher water content to facilitate enzyme turnover. We thus established two different methods , re-dissolved samples and concentrated solutions, for the study of Xenopus (6-4) PHR  and E. coli CPD PHR , respectively. Our FTIR results will be presented including the trial of functional conversion between CPD and (6-4) PHRs. References  H. Kandori, Biochim. Biophys. Acta 1460, 177-191 (2000).  D. Yamada and H. Kandori, Methods Mol. Biol. 1148, 361-376 (2014).  Y. Zhang Y. et al. Biochemistry 50, 3591-3598 (2011); Y. Zhang Y. et al. J. Phys. Chem. Lett. 2, 2774-2777 (2011); D. Yamada et al. Biochemistry 51, 5774-5783 (2012); D. Yamada Biophys. Physicobiol. 12, 139-144 (2015); D. Yamada et al. Biochemistry 55, 715-723 (2016).  I M. M. Wijaya et al. Biochemistry 52, 1019-1027 (2013); I M. M. Wijaya et al. Biochemistry 53, 5864-5875 (2014); I M. M. Wijaya et al. BIOPHYSICS 11, 39-45 (2015); I M. M. Wijaya et al. J. Am. Chem. Soc. in press (2016).
11:30 Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme " the spore photoproduct lyase Linlin Yang, Indiana University Purdue University Indianapolis (IUPUI)
; Yajun Jian, Indiana University Purdue University Indianapolis (IUPUI); Peter Setlow, UConn Health; Lei Li*, Indiana University Purdue University Indianapolis (IUPUI)
Abstract: UV radiation of DNA generates three pyrimidine dimers, cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP). All these dimers can be repaired by direct reversal enzymes where CPDs and 6-4PPs are repaired by the respective photolyase and SP is repaired by spore photoproduct lyase (SPL). All these dimers can also be repaired by the general but relatively inefficient nucleotide excision repair (NER) pathway. Prof. Sancar's earlier seminal work demonstrated that the CPD photolyase exhibits roughly 105-fold higher affinity toward CPD than undamaged DNA, explaining the highly efficient CPD repair in duplex DNA by the enzyme. It was widely expected that SPL may exhibit a similarly high affinity toward SP in duplex DNA leading to an efficient SP repair. Surprisingly, our latest in vitro studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01-0.2 min-1. The faster turnover numbers are reached under single turnover conditions, and SPL activity is low with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3 ~ 0.4 min-1, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from DNA photolyases. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins may have to cooperate with SPL to enable the efficient SP repair in germinating spores. Moreover, SP can be produced in non-spore-forming microorganisms; our results indicate that the SP repair in these species may be slow and potentially problematic.