Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Dynamics and mechanism of repair of ultraviolet-induced (6–4) photoproduct by photolyase

This article has been updated


One of the detrimental effects of ultraviolet radiation on DNA is the formation of the (6–4) photoproduct, 6–4PP, between two adjacent pyrimidine rings1. This lesion interferes with replication and transcription, and may result in mutation and cell death2. In many organisms, a flavoenzyme called photolyase uses blue light energy to repair the 6–4PP (ref. 3). The molecular mechanism of the repair reaction is poorly understood. Here, we use ultrafast spectroscopy to show that the key step in the repair photocycle is a cyclic proton transfer between the enzyme and the substrate. By femtosecond synchronization of the enzymatic dynamics with the repair function, we followed the function evolution and observed direct electron transfer from the excited flavin cofactor to the 6–4PP in 225 picoseconds, but surprisingly fast back electron transfer in 50 picoseconds without repair. We found that the catalytic proton transfer between a histidine residue in the active site and the 6–4PP, induced by the initial photoinduced electron transfer from the excited flavin cofactor to 6–4PP, occurs in 425 picoseconds and leads to 6–4PP repair in tens of nanoseconds. These key dynamics define the repair photocycle and explain the underlying molecular mechanism of the enzyme’s modest efficiency.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Enzyme–substrate complex structure and a possible repair scheme.
Figure 2: Femtosecond-resolved dynamics of flavin species involved in the repair of damaged DNA by (6–4) photolyase enzyme (E6–4PL).
Figure 3: Femtosecond-resolved transient absorption dynamics of various species involved in the damaged DNA repair.
Figure 4: Repair photocycle of (6–4) thymine photoproduct by (6–4) photolyase.

Change history

  • 11 August 2010

    A correction was made to the x-axis labels of the Fig. 2a inset.


  1. Mitchell, D. L. The relative cytotoxicity of (6–4) photoproduct and cyclobutane dimers in mammalian cells. Photochem. Photobiol. 48, 51–57 (1988)

    Article  CAS  Google Scholar 

  2. Taylor, J. S. Unraveling the molecular pathway from sunlight to skin cancer. Acc. Chem. Res. 27, 76–82 (1994)

    Article  CAS  Google Scholar 

  3. Todo, T. et al. A new photoreactivating enzyme that specifically repairs ultraviolet light-induced (6–4) photoproducts. Nature 361, 371–374 (1993)

    Article  ADS  CAS  Google Scholar 

  4. Sancar, A. Structure and function of DNA photolyase and cryptochrome blue-light photoreceptors. Chem. Rev. 103, 2203–2237 (2003)

    Article  CAS  Google Scholar 

  5. Hitomi, K. et al. Functional motifs in the (6–4) photolyase crystal structure make a comparative framework for DNA repair photolyases and clock cryptochromes. Proc. Natl Acad. Sci. USA 106, 6962–6967 (2009)

    Article  ADS  CAS  Google Scholar 

  6. Maul, M. J. et al. Crystal structure and mechanism of a DNA (6–4) photolyase. Angew. Chem. Int. Edn Engl. 47, 10076–10080 (2008)

    Article  CAS  Google Scholar 

  7. Kao, Y.-T., Saxena, C., Wang, L., Sancar, A. & Zhong, D. Direct observation of thymine dimer repair in DNA by photolyase. Proc. Natl Acad. Sci. USA 102, 16128–16132 (2005)

    Article  ADS  CAS  Google Scholar 

  8. Zhong, D. Ultrafast catalytic processes in enzymes. Curr. Opin. Chem. Biol. 11, 174–181 (2007)

    Article  CAS  Google Scholar 

  9. Zhao, X. et al. Reaction mechanism of (6–4) photolyase. J. Biol. Chem. 272, 32580–32590 (1997)

    Article  CAS  Google Scholar 

  10. Hitomi, K. et al. Role of two histidines in the (6–4) photolyase reaction. J. Biol. Chem. 276, 10103–10109 (2001)

    Article  CAS  Google Scholar 

  11. Glas, A. F., Schneider, S., Maul, M. J., Hennecke, U. & Carell, T. Crystal structure of the T(6–4)C lesion in complex with a (6–4) DNA photolyase and repair of UV-induced (6–4) and dewar photolesions. Chem.-Eur. J. 15, 10387–10396 (2009)

    Article  CAS  Google Scholar 

  12. Joseph, A., Prakash, G. & Falvey, D. E. Model studies of the (6–4) photoproduct photolyase enzyme: laser flash photolysis experiments confirm radical ion intermediates in the sensitized repair of thymine oxetane adducts. J. Am. Chem. Soc. 122, 11219–11225 (2000)

    Article  CAS  Google Scholar 

  13. Borg, O. A., Eriksson, L. A. & Durbeej, B. Electron-transfer induced repair of 6–4 photoproducts in DNA: a computational study. J. Phys. Chem. A 111, 2351–2361 (2007)

    Article  CAS  Google Scholar 

  14. Yamamoto, J., Hitomi, K., Hayashi, R., Getzoff, E. D. & Iwai, S. Role of the carbonyl group of the (6–4) photoproduct in the (6–4) photolyase reaction. Biochemistry 48, 9306–9312 (2009)

    Article  CAS  Google Scholar 

  15. Domratcheva, T. & Schlichting, I. Electronic structure of (6–4) DNA photoproduct repair involving a non-oxetane pathway. J. Am. Chem. Soc. 131, 17793–17799 (2009)

    Article  CAS  Google Scholar 

  16. Schleicher, E. et al. Electron nuclear double resonance differentiates complementary roles for active site histidines in (6–4) photolyase. J. Biol. Chem. 282, 4738–4747 (2007)

    Article  CAS  Google Scholar 

  17. Kao, Y.-T., Saxena, C., Wang, L., Sancar, A. & Zhong, D. Femtochemistry in enzyme catalysis: DNA photolyase. Cell Biochem. Biophys. 48, 32–44 (2007)

    Article  CAS  Google Scholar 

  18. Chang, C.-W. et al. Ultrafast solvation dynamics at binding and active sites of photolyases. Proc. Natl Acad. Sci. USA 107, 2914–2919 (2010)

    Article  ADS  CAS  Google Scholar 

  19. Wang, H. et al. Protein dynamics control the kinetics of initial electron transfer in photosynthesis. Science 316, 747–750 (2007)

    Article  ADS  CAS  Google Scholar 

  20. Hitomi, K. et al. Binding and catalytic properties of Xenopus (6–4) photolyase. J. Biol. Chem. 272, 32591–32598 (1997)

    Article  CAS  Google Scholar 

  21. Marcus, R. A. Summarizing lecture: factors influencing enzymatic H-transfers, analysis of nuclear tunnelling isotope effects and thermodynamic versus specific effects. Phil. Trans. R. Soc. B 361, 1445–1455 (2006)

    Article  CAS  Google Scholar 

  22. Yamamoto, J., Tanaka, Y. & Iwai, S. Spectroscopic analysis of the pyrimidine(6–4)pyrimidone photoproduct: insights into the (6–4) photolyase reaction. Org. Biomol. Chem. 7, 161–166 (2009)

    Article  CAS  Google Scholar 

  23. Li, J., Uchida, T., Ohta, T., Todo, T. & Kitagawa, T. Characteristic structure and environment in FAD cofactor of (6–4) photolyase along function revealed by resonance Raman spectroscopy. J. Phys. Chem. B 110, 16724–16732 (2006)

    Article  CAS  Google Scholar 

  24. LeClerc, J. E., Borden, A. & Lawrence, C. W. The thymine-thymine pyrimidine-pyrimidone(6–4) ultraviolet light photoproduct is highly mutagenic and specifically induces 3′ thymine-to-cytosine transitions in Escherichia coli. Proc. Natl Acad. Sci. USA 88, 9685–9689 (1991)

    Article  ADS  CAS  Google Scholar 

  25. Saxena, C., Sancar, A. & Zhong, D. Femtosecond dynamics of DNA photolyase: Energy transfer of antenna initiation and electron transfer of cofactor reduction. J. Phys. Chem. B 108, 18026–18033 (2004)

    Article  CAS  Google Scholar 

  26. Kao, Y.-T. et al. Ultrafast dynamics and anionic active states of the flavin cofactor in cryptochrome and photolyase. J. Am. Chem. Soc. 130, 7695–7701 (2008)

    Article  CAS  Google Scholar 

Download references


We thank T. Todo and T. Kitagawa for the generous gift of Arabidopsis thaliana (6–4) photolyase plasmid; C. Forsyth for discussions; and Y.-T. Kao and C.-W. Chang for help during the experiments. This work is supported in part by the National Institutes of Health (research grant GM074813) and the Packard fellowship.

Author information

Authors and Affiliations



D.Z. designed the research. J.L., Z.L., C.T., X.G. and L.W. performed the research. J.L., Z.L., C.T. and D.Z. analysed the data. J.L., Z.L., A.S. and D.Z. wrote the paper. All authors discussed and edited the manuscript.

Corresponding author

Correspondence to Dongping Zhong.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Kinetic Data Analysis, Supplementary Table 1 and Supplementary Figures 1S-3S with legends. (PDF 151 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Li, J., Liu, Z., Tan, C. et al. Dynamics and mechanism of repair of ultraviolet-induced (6–4) photoproduct by photolyase. Nature 466, 887–890 (2010).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing