Functional Conversion of CPD and (6-4) Photolyases by Mutation

Biochemistry. 2016 Aug 2;55(30):4173-83. doi: 10.1021/acs.biochem.6b00361. Epub 2016 Jul 19.


Ultraviolet (UV) light from the sun damages DNA by forming a cyclobutane pyrimidine dimer (CPD) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) PP]. Photolyase (PHR) enzymes utilize near-UV/blue light for DNA repair, which is initiated by light-induced electron transfer from the fully reduced flavin adenine dinucleotide chromophore. Despite similar structures and repair mechanisms, the functions of PHR are highly selective; CPD PHR repairs CPD, but not (6-4) PP, and vice versa. In this study, we attempted functional conversion between CPD and (6-4) PHRs. We found that a triple mutant of (6-4) PHR is able to repair the CPD photoproduct, though the repair efficiency is 1 order of magnitude lower than that of wild-type CPD PHR. Difference Fourier transform infrared spectra for repair demonstrate the lack of secondary structural alteration in the mutant, suggesting that the triple mutant gains substrate binding ability while it does not gain the optimized conformational changes from light-induced electron transfer to the release of the repaired DNA. Interestingly, the (6-4) photoproduct is not repaired by the reverse mutation of CPD PHR, and eight additional mutations (total of 11 mutations) introduced into CPD PHR are not sufficient. The observed asymmetric functional conversion is interpreted in terms of a more complex repair mechanism for (6-4) repair, which was supported by quantum chemical/molecular mechanical calculation. These results suggest that CPD PHR may represent an evolutionary origin for photolyase family proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Animals
  • Catalytic Domain / genetics
  • Crystallography, X-Ray
  • DNA Damage
  • DNA Repair
  • Deoxyribodipyrimidine Photo-Lyase / chemistry
  • Deoxyribodipyrimidine Photo-Lyase / genetics*
  • Deoxyribodipyrimidine Photo-Lyase / metabolism*
  • Electron Transport
  • Models, Molecular
  • Molecular Dynamics Simulation
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Pyrimidine Dimers / chemistry
  • Pyrimidine Dimers / metabolism*
  • Pyrimidine Dimers / radiation effects
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Spectroscopy, Fourier Transform Infrared
  • Ultraviolet Rays
  • Xenopus laevis


  • Pyrimidine Dimers
  • Recombinant Proteins
  • pyrimidine(6-4)pyrimidone photolyase
  • Deoxyribodipyrimidine Photo-Lyase