FTIR study of CPD photolyase with substrate in single strand DNA.

Abstract

Photolyases (PHRs) utilize near UV/blue light to specifically repair the major photoproducts (PPs) of UV-induced damaged DNA. The cyclobutane pyrimidine dimer (CPD)-PHR binds flavin adenine dinucleotide (FAD) as a cofactor and repairs CPD lesions in double-stranded DNA. To understand the activation and repair mechanism of CPD-PHR, we applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to CPD-PHR, whose signals were identified by use of isotope-labeling. To further investigate the enzymatic function, here we study the activation and repair mechanism of CPD-PHR with the substrate in single strand DNA, and the obtained FTIR spectra are compared with those in double-stranded DNA, the natural substrate. The difference spectra of photoactivation, the fully-reduced (FADH(-)) minus semiquinone (FADH(•)) spectra, are almost identical in the presence of single strand and double-stranded DNA, except for slight spectral modification in the amide-I region. On the other hand, the difference spectra of photorepair were highly substrate dependent. Strong bands of the C=O stretch (1,720-1,690 cm(-1)) and phosphate vibrations (1,090-1,060 cm(-1)) of double-stranded DNA may have disappeared in the case of single strand DNA. However, an isotope-labeled enzyme study revealed that spectral features upon DNA repair are similar between both substrates, and the main reason for the apparent spectral difference originates from structural flexibility of DNA after repair.