Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses

Takuya Torizawa,Takumi Ueda,Seiki Kuramitsu,Kenichi Hitomi,Takeshi Todo,Shigenori Iwai,Kosuke Morikawa,Ichio Shimada

doi:10.1074/jbc.m404536200

Abstract

The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from (31)P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.

Highlights

Irradiation of DNA with ultraviolet (UV) light produces various damaged bases, leading to cellular transformation and cell death [1,2,3]
Based upon results from 31P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of cyclobutane pyrimidine dimer (CPD)-containing single-stranded DNA on the CPD photolyase
Higher NaCl concentrations resulted in significant reductions of the affinity, suggesting that electrostatic interactions contribute to the CPD binding

Summary

EXPERIMENTAL PROCEDURES

Preparation of Oligonucleotides and CPD Photolyase from T. thermophilus—Oligonucleotides labeled with 13C and 15N were prepared according to a published method [16]. One-dimensional 1H, 1H-13C heteronuclear single quantum coherence (HSQC) [20], HCCH type- [21], HCN [22], HCP [23], and 13C heteronuclear single quantum coherence-nuclear Overhauser effect spectroscopy (HSQC-NOESY) [20] spectra were used for the detection and assignments of the resonances from CPD labeled with stable isotopes. In these experiments, the NMR samples were dissolved in Buffer III. One-dimensional 1H, 1H-15N HSQC, and 15N NOESY-HSQC [20] were used for the detection and assignments of the imino resonances of dsDNA

RESULTS

Ϫb Ϫb

DISCUSSION