Crystal structure of thermostable DNA photolyase: Pyrimidine-dimer recognition mechanism

Abstract

DNA photolyase is a pyrimidine-dimer repair enzyme that uses visible light. Photolyase generally contains two chromophore cofactors. One is a catalytic cofactor directly contributing to the repair of a pyrimidine-dimer. The other is a light-harvesting cofactor, which absorbs visible light and transfers energy to the catalytic cofactor. Photolyases are classified according to their second cofactor into either a folate- or deazaflavin-type. The native structures of both types of photolyases have already been determined, but the mechanism of substrate recognition remains largely unclear because of the lack of structural information regarding the photolyase-substrate complex. Photolyase from Thermus thermophilus , the first thermostable class I photolyase found, is favorable for function analysis, but even the type of the second cofactor has not been identified. Here, we report the crystal structures of T. thermophilus photolyase in both forms of the native enzyme and the complex along with a part of its substrate, thymine. A structural comparison with other photolyases suggests that T. thermophilus photolyase has structural features allowing for thermostability and that its light-harvesting cofactor binding site bears a close resemblance to a deazaflavin-type photolyase. One thymine base is found at the hole, a putative substrate-binding site near the catalytic cofactor in the complex form. This structural data for the photolyase-thymine complex allow us to propose a detailed model for the pyrimidine-dimer recognition mechanism.

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

“…Preparation of Oligonucleotides and CPD Photolyase from T. thermophilus-Oligonucleotides labeled with 13 C and 15 N were prepared according to a published method (16). The other oligonucleotides without the CPD lesion were purchased from ESPEC Oligo Service Corp. d(CGCAAT[CPD]TAAGCCG) was chemically synthesized as previously reported (17).…”

“…The experiments described above were performed on a Bruker DRX400 spectrometer, and those described below were monitored on a Bruker DRX600 spectrometer. One-dimensional 1 H, 1 H-13 C heteronuclear single quantum coherence (HSQC) (20), HCCH type-(21), HCN (22), HCP (23), and 13 C 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.…”

“…The helical domain is composed of clusters I and II, and a cavity is formed between the clusters, where the FAD is deeply buried. It has been suggested that the cavity is used for the CPD binding, because the asymmetric polarity of the cavity fits well with that of the CPD (11)(12)(13)(14)(15). Based upon the crystal structures without the substrates, two research groups have proposed computer models of the DNA-CPD photolyase complex, in which the relative orientations of the DNA chain are different from each other (11)(12)(13)(14)(15).…”

“…In order to understand the mechanism based on structural analyses, the crystal structures of the CPD photolyases from Escherichia coli, Anacystis nidulans, and Thermus thermophilus have been solved without the substrates, i.e. CPD-containing DNAs (11)(12)(13). The x-ray studies revealed that the enzymes share a similar global fold, which consists of an ␣/␤ domain and a helical domain.…”

Systems Level Insights Into the Stress Response to UV Radiation in the Halophilic ArchaeonHalobacterium NRC-1

“…Classification of photolyases into class I or class II photolyases is not possible through analysis of amino acid sequence alone (Komori et al 2001). To further investigate the role of each putative photolyase in photoreactivation suggested by the survival experiments, we constructed strains of Halobacterium NRC-1 with single (phr1 or phr2) and double (phr1 and phr2) in frame deletions in the two genes.…”