title: Photoreactivation of Escherichia coli is impaired at high growth temperatures.
abstract: Photolyase repairs UV-induced lesions in DNA using light energy, which is the principle of photoreactivation. Active photolyase contains the two-electron-reduced flavin cofactor. We observed that photoreactivation of Escherichia coli was impaired at growth temperatures ⩾37°C, and growth in this temperature range also resulted in decreased photolyase protein levels in the cells. However, the levels of phr transcripts (encoding photolyase) were almost unchanged at the various growth temperatures. A lacZ-reporter under transcriptional control of the phr promoter showed no temperature-dependent expression. However, a translational reporter consisting of the photolyase N-terminal α/β domain-LacZ fusion protein exhibited lower β-galactosidase activity at high growth temperatures (37-42°C). These results indicated that the change in photolyase levels at different growth temperatures is post-transcriptional in nature. Limited proteolysis identified several susceptible cleavage sites in E. coli photolyase. In vitro differential scanning calorimetry and activity assays revealed that denaturation of active photolyase occurs at temperatures ⩾37°C, while apo-photolyase unfolds at temperatures ⩾25°C. Evidence from temperature-shift experiments also implies that active photolyase is protected from thermal unfolding and proteolysis in vivo, even at 42°C. These results suggest that thermal unfolding and proteolysis of newly synthesized apo-photolyase, but not active photolyase, is responsible for the impaired photoreactivation at high growth temperatures (37-42°C).
authors.affiliation: Institute of Molecular Biology and Biotechnology, Anhui Normal University, Wuhu, Anhui, China; Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Xu
authors.affiliation: Institute of Molecular Biology and Biotechnology, Anhui Normal University, Wuhu, Anhui, China.
authors.forename: Changqing
authors.initials: C
authors.lastname: Tian
authors.affiliation: Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Lu
authors.affiliation: Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
authors.forename: Liefeng
authors.initials: L
authors.lastname: Ling
authors.affiliation: Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Lv
authors.affiliation: Institute of Molecular Biology and Biotechnology, Anhui Normal University, Wuhu, Anhui, China; Anhui Province Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu, Anhui, China.
authors.forename: Mingcai
authors.initials: M
authors.lastname: Wu
authors.affiliation: Institute of Molecular Biology and Biotechnology, Anhui Normal University, Wuhu, Anhui, China. Electronic address: gpz1996@yahoo.com.
authors.forename: Guoping
authors.initials: G
authors.lastname: Zhu
mesh_terms: D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development* / Q000502:physiology / Q000528:radiation effects; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D055668:Photochemical Processes; D011401:Promoter Regions, Genetic; D055550:Protein Stability; D013696:Temperature; D014466:Ultraviolet Rays
chemical_list: D029968:Escherichia coli Proteins; D004255:Deoxyribodipyrimidine Photo-Lyase