Photolyases Characterization In The Cyanobacteria Synechococcus Pcc 7335 Exposed To Ultraviolet-B Radiation

SOTO, DÉBORA; CASSIA, RAÚL; FERNÁNDEZ, MARÍA BELÉN

Chapadmalal

Congreso; XVIII Congreso de la Sociedad Argentina de Microbiología General;; 2023

Sociedad Argentina de Microbiología General

Ultraviolet- B radiation (UV-B) constitutes a small portion of the solar spectrum that reaches the Earth´s surface and causes biochemical and morphological changes in photosynthetic organisms. Cyanobacteria are photoautotrophic organisms that depend on solar energy. Solar UV-B affects the DNA-inducing cyclobutane pyrimidine dimers (CPDs, 75%) as the major damage, and pyrimidine (6–4) pyrimidone photoproducts (6–4PPs, 10–30%), among others. Because these lesions block DNA replication and translation, they must be repaired for survival. Photoreactivation is a light-dependent mechanism in which DNA damages are repaired by photolyases (PHRs) in most organisms, except for placental mammals. PHRs repair either CPDs or 6-4PPs. However, a bifunctional PHR able to repair both damages (PhrSph98) was reported in Sphingomonas. Synechococcus PCC 7335 is a unicellular cyanobacterium that possesses several adaptations to light, including 1) chromatic complementary adaptation and 2) far-red light photoacclimation, allowing this strain to inhabit a wide range of niches. Regarding UV-B tolerance, we previously described that S. PCC 7335 encodes a UV-B inducible operon of a pair of PHRs. The downstream gene encodes a protein with structural homology to PhrSph98. This work aimed to characterize the photoreactivation mechanism in S. PCC 7335 during UV-B exposition. Bioinformatic homology search revealed that S. PCC 7335 encodes for three putative PHRs, one as a monocistronic transcript and the other as an operon. We analyzed the expression pattern of these transcripts by RT-qPCR. Results obtained show that all PHRs were induced by UV-B and that the monocistronic one had the greater induction compared to the operon-encoded ones. Also, CPD damages increased after UV-B irradiation of S. PCC 7335 cultures, as detected by immunochemistry using the TDM- 2 antibody. In addition, 1 h of white light recovery post-irradiation was not sufficient to reduce the CPD damages compared to the amount detected immediately after UV-B exposition. To assess the potential bifunctional activity in the repair of CPDs and 6–4PPs we cloned the predicted bifunctional PHR from S. PCC 7335. To analyze the role of specific amino acids, site-directed mutagenesis was performed. The replacement of Trp by Phe was made in residues expected to be important for electron transfer and activity, or possibly involved in DNA lesion binding. The mutant and wildtype enzymes were cloned, heterologously expressed in E. coli, and purified by immobilized metal affinity chromatography. The photo repair activity of the recombinant proteins was tested by repair assays in vitro using a UVC-damaged substrate. Finally, we conclude that this strain has a great capacity to cope with elevated doses of UV-B as a consequence of a highly efficient mechanism of DNA repair. This work was supported by ANPCyT, CONICET and UNMdP.