Overexpression and functional characterization of two members of the photolyase-cryptochrome family from the UV-resistant Gamma-proteobacteria, Acinetobacter sp. Ver3

ALBARRACÍN V.H.; VALLE L.; PATHAK G.; FARIAS M.E.; BORSARELLI C.D.; GÄRTNER W.

Montreal

Congreso; AMERICAN SOCIETY OF PHOTOBIOLOGY MEETING; 2012

American Society of Photobiology

Photolyases/cryptochromes comprise a diversified gene family of photoactive molecules present throughout the three biological domains including the cyclobutane pyrimidine dimer photolyases (CPD), the (6?4) pyrimidine-pyrimidone adduct photolyases, and the cryptochromes. Photolyases utilize light energy for repair UV damaged DNA while cryptochromes function as signalling molecules that regulate diverse biological responses. Acinetobacter sp. Ver3 isolated from Lake Verde (4,400 m asl in Catamarca, Northwest Argentina) has been previously characterized by its outstanding UV-B resistance and efficient photorepairing ability. In this work, we go further by characterizing their photolyase/cryptochromes that can be involved it its UV-resistance mechanisms. Acinetobacter sp. Ver3 was subjected to genome pyro-sequencing. Two different photolyase-coding sequences were found and overexpressed by using standard cloning protocols: i.e. expression vectors (pET52b) in E.coli BL21 host strains. The N-terminal His-tag overexpressed proteins were purified by Ni-NTA affinity chromatography and subjected to spectroscopic measurements. E. coli strains harbouring the PL1 gene were subjected to UV-B irradiation (10 kJ/m2) and then exposed to light (PR) and dark repair (DR). Survival was assessed by plating aliquots of all the treatments in LB agar plates. For in vitro analyses, overexpressed protein was mixed with a UV damaged poly-dT-oligo and photoinduced. Activity was evaluated from the increase of absorbance at 260 nm. Upon BLAST and phylogenetic analysis one of the photolyases (PL1) was assigned to the Class I CPD-photolyase while the second one, PL2, was more related to the recently described Class III-photolyase. Clear enzymatic function was assessed for PL1, both in vivo and in vitro. E. coli cells harbouring the recombinant plasmid carrying PL1 survived after UV exposure while cells with the empty vector were completely depleted after the treatment. The recovery after PR in the cells carrying the PL1 was three orders of magnitudes greater that that observed in the control. PL1 was also able to decrease T-T dimers: when mixed with a single strand damaged oligo-DNA 40% of recovery of the absorbance at 260 nm was observed in the photoinduced treatment, while in the dark control this was not detected, thus probing the functional and efficient activity of PL1.