Functional analyses of novel photolyases from extreme microbes

SIMON, J. ; ALBARRACÍN, V.H.; FARIAS M.E.; GÄRTNER W.

Dortmund

Simposio; 3TH JUNGES CHEMIE SYMPOSIUM RUHR; 2012

High-Altitude Andean Lakes (HAAL) are shallow lakes allocated in the South American Puna-High Andes region. Caused by the extreme environmental conditions of those habitats, e.g., significantly high UV-exposition and heavy metal contamination, outstanding and highly efficient mechanisms have evolved, which protect microorganisms living under such harmful influences. One of these protection mechanisms comprises photolyases, which are able to repair UV-induced damages in DNA. The complete genome sequence of Acinetobacter sp. Ver3, an extremophilic bacterium isolated from HAAL, revealed two genes encoding photolyases (PL1 and PL2)[1]. During previous work these two genes had been cloned into E. coli BL21. The aim of this work is to functionally characterize these novel photoreceptors. The N-terminal His-tag overexpressed proteins were purified by IMAC and subjected to spectroscopic measurements. E. coli strains harbouring the PL1 gene were subjected to UV-B irradiation 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. All the obtained results were compared to the photolyase of E. coli (EcPHR) for a direct functional comparison of the novel photolyases from the Andean Lakes. 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 than observed in the control. PL1 was also able to decrease T-T dimers: when mixed with a damaged oligo-ssDNA 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.