Characterization of photolyases from Sulfolobus solfataricus

Abstract

Ultraviolet light induces DNA damage in the form of photoproducts, leading to increased genomic instability, if left unrepaired. Photoreactivation, catalysed by the enzyme photolyase, is the simplest and most efficient mechanism of repairing UV-induced DNA damage. Interestingly however, the gene encoding photolyase has been lost through evolution in all placental mammals including humans. Sulfolobus solfataricus, is a thermophilic archaeon that is highly tolerant to high doses of UV light in their natural environment. However, the mechanism underlying this tolerance or the role of photoreactivation remains uncharacterised.This study, identifies a novel putative S. solfataricus phr gene, and its expression was functionally characterised in E.coli both in vivo and in vitro. The optimum temperature for the activity of the enzyme was determined to be 90 ºC.The archaeal phr gene was also transfected in a photolyase-deficient human cell line, HeLa cells, using a mammalian expression vector (pDs-Red Monomer as a fusion tag). In parallel, two photolyase genes from E.coli and rat kangaroo (closest non-placental mammal which expresses photolyase) were used in similar systems as positive controls. The results showed no photoreactivation of UV treated cells following illumination with white light in the transfected HeLa cells with phr genes from the three organisms. This suggests that either the proteins were not expressed or not folded successfully in the cells. The results and findings are discussed in the context and compared with the other findings in the literature on the investigation of photoreactivation system in S. solfataricus and other organisms

Date of Award	19 Jun 2013
Original language	English
Awarding Institution	University of Bath
Supervisor	Momna Hejmadi (Supervisor), David Hough (Supervisor) & Michael Danson (Supervisor)