Vibrio cholerae fotoliaz enzimi ifadesinin mavi ışığa göre değişimi

The photolyase/cryptochrome (phrs) family is blue-light sensing flavoproteins. Although photolyases and cryptochromes are structurally similar proteins, their functions are different depending on organisms. V. cholerae possess one photolyase and two cryptochrome genes. Photolyase (VcPhr) repairs UV-damaged DNA using energy from the blue light while Cry1, belongs to DASH family, responsible for the single strand DNA repair. In this thesis we wished to identify blue-light dependent expression of phrs genes in V. cholerae using Real-Time PCR. To this end, the cells were exposed to blue light and the quantities of cry1, cry2, phr expressions were determined. Analysis of RT-PCR results indicated that only phr expression was significantly increased when cells were exposed to blue light compared with the control samples. To see this effect is not due to the free-radical formation upon exposure cells to the blue-light, cells were subjected to the hydrogen peroxide and methylene blue and phr gene expression level was monitored by Real-Time PCR. We observed that there is no induction of phr gene expression compared with control genes that are induced under the free radicals. Next we wish to identify the gene that is responsible for induction of the expression level of phr. We performed a genome wide analysis on V. cholerae to detect possible blue-light receptors and analysis end up with three candidates, which are Cry1, Cry2, and VcPhr proteins. To see phr induction is mediated by VcPhr, Cry1 or Cry2, we first over-expressed Cry1 protein in bacteria by transferring the plasmid that carry cry1 gene, the result indicated a significant increase of phr gene compared with untransformed cells under the blue light condition. However Cry2 overexpression did not affect phr expression under the same conditions. Also to see the effect of photolyase on its own expression, similar experiments were performed on phr knockout V. cholerae. We did not observe a significant induction of phr gene by blue light when there is no functional VcPhr protein in the cell. All these results suggest that both VcPhr and Cry1 are involved in the induction of phr expression under blue light. The present study shows blue light dependency of repair activity in V. cholerae and it is possible that Cry1 and VcPhr take role in induction of phr expression and make cells ready to repair DNA damage, caused by UV light.