| Archaeal Cdc6 proteins show a high sequence similarity to the largest subunit of origin recognition complex(ORC1)and to the cell division control protein 6(Cdc6),both of which are involved in controlling initiation of chromosome replication in eukaryotes.Except for Methanococcales and Methanopyrales,all known archaeal genomes contain at least one cdc6 genes,and most of them have two cdc6 genes.Sulfolobus islandicus contain three cdc6 genes(cdc6-1,cdc6-2,cdc6-3)and strikingly ,only two of them(cdc6-l and cdc6-3)function in DNA replication whereas the third(cdc6-2)does not(Samson et al 2013).Indeed,the three genes show differential expression in Sulfolobus solfataricus upon UV irradiation:the expression of the initiator cdc6(cdc6-l and cdc6-3)genes are down regulated,while the expression of the non-initiator cdc6-2 is up regulated(FroLs et al 2008;Gotz et al 2007).This suggests that Cdc6-2 is a paralog of the classical replication initiators and it could play an important role in archaeal DNA damage repair.The main aim of this thesis is to conduct genetic and biochemical analyses on the S.islandicus cdc6-2 gene to yield an insight into its function.Some results acquired in past few years are as follow:(1)A mutant of S.islandicus REY15A mutant lacking Cdc6-2(Acdc6-2)constructed previously was grown in a SCVyU medium containing different doses of 4-nitroquinoline 1-oxide(4-NQO),a UV-mimicking agent,with the genetic host S.islandicus E233S as the reference strain(wt).This showed depletion of Cdc6-2 by gene deletion rendered the mutant hypersensitive to 4-NQO compared to the wild-type strain.Furthermore,genetic complementation of the deficiency by episomic expression of the cdc6-2 gene from a plasmid increased the drug tolerance but overexpression of Cdc6-2 in Acdc6-2 inhibited culture growth.(2)The effect of 4-NQO on cdc6-2 expression in S.islandicus was investigated by using RT-qPCR and Western-blot.The results show the relative level of cdc6-2 mRNA increased by 25 fold and the protein level was elevated for 16 fold after 5 h of 4-NQO treatment,indicating that cdc6-2 exhibits a strong DNA-damage responsive expression in this archaeon.(3)To gain an insight into the molecular mechanisms of up regulation of Cdc6-2,a putative promoter of the cdc6-2 gene was identified by analyzing the upstream sequence of the gene by which a 200-bp DNA fragment was defined as a completed promoter fragment.To identify DNA elements presented in the cdc6-2 promoter,three shortened promoter fragments were generated.All four promoter fragments were then cloned to a reporter gene vector carrying a lacS gene individually,yielding reporter gene plasmids.These plasmids were transformed into the wild type strain as well as in Acdc6-2,and their promoter activity was estimated by measuring ?-glycosidase activity in the transformants.This led to the identification of a DNA-damage responsive element(DDRE1)in the cdc6-2 promoter.Furthermore,the 4-NQO-induced expression of Cdc6-2 requires both DDRE1 and Cdc6-2 protein,and this suggests that Cdc6-2 could bind to DDRE1 and regulate its own expression.(4)Electrophoretic mobility shift assay(EMSA)and DNase I footprinting were employed to test if Cdc6-2 could bind to its own promoter and regulate the expression.We found that Cdc6-2 recombinant protein binds specifically to its own promoter and DNase I footprinting has identified 29-bp long stretch of DNA(-66 to-38),which could be the core sequence of the DDRE1 element.(5)The importance of the DDRE1 motif was investigated by transversion mutagenesis in which 10 different mutant promoters were constructed and analyzed as for the wild-type cdc6-2 promoter.A core binding site(-58 to-46)has been identified,which is sufficient to support the DNA binding by Cdc6-2 in vitro and the DNA damage-inducible expression in vivo.Together,these results indicate that Cdc6-2 plays a very important role in mediating DNA tolerance in S.islandicus and the regulation involves the binding of Cdc6-2 to its own promoter. |
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