부산대학교 도서관

The RECQ helicases are conserved throughout evolution and have roles in DNA replication, recombination and repair. Members of the family include BLM, WRN, RECQL1, RECQL4 and RECQL5. Defects in BLM, WRN and RECQL4 are associated with genetically inherited syndromes with common symptoms of premature ageing, genomic instability, and a predisposition to cancers. RECQL5 has not been linked to any genetic syndromes but RECQL5 null mice are more cancer-prone and it has been shown to have a lower protein expression level in colorectal cancers. RECQL5's function has been linked to protecting cells from replication stress, with cells depleted of RECQL5 more sensitive to camptothecin and cells over-expressing RECQL5 more resistant to thymidine. This thesis investigates the expression of RECQL5 in bladder cancer and the role of RECQL5 in replication stress. Here, using Western blotting, immunohistochemistry and qRT-PCR, RECQL5β protein has been shown to be over-expressed in bladder cancer compared to normal bladder, whereas RECQL5β mRNA has been shown to be under-expressed. Further work has shown that this protein regulation was not due to differences in cell cycle, miRNAs or protein stability, but is likely to be controlled by phosphorylation of eIF2α at translation initiation, changing global translation rates. Using DNA fibre analysis, over-expression of RECQL5β was shown to increase replication fork speed and protect replication forks from camptothecin and DRB induced slowing. Where wild-type MRC5V2 cells had slower replication fork speed in camptothecin and DRB treatments individually, adding both treatments together did not slow replication, suggesting both drugs slow replication in a manner dependent on either replication or transcription. Interestingly, expressing a helicase-dead RECQL5β in MRC5V2 cells slowed endogenous replication but speed was increased in the presence of camptothecin or DRB, but not together, suggesting RECQL5 has a role in maintaining replication at replication-transcription collisions. Depleting bladder cancer cells of RECQL5β in clonogenic toxicity assays did sensitise to camptothecin but this did not occur in MRC5V2 cells, implying that cancer cells may be more dependent on the RECQL5β protein. Finally, work here has shown that the difference in RECQL5 mRNA expression between normal bladder and bladder cancer tissues is dependent on the expression of MRE11.