| Research Abstract |
In this research, I have aimed to understand pathways for recognition and repair of DNA damages in a biochemical aspect using Xenopus egg extract cell-free experimental system. During the term of this project, the analysis was focused on DNA damage-induced behaviors and functions of RecQ helicases, such as BLM, WRN and RecQL4, which are thought to play central roles in the maintenance of genomic integrity. BLM is a product of a causative gene for Bloom syndrome that is characterized by higher susceptibility to varieties of tumors. After induction of double-strand DNA breaks (DSB), this helicase appeared to be serially phosphorylated by DNA-dependent protein kinase (DNA-PK) and ATM. On the other hand, WRN and RecQL4, which defects lead to progeroid Werner and Rothmund-Thomson syndromes, respectively, were accumulated in the chromatin fraction by an induction of DSB. Both of the chromatin associations were dependent on single-stranded DNA binding protein complex, RPA, but not on the function of one of important factors for DNA recombination, Rad51. Further analysis for the function of RecQL4 revealed that a depletion of this protein resulted in a reduced rate of a disappearance of phosphorylated H2AX (γH2AX), suggesting that DSB-repair is impaired by the removal of RecQL4. Furthermore, the chromatin association of RecQL4 was moderately or severely suppressed by an addition of caffeine or wortomannin, an inhibitor of ATM and ATR, or ATM and DNA-PK, respectively, suggesting that RecQL4 functions downstream of DNA-PK-dependent non-homologous end-joining (NHEJ) in addition to ATM-induced checkpoint pathway. Supporting the probable functional relationships between DNA-PK and RecQL4, chromatin association of RecQL4 was suggested to take place in the proximity of the binding site of a subunit of DNA-PK, Ku70, but not Rad51, by an immunoprecipitation analysis after fragmentation of the damaged chromatin.
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