DNA 손상 반응과정 중 Protein Phosphatase 5 에 의한 53BP1 조절기작에 대한 연구

Abstract

DNA double-strand break (DNA 이중나선절단, DSB)은 주로 방사선과 방사선 유사물질에 의해 발생한다. 또한, 염색체에서 기계적인 스트레스와 복제과정에 DNA 중합효소가 (DNA single-strand break )DNA 단일나선절단 같은 DNA 손상을 만났을 때 발생할 수 있다. DSB의 부정확한 복구와 복구 결핍은 돌연변이 및 유전자 불안정성을 일으키고, 나아가 세포사멸사 및 암 유발을 유도한다. DNA 손상반응에서 초기현상중의 하나인 γ-H2AX의 형성은 ataxia telangiectasia mutated(ATM), ataxia telangiectasia related(Rad 3, ATR), ATM related kinase(ATX), DNA dependant protein kinase(DNA-PK)같은 phosphatidylinositol-3 kinase-like family of kinases(PIKK)에 의해 histone H2AX가 인산화된다. DNA 손상 수분 안에, γ-H2AX는 MRN complex, 53BP1, BRCA1, and MDC1같은 DNA 손상복구요소들을 포함한 뚜렷한 foci를 형성한다. Yeast two hybrid 시스템을 통하여, 53BP1과 상호작용하는 몇 개의 유전자 중 serine/threonine phosphatase family인 PP5 유전자를 확인하였다. PP5와 53BP1의 결합은 DNA 손상 후 크게 증가하였다. PP5와 53BP1의 상호작용을 확인하기 위하여, PP5 과발현 세포주와 억제된 세포주를 만들고 DNA 손상후 Western blotting and immunofluorescense analysis을 수행하였다. PP5는 53BP1의 탈 인산화를 조절하고, 또한 53BP1 foci의 축적 및 유지에 영향을 미치는 것으로 사료된다.|The DNA double-strand break (DSB) is the principle cytotoxic lesion for ionizing radiation and radio-mimetic chemicals but can also be caused by mechanical stress on chromosomes or when a replicative DNA polymerase encounters a DNA single-strand break or other type of DNA lesion. Inaccurate repair or lack of repair of a DSB can lead to mutations or to genomic instability, cancer development or cell death according to apoptosis. One of the earliest events in the damage response is phosphorylation of histone H2AX at Ser139 by members of the phosphatidylinositol-3 kinase-like family of kinases such as ataxia telangiectasia mutated(ATM), ataxia telangiectasia related(Rad 3, ATR), ATM related kinase(ATX), DNA dependant protein kinase(DNA-PK) to create γ-H2AX. Within minutes of DNA damage, γ-H2AX appears at discrete nuclear foci that contain DNA repair factors like the MRN repair complex, 53BP1, BRCA1, and MDC1. Through yeast two-hybridization system we could find several cDNAs putative interacting with 53BP1. After DNA sequencing, we found that one is a cDNA for PP5, serine/threonine phosphatase family. The binding of PP5 with 53Bp1 was highly increased after DNA damage. To evaluate the interaction of PP5 and 53BP1, we made overexpressed- and suppressed-PP5 cell lines and confirmed the effects by Western blotting and immunofluorescense analysis after DNA injury. So, we could investigate that the dephosphorylation of 53BP1 was regulated by PP5 and also influenced the maintenance and accumulation of 53BP1 foci.