The Regulatory Mechanism of DNA Damage Checkpoint Protein MDC1

Abstract

Mediator of DNA damage checkpoint protein 1(MDC1) plays a vital in DNA damage response (DDR) to repair DNA damages, especially DNA double strand breaks (DSBs). In response to DSBs, MDC1 relocates to the damaged site to mediate recruitment of DNA repair proteins. Any delay or impairment in DSB induced translocation of MDC1 to the nucleus leads to disorganized DNA repair results in accumulation of DNA damages, carcinogenesis, chromatin instability and ultimately cell death. Here, we found Karyopherin α-2 (KPNA2, a member of importin-α family responsible for nuclear transport of proteins bearing Nuclear Localization Sequence) as MDC1 interacting protein in yeast two-hybrid screening and further confirmed by immunoprecipitation assay. In absence of KPNA2, DSB induced nuclear transport and nuclear foci formation of MDC1 was reduced which affected the recruitment of downstream repair proteins RAD51, 53BP1 and BRCA1. We also showed that KPNA2 depleted cells accumulate DSBs which is exposed by detecting increased number of late γH2AX foci after DSB and hyper tail movement in neutral comet assay. Furthermore, DR-GFP reporter assay revealed a lesser amount of Homologous Recombination (HR) activity in KPNA2i condition. Cells showed hypersensitivity to IR-induced cell death in absence of KPNA2, as disclosed by clonogenic cell survival assay. These results suggest that KPNA2 is important for DSB induced nuclear translocation and proper functioning of MDC1 in DDR pathway, especially in HR. We also did an initial screening to elucidate the novel functions of ZNF114, PHB2 and FHL2 in DNA damage repair.|MDC1은 DNA double strand breaks 같은 DNA 손상 자극시 손상복구에 관여하는 중요한 단백질로 알려져 있다. 그러나 MDC1 기능을 조절하는 조절단백질과 그 자세한 기전에 대한 연구는 미흡한 실정이다. 따라서 본 연구논문에서는 MDC1의 활성을 조절하는 새로운 조절단백질을 밝히고자, MDC1의 yeast two hybrid assay를 실시하여 MDC1와 결합하는 몇몇 단백질 (KPNA2, ZNF114, PHB2, FHL2)들을 동정하였고, 그들 후보 조절유전자들의 결핍을 통해 MDC1활성에 미치는 영향을 조사하였다. 각 후보 결합유전자들이 결핍된 세포에서 MDC1의 DNA손상 foci, 상동재결합 활성이 감소함을 확인하였다. 또한 clonal survival assay를 통해 후보유전자들이 결핍된 세포는 방사선조사에 더 민감함을 확인하였다. 따라서 본 연구결과는 MDC1에 결합해 MDC1 활성을 조절하는 새로운 단백질들을 동정하였고, MDC1 활성조절의 새로운 분자적 기전을 제시한다.