Abstract
RAD9 participates in DNA damage-induced cell cycle checkpoints and DNA repair. As a member of the RAD9-HUS1-RAD1 (9-1-1) complex, it can sense DNA damage and recruit ATR to damage sites. RAD9 binding can enhance activities of members of different DNA repair pathways, including NEIL1 DNA glycosylase, which initiates base excision repair (BER) by removing damaged DNA bases. Moreover, RAD9 can act independently of 9-1-1 as a gene-specific transcription factor. Herein, we show that mouse Rad9−/− relative to Rad9+/+ embryonic stem (ES) cells have reduced levels of Neil1 protein. Also, human prostate cancer cells, DU145 and PC-3, knocked down for RAD9 demonstrate reduced NEIL1 abundance relative to controls. We found that Rad9 is required for Neil1 protein stability in mouse ES cells, whereas it regulates NEIL1 transcription in the human cells. RAD9 depletion enhances sensitivity to UV, gamma rays and menadione, but ectopic expression of RAD9 or NEIL1 restores resistance. Glycosylase/apurinic lyase activity was reduced in Rad9−/− mouse ES and RAD9 knocked-down human prostate cancer whole cell extracts, relative to controls. Neil1 or Rad9 addition restored this incision activity. Thus, we demonstrate that RAD9 regulates BER by controlling NEIL1 protein levels, albeit by different mechanisms in human prostate cancer versus mouse ES cells.
Highlights
The genomic integrity of cells is always challenged because of exposure to DNA damaging agents, either from exogenous sources, including radiations and chemicals, or endogenous toxic metabolites such as reactive oxygen species and free radicals [1]
We observed that when RAD9 level was lowered, NEIL1 protein quantity was reduced by 51% in DU145 and by 43% in the PC-3 populations
We show that NEIL1 protein levels are reduced in response to RAD9 depletion, and that RAD9-mediated NEIL1 protein level regulation is transcriptional in human prostate cancer cells but post-transcriptional in Mouse embryonic stem (mES) cells
Summary
The genomic integrity of cells is always challenged because of exposure to DNA damaging agents, either from exogenous sources, including radiations and chemicals, or endogenous toxic metabolites such as reactive oxygen species and free radicals [1]. DNA damage-induced cell cycle checkpoints promote genome stability through transient delays in cell cycle progression that allow cells to repair DNA lesions before entering critical phases of the cell cycle. Proteins involved in this pathway are regulated through a wide range of processes, including transcriptional and posttranscriptional control [3], protein–protein interactions [4] and subcellular localization [5]. Aberration in these processes can lead to cancer, immunodeficiency and neurological disorders [6,7]
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