LB4
An siRNA screen to identify phosphatases that modulate the DNA damage response
Konstantina Marinoglou, Matthias Dobbelstein
University of Goettingen, Germany
The response of cells to damaged DNA can determine whether the affected cell will survive or die; in the first case, if the DNA is not properly repaired, genomic instability may lead to transformation of the cell and tumour induction. The signal transduction from the damaged DNA to the effector proteins depends on a series of phosphorylation events. The tumour suppressor p53 is a major effector protein that can activate DNA repair and induce cell cycle arrest or apoptosis upon DNA damage. Several kinases have been shown to play important roles in the DNA damage signalling cascade; however, little is known about the role of phosphatases in the regulation of DNA damage signalling. Our aim is to identify phosphatases that modulate this DNA damage response, focusing on the early steps of the signalling cascade and on the tumour suppressor p53. For this purpose, 298 human phosphatase subunits were knocked down in osteosarcoma U2OS cells (wild type p53). 48h after transfection with the human phosphatase siRNA library, DNA damage was induced by exposing the cells to 20 J/m² UVC. An early DNA damage marker protein, γH2Ax, and p53 were detected by immunofluorescence 2,5h after UVC exposure. Automated microscopy and single cell - based image analysis was used to quantify the DNA damage response. The knockdown of 37 phosphatases had a strong effect on p53 and/ or γH2Ax levels. These phosphatases will be further validated and analysed for their role in cell cycle, apoptosis induction and p53 regulation.
