Dynamics of GFP tagged chromatin factors RSF1, CENPS and CENPX at microirradiation induced DNA damage sites
Tidke, Pritishkumar
Tidke, Pritishkumar
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Publication Date
2022-02-22
Type
Thesis
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Abstract
Chromatin has a major influence on the DNA damage response (DDR). Several chromatin-related factors contribute to the DDR including the CENPS and CENPX histone fold proteins, also known as MHF1 and MHF2, and the chromatin remodelling factor RSF1. To quantitate the dynamic exchanges of these factors during the DDR we established cell lines stably expressing GFP tagged RSF1, CENPS and CENPX with moderate over-expression. We then identified conditions for point microirradiation to induce double strand break (DSB) responses by using Hoechst-33342 and a 405 nm laser at powers 100 fold lower than in the absence of sensitiser. As a quantitative validation we reproduced published results for half times of recruitment and removal of MRE11-GFP and RFP-PCNA. We also confirmed the expected recruitment of MRE11-GFP, RFP-PCNA, GFP-CENPS, GFP-CENPX and GFP-RSF1 to stripe and point microirradition DSB sites identified by γH2AX immunofluorescence. We then measured GFP tagged RSF1, CENPS and CENPX kinetics in real time and used curve fitting to derive half times for DSB site recruitment of ~100 s and removal of ~2000 s. To determine cell cycle phase specific behaviours we introduced stably expressing red fluorophore tagged S and G1 phase specific markers PCNA and CDT1 into the cell lines and enriched for cells in G1, S and G2 after release from double thymidine blocks. This revealed that GFP-CENPS and GFP-CENPX recruitment occurs in all cell cycle phases, but that the half times of recruitment and removal in G2 are delayed and involve increased abundance relative to G1. GFP-CENPS and GFP-CENPX recruitment to DSBs occurs simultaneously and slightly after GFP-RSF1, at a time consistent with published estimates for ATM activation that initiates repair by NHEJ and HR. The removal of GFP-CENPS and GFP-CENPX are at a similar time to the recruitment of RAD51. This places CENPS and CENPX in the vicinity of DSBs at the time when nucleosomes are being actively remodelled to enable resection, and their increased abundance at DSBs in G2 correlates with extended resection for HR.
Publisher
NUI Galway