Abstract
The proper cellular response to DNA double-strand breaks (DSBs) is critical for maintaining the integrity of the genome. RecQL4, a DNA helicase of which mutations are associated with Rothmund–Thomson syndrome (RTS), is required for the DNA DSB response. However, the mechanism by which RecQL4 performs these essential roles in the DSB response remains unknown. Here, we show that RecQL4 and its helicase activity are required for maintaining the stability of the Mre11-Rad50-Nbs1 (MRN) complex on DSB sites during a DSB response. We found using immunocytochemistry and live-cell imaging that the MRN complex is prematurely disassembled from DSB sites in a manner dependent upon Skp2-mediated ubiquitination of Nbs1 in RecQL4-defective cells. This early disassembly of the MRN complex could be prevented by altering the ubiquitination site of Nbs1 or by expressing a deubiquitinase, Usp28, which sufficiently restored homologous recombination repair and ATM, a major checkpoint kinase against DNA DSBs, activation abilities in RTS, and RecQL4-depleted cells. These results suggest that the essential role of RecQL4 in the DSB response is to maintain the stability of the MRN complex on DSB sites and that defects in the DSB response in cells of patients with RTS can be recovered by controlling the stability of the MRN complex.
Highlights
Proper cellular response to DNA double-strand breaks (DSBs), the most cytotoxic type of DNA damage, is critical for maintaining the integrity of genome to avoid instability that may result in genetic diseases, carcinogenesis, and cell death [1, 2]
Because both homologous recombination (HR) repair and ataxia telangiectasia mutated (ATM) activation were reported to be impaired in RecQL4-defective cells [22,23,24], we reasoned that factors commonly involved in both these processes may be affected by the defects in RecQL4
All RecQ helicases in mammalian cells have been shown to be involved in DNA DSB responses [14, 15], and direct participation of WRN and BLM have been well documented in many studies; WRN stimulates nonhomologous end joining (NHEJ) by its helicase and exonuclease activities [19], and BLM plays both pro- and antirecombination roles by stimulating the end resection activity of Dna2 [27] and by the displacement of Rad51 from resected DNA intermediates [20]
Summary
Hyunsup Kim1,‡, Hyemin Choi2,‡, Jun-Sub Im2, Soon-Young Park, Gwangsu Shin, Jung-Ho Yoo, Gyungmin Kim, and Joon-Kyu Lee1,2,* From the 1Interdisciplinary Graduate Program in Genetic Engineering, 2Department of Biology Education, Seoul National University, Seoul, Korea
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