Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. Synopsis The S. cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate Mec1/ATR activation by promoting the formation of RPA-coated ssDNA at dsDNA breaks. Xrn1 promotes the formation of single-stranded DNA at the ends of double-stranded DNA breaks. Rrp6 and Trf4 contribute to the recruitment of RPA and Mec1/ATR to the single-stranded DNA ends. The S. cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate Mec1/ATR activation by promoting the formation of RPA-coated ssDNA at dsDNA breaks.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA / N. Manfrini, C. Trovesi, M. Wery, M. Martina, D. Cesena, M. Descrimes, A. Morillon, F. D'Adda Di Fagagna, M.P. Longhese. - In: EMBO REPORTS. - ISSN 1469-221X. - 16:2(2015 Feb), pp. 221-231. [10.15252/embr.201439458]

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA

Manfrini N.;Trovesi C.;
2015-02

Abstract

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability. Synopsis The S. cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate Mec1/ATR activation by promoting the formation of RPA-coated ssDNA at dsDNA breaks. Xrn1 promotes the formation of single-stranded DNA at the ends of double-stranded DNA breaks. Rrp6 and Trf4 contribute to the recruitment of RPA and Mec1/ATR to the single-stranded DNA ends. The S. cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 facilitate Mec1/ATR activation by promoting the formation of RPA-coated ssDNA at dsDNA breaks.
DNA damage checkpoint; DNA double-strand breaks; Rrp6; Trf4; Xrn1; DNA Breaks, Double-Stranded; DNA Repair; DNA, Single-Stranded; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Exoribonucleases; Exosome Multienzyme Ribonuclease Complex; Intracellular Signaling Peptides and Proteins; Protein-Serine-Threonine Kinases; Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
Settore BIO/06 - Anatomia Comparata e Citologia
Settore BIO/18 - Genetica
19-dic-2014
Article (author)
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/861034
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