XPG-related nucleases are hierarchically recruited for double-stranded rDNA break resection

Kevin J Barnum,Y Tram Nguyen,Matthew J O’Connell

doi:10.1074/jbc.ra118.005415

Kevin J Barnum, Y Tram Nguyen + Show 1 more

Open Access

https://doi.org/10.1074/jbc.ra118.005415

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Abstract

dsDNA breaks (DSBs) are resected in a 5'→3' direction, generating single-stranded DNA (ssDNA). This promotes DNA repair by homologous recombination and also assembly of signaling complexes that activate the DNA damage checkpoint effector kinase Chk1. In fission yeast (Schizosaccharomyces pombe), genetic screens have previously uncovered a family of three xeroderma pigmentosum G (XPG)-related nucleases (XRNs), known as Ast1, Exo1, and Rad2. Collectively, these XRNs are recruited to a euchromatic DSB and are required for ssDNA production and end resection across the genome. Here, we studied why there are three related but distinct XRN enzymes that are all conserved across a range of species, including humans, whereas all other DSB response proteins are present as single species. Using S. pombe as a model, ChIP and DSB resection analysis assays, and highly efficient I-PpoI-induced DSBs in the 28S rDNA gene, we observed a hierarchy of recruitment for each XRN, with a progressive compensatory recruitment of the other XRNs as the responding enzymes are deleted. Importantly, we found that this hierarchy reflects the requirement for different XRNs to effect efficient DSB resection in the rDNA, demonstrating that the presence of three XRN enzymes is not a simple division of labor. Furthermore, we uncovered a specificity of XRN function with regard to the direction of transcription. We conclude that the DSB-resection machinery is complex, is nonuniform across the genome, and has built-in fail-safe mechanisms, features that are in keeping with the highly pathological nature of DSB lesions.

Highlights

Edited by Patrick Sung dsDNA breaks (DSBs) are resected in a 5؅33؅ direction, generating single-stranded DNA
We have previously used the formation of replication protein A (RPA) and Rad51 foci at bleomycin-induced DSBs as a marker for single-stranded DNA (ssDNA) production at lesions [34]
Forming defect (60 –70% lower than WT, p ϭ 0.002– 0.0004), only the triple mutant cells are defective in activating the Chk1-dependent cell cycle arrest, albeit not quite as defective as spores derived from the triple null mutant deleted for all three genes, i.e. Rad2 is critical for end resection in the absence of Exo1

Summary

Introduction

Edited by Patrick Sung dsDNA breaks (DSBs) are resected in a 5؅33؅ direction, generating single-stranded DNA (ssDNA) This promotes DNA repair by homologous recombination and assembly of signaling complexes that activate the DNA damage checkpoint effector kinase Chk. In fission yeast (Schizosaccharomyces pombe), genetic screens have previously uncovered a family of three xeroderma pigmentosum G (XPG)–related nucleases (XRNs), known as Ast, Exo, and Rad2 These XRNs are recruited to a euchromatic DSB and are required for ssDNA production and end resection across the genome. During S- and G2-phases, the presence of an identical sister chromatid provides a template for error-free repair by homologous recombination (HR) For this to occur, and for the associated DNA damage checkpoint to be initiated, the lesion must be processed to generate single-stranded DNA (ssDNA) with an exposed 3Ј-OH group and a double-stranded junction with a 5Ј-PO4. RPAcoated ssDNA recruits Cdc24/Dna, a helicase/nuclease implicated in replication fork processing [16] and subsequent protein A; HR, homologous recombination; SSA, single-stranded annealing; DAPI, 4Ј,6-diamino-2-phenylindole; OF, Okazaki fragment; ATR, ATM and Rad3-related

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