DDR regulators accumulate at the 3’ end of genes after XRN2 loss.

<p>(<b>A-E</b>) Accumulation of the DNA damage regulators, ATM, BRCA1, γ-H2AX, 53BP1, and CtIP, was monitored at the 3’ pause sites of the (<b>A</b>) <i>ENSA</i>, (<b>B</b>) <i>β-actin</i>, (<b>C</b>) <i>Akirin</i>,<i>1</i> (<b>D</b>) <i>Gemin7</i> genes and (<b>E</b>) an intronic region of <i>Gemin7</i> by chromatin immunoprecipitation. n = 3, S.E. is indicated. (<b>F</b>) Model for XRN2 functions in DNA repair pathway choice. In normal conditions, XRN2 binds to the NHEJ factor 53BP1 promoting DSB repair via the NHEJ pathway. In the absence of XRN2, NHEJ is inhibited downstream of 53BP1, allowing DSB repair via the HR pathway.</p

XRN2-deficient cells are hypersensitive to various chemotherapeutic agents.

<p>(<b>A-D</b>) shScr and shXRN2 fibroblasts and MDA-MB-231 cells transfected with a siRNA control or targeting XRN2 were either mock-treated or exposed to: (<b>A, B</b>) ionizing radiation (IR); (<b>C, D</b>) ultraviolet light (UV). (<b>E-F</b>) shScr and shXRN2 fibroblasts were either mock-treated or exposed to: (<b>E</b>) H₂O₂ or (<b>F</b>) Aphidicolin (APH). Cells were then monitored for survival using colony forming assays. Colonies of >50 normal-appearing cells were quantified for mock- versus agent-exposed cells. (**p<0.01).</p

Loss of XRN2 leads to increased DSB formation and genomic instability.

<p>(<b>A</b>) Steady state levels of XRN2 protein in shScr (+) compared to shXRN2 (-) fibroblast cells were monitored by western blotting and immunofluorescence. (<b>B, C</b>) Basal levels of 53BP1, pATM^ser1981, γ-H2AX, and BRCA1 foci/nuclei were quantitated in shScr, shXRN2, and MCF-7 cells treated with control and XRN2 specific siRNA. (<b>D, E</b>) Genomic aberrations were quantified in shScr, shXRN2, and shk-h cells using derived metaphase spreads. (**p<0.01).</p

R-loop formation and transcription contribute to delayed DSB repair kinetics in XRN2-deficient cells.

<p><b>(A)</b> Levels of R loops were monitored in mock- or IR (1 Gy)-exposed shXRN2 compared to shScr fibroblast cells by immunofluorescence. <b>(B-C)</b> Regression of 53BP1 foci/nucleus was monitored by IF in IR (1 Gy)-treated shXRN2 and shScr cells that were exposed to α-amanitin (α-aman) or transfected with GFP-RNaseH. (*p<0.05).</p

XRN2 is a DNA damage-responsive factor.

<p>(<b>A</b>) To interrogate potential <i>in vivo</i> XRN2-interacting partners, exponentially growing HeLa cells were collected and lysed for FPLC analyses. Individual fractions were separated by SDS-PAGE and indicated proteins visualized by immunoblot (IB). (<b>B</b>) Interactions between XRN2 and the DNA damage regulators Ku80, 53BP1, and BRCA1 were interrogated by immunoprecipitation (IP). (<b>C</b>) XRN2 foci were quantitated in human fibroblasts stably expressing an shRNA control (shScr) or a Kub5-Hera shRNA (shk-h) in mock (unt)-, IR (1 Gy)-, and UV (20 J/m²)-treated cells. (<b>D</b>) Sub-cellular XRN2 localization and S9.6 foci were visualized in mock-, UV (20 J/m²)-, or UV (20 J/m²)- + α amanitin (α-aman)-treated shScr fibroblast cells by immunofluorescence. (*p<0.05).</p

Loss of XRN2 leads to increase amounts of replication stress.

<p>(<b>A</b>) Basal levels of 53BP1 were monitored in PCNA positive cells, an S-phase indicator, in shXRN2 and shScr cells by immunofluorescence (IF). (<b>B, C</b>) Basal levels of phosphorylated RPA, were monitored by IF in shXRN2, shScr, shk-h fibroblasts and MCF-7 cells transfected with control or XRN2 specific siRNAs. (<b>D</b>) Basal levels of phosphorylated ATR were monitored by IF in MCF-7 cells transfected with a siRNA control and a siXRN2. (<b>E</b>) Basal levels of phosphorylated Chk1 were monitored in.shScr, shXRN2, and shk-h fibroblasts by IF. (<b>F)</b> Basal levels of phospho-Chk1-pS-317 and RPA32-pS-(4/8), replication stress indicators, in shXRN2 (-) versus shScr (+) cells were monitored by western blotting. (<b>G</b>) DNA replication elongation was monitored in log-phase shScr, shXRN2, or shk-h fibroblasts by DNA fiber analyses. (**p<0.01).</p