WRN protects against topo I but not topo II inhibitors by preventing DNA break formation

Abstract

The Werner syndrome helicase/3′-exonuclease (WRN) is a major component of the DNA repair and replication machinery. To analyze whether WRN is involved in the repair of topoisomerase-induced DNA damage we utilized U2-OS cells, in which WRN is stably down-regulated (wrn-kd), and the corresponding wild-type cells (wrn-wt). We show that cells not expressing WRN are hypersensitive to the toxic effect of the topoisomerase I inhibitor topotecan, but not to the topoisomerase II inhibitor etoposide. This was shown by mass survival assays, colony formation and induction of apoptosis. Upon topotecan treatment WRN deficient cells showed enhanced DNA replication inhibition and Sphase arrest, whereas after treatment with etoposide they showed the same cell cycle response as the wild-type. A considerable difference between WRN and wild-type cells was also observed for DNA single-and double-strand break formation in response to topotecan. Topotecan induced most DNA single-strand breaks 6 h after treatment. In both wrn-wt and wrn-kd cells these breaks were repaired at similar kinetics. However, in wrn-kd but not wrn-wt cells they were converted into DNA doublestrand breaks (DSBs) at high frequency, as shown by neutral comet assay and phosphorylation of H2AX. Our data provide evidence that WRN is involved in the repair of topoisomerase I, but not topoisomerase II-induced DNA damage, most likely via preventing the conversion of DNA singlestrand breaks into DSBs during the resolution of stalled replication forks at topo I-DNA complexes. We suggest that the WRN status of tumor cells impacts anticancer therapy with topoisomerase I, but not topoisomerase II inhibitors.

The Drosophila Werner Exonuclease Participates in an Exonuclease-Independent Response to Replication Stress

“…Many studies have demonstrated an abundance of DSBs in the absence of WRN either during normal cell growth or following treatment with a replication fork-stalling reagent (Christmann et al 2008;Franchitto et al 2008;Liu et al 2009;Mao et al 2010;Murfuni et al 2012). To investigate whether the embryonic nuclear defects we observed were due to an accumulation of DSBs, we stained WRNexo D embryos for phosphorylated histone 2Av (g-H2Av).…”

Artesunate Induces Oxidative DNA Damage, Sustained DNA Double-Strand Breaks, and the ATM/ATR Damage Response in Cancer Cells

“…Alkaline cell lysis and electrophoresis was essentially carried out as described previously (18). Formamidopyrimidine DNA glycosylase (FPG; generous gift from Bernd Epe, University of Mainz, Mainz, Germany) was added to the agarose gel on the slide at a dilution of 1 mg/mL.…”

Recapitulation of Werner syndrome sensitivity to camptothecin by limited knockdown of the WRN helicase/exonuclease

“…WS patient fibroblasts show rapid replicative senescence, a mutator phenotype and hypersensitivity to certain DNA-damaging agents including the topoisomerase poison camptothecin (CPT) (Christmann et al 2008;Lebel and Leder 1998;Ogburn et al 1997;Okada et al 1998;Pichierri et al 2000;Poot et al 1999). WS results from loss of function mutations in the WRN gene (Yu et al, 1996), which encodes a RecQlike helicase with a unique 3 0 -5 0 exonuclease domain (Gray et al 1997;Huang et al 1998;Shen et al 1998;Suzuki et al 1997).…”

“…The importance of HJ resolution in overcoming the effects of camptothecin-mediated arrest has been demonstrated using a Holliday junction endonuclease RusA, that reduces the hypersensitivity of WS cells to CPT (Rodriguez-Lopez et al 2007). The hypersensitivity of WS patient cells to CPT thus presumably occurs because WS cells are defective in processing and/or restarting stalled or collapsed replication forks (Franchitto and Pichierri 2004;Machwe et al 2007;Rodriguez-Lopez et al 2002;Sidorova et al 2008), since WRN usually prevents conversion of single strand to double strand DNA breaks (Christmann et al 2008). …”