The Werner's Syndrome protein (WRN) is a DNA helicase/exonuclease that is believed to be active at arrested DNA replication forks. In previous experiments we have shown that WRN promotes resistance to DNA methylating agents in human glioma cells lacking O6-methylguanine-DNA-methyltransferase (MGMT). MGMT-deficient cells cannot remove O6-methylguanine (O6-meG) adducts, which are unable to properly base-pair with any dNTP. They are thought to stall DNA replication due to the inability of exonucleases and/or mismatch repair (MMR) proteins to resolve the mis-pair. The resulting single-stranded discontinuity is hypothesized to be a substrate for a repair pathway involving WRN. MMR is involved in the lethality of some types of DNA damage, specifically O6-meG adducts caused by the DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanadine (MNNG). MMR-deficient cells therefore exhibit methylation tolerance. We hypothesized that tolerance of unrepaired O6-meG conferred by MMR deficiency would eliminate the requirement for WRN in methylator resistance by preventing the formation of single-stranded DNA discontinuities in newly replicated DNA. We suppressed WRN using antisense oligonucleotides (ASO) in MMR-proficient and deficient human glioma cell lines lacking MGMT. Using clonogenic survival assays, we examined the sensitivity of both cell lines to the methylator MNNG with and without previous ASO treatment. Assays using 4-nitroquinoline 1-oxide (4NQO), a DNA damaging agent with different specificity, and cells treated with sense oligonucleotides (SO; complementary to the ASO used) provided controls. MMR-proficient cells showed a significant increase in sensitivity to MNNG when treated with ASO to WRN. LD10 for SO- and ASO- treated cells was 0.21μM and 0.14 μM respectively, representing a 1.5-fold difference. MMR-deficient cells, however, did not exhibit an increase in sensitivity when WRN was suppressed (LD10 for SO-treated cells was 9.7 μM and for ASO-treated cells was 9.3 μM). These results show that MMR deficiency eliminates the need for WRN in methylator resistance, and therefore provides evidence that WRN participates in a post-replication repair pathway that promotes resistance to O6-meG.
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