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In response to replication stress, replication fork stalls, and cells activate replication stress response (RSR) pathways to repair and restart replications. Among these RSR pathways, translesion synthesis is performed by translesion synthesis polymerases. The human Y-family translesion polymerase kappa (pol k) is overexpressed in glioblastoma multiforme (GBM), the most aggressive, invasive, and heterogenous form of brain cancer. GBM tumors typically have a high degree of resistance to standard therapeutics, which contributes to poor prognosis. In this study, we explore the mechanism by which pol k acts as a barrier to protect tumor cells from DNA damaging effects of chemotherapeutics. Results from our laboratory demonstrated a role for pol k in controlling fork speed and suppression of genomic single-stranded DNA (ssDNA) gap formation in GBM cells. By performing DNA fiber experiments (± S1 nuclease that cleaves single stranded DNA allowing determination of ssDNA gaps) in multiple cell lines, it was determined that pol k slows fork elongation rate and prevents ssDNA gaps accumulation in GBM-derived cells (e.g., T98G, U118-MG, and primary cultures from GBM specimens) with no considerable effect on several non-GBM cell lines, including HAP-1 cells, a commercially available near-haploid cell line derived from chronic myeloid leukemia patient. Results with cells co-depleted of pol k and another y family translesion polymerase Rev1 support the idea that these TLS enzymes both function to promote replication gap suppression in GBM cells, with pol k exerting a slightly more pronounced effect on fork speed and gap formation than Rev1. Conversely, loss of pol k is dispensable for replication gap suppression in HAP-1 cells unless Rev1 is also depleted. To begin to understand the source of ssDNA formation in pol k-deficient GBM cells, I used RNA interference to deplete PrimPol expression and then performed DNA fiber analysis. My results are supportive of the idea that ssDNA gap accumulation in pol k-depleted GBM cells was dependent upon repriming mediated by PrimPol, with no discernable impact of PrimPol depletion on gaps formation in either WT or POLK-KO HAP-1 cells. To explore the possibility that pol k could also be preventing ssDNA formation through its fork protection activity, I performed a modified DNA fiber assay that includes treatment with high dose hydroxyurea (HU) after labeling with CldU and IdU. Depletion of pol k led to degradation of nascent strand DNA in both GBM and non GBM cells. Therefore, it seems unlikely that this function of pol k is what aids in control of replication fork elongation and prevention of ssDNA gap formation in GBM cells. In conclusion, pol k has a heightened impact on controlling fork speed and ssDNA gap formation in GBM cells, a role that is not apparent in HAP-1 cells unless cells are also depleted of Rev1. Importantly, pol k suppresses ssDNA gaps in GBM cells by opposing repriming mediated by PrimPol, but the exact mechanistic basis of pol k-mediated fork slowing remains unknown. In contrast to the GBM-specific role in replication gap suppression, pol k was found to have a general role in replication fork protection in cell lines originating from different tumor types. These findings have important implications for understanding how tumor-specific mechanisms promote tolerance of DNA damage and survival of genotoxic therapies. This work was supported by a grant from the National Science Foundation (MCB 1903357 to R.L.E) along with a Barton Bridging Award from the UAMS College of Medicine to R.L.E.
Sewilam et al. (Fri,) studied this question.