The dominant role of proofreading exonuclease activity of replicative polymerase ε in cellular tolerance to cytarabine (Ara-C)

Abstract

Chemotherapeutic nucleoside analogs, such as Ara-C, 5-Fluorouracil (5-FU) and Trifluridine (FTD), are frequently incorporated into DNA by the replicative DNA polymerases. However, it remains unclear how this incorporation kills cycling cells. There are two possibilities: Nucleoside analog triphosphates inhibit the replicative DNA polymerases, and/or nucleotide analogs mis-incorporated into genomic DNA interfere with the next round of DNA synthesis as replicative DNA polymerases recognize them as template DNA lesions, arresting synthesis. To address the first possibility, we selectively disrupted the proofreading exonuclease activity of DNA polymerase ε (Polε), the leading-strand replicative polymerase in avian DT40 and human TK6 cell lines. To address the second, we disrupted RAD18, a gene involved in translesion DNA synthesis, a mechanism that relieves stalled replication. Strikingly, POLE1exo−/− cells, but not RAD18−/− cells, were hypersensitive to Ara-C, while RAD18−/− cells were hypersensitive to FTD. gH2AX focus formation following a pulse of Ara-C was immediate and did not progress into the next round of replication, while gH2AX focus formation following a pulse of 5-FU and FTD was delayed to the next round of replication. Biochemical studies indicate that human proofreading-deficient Polε-exo− holoenzyme incorporates Ara-CTP, but subsequently extend from this base several times less efficiently than from intact nucleotides. Together our results suggest that Ara-C acts by blocking extension of the nascent DNA strand and is counteracted by the proofreading activity of Polε, while 5-FU and FTD are efficiently incorporated but act as replication fork blocks in the subsequent S phase, which is counteracted by translesion synthesis.

TDP1 is Critical for the Repair of DNA Breaks Induced by Sapacitabine, a Nucleoside also Targeting ATM- and BRCA-Deficient Tumors

“…We prepared the methylcellulose medium as described in (23). After incubating the cells for 12 days at 37°C with 5% CO2, we counted the number of colonies in each well.…”

DNA Damage Does Not Cause BrdU Labeling of Mouse or Human β-Cells

“…This study does not address the extent to which BrdU, marking S-phase entry, predicts completion of the cell cycle and production of two daughter cells. Evidence supporting the conclusion that some BrdU-labeled b-cells do successfully complete cell division include the many studies showing that BrdU labeling correlates with other proliferation markers such as Ki67, PCNA (proliferating cell nuclear antigen), and pHH3 (17,34,35), the documented increase in b-cell number under conditions when BrdU labeling is increased in vitro (17,36,37) and in vivo (33), and our current data showing BrdU(+) nuclei in mitosis ( Fig. 4G) and colabeling with pHH3 (Supplementary Fig.…”

“…BrdU exposure may or may not induce sister chromatid exchanges, a marker of genome instability (32). Triphosphate nucleoside analogs such as 5-fluorouracil act as replication fork blocks; however, these induce gH2AX not at the time of initial incorporation, but rather during S-phase of a subsequent cell cycle event (33). Extensive colabeling of gH2AX and pHH3 in the absence of BrdU exposure argue against a primary role for BrdU toxicity in the observed gH2AX population, but rather that gH2AX staining is associated with cycling cells.…”

Nucleobase and Nucleoside Analogues: Resistance and Re-Sensitisation at the Level of Pharmacokinetics, Pharmacodynamics and Metabolism

“…For instance, the triphosphate metabolite of cytarabine (ara-C) is readily incorporated into DNA by cellular replicases in vitro, with an efficiency comparable with dCTP. However, the extension from the mis-incorporated ara-CMP terminus by these enzymes occurs with a significantly reduced efficiency [ 139 , 140 , 141 , 142 ]. In line with this, ara-C treated cells quickly accumulate genomic ara-CMP, and this coincides with decreased DNA synthesis and activation of the intra-S-phase checkpoint [ 117 , 143 ], indicating that it is likely this delayed extension, slowing nascent chain synthesis, and the resulting replication fork stalling, that contributes to cancer cell death.…”