Translesion Synthesis DNA Polymerase Kappa Is Indispensable for DNA Repair Synthesis in Cisplatin Exposed Dorsal Root Ganglion Neurons

Abstract

In the peripheral nervous system (PNS) in the absence of tight blood barrier, neurons are at increased risk of DNA damage, yet the question of how effectively PNS neurons manage DNA damage remains largely unanswered. Genotoxins in systemic circulation include chemotherapeutic drugs that reach peripheral neurons and damage their DNA. Because neurotoxicity of platinum-based class of chemotherapeutic drugs has been implicated in PNS neuropathies, we utilized an in vitro model of Dorsal Root Ganglia (DRGs) to investigate how peripheral neurons respond to cisplatin that forms intra- and interstrand crosslinks with their DNA. Our data revealed strong transcriptional upregulation of the translesion synthesis DNA polymerase kappa (Pol κ), while expression of other DNA polymerases remained unchanged. DNA Pol κ is involved in bypass synthesis of diverse DNA lesions and considered a vital player in cellular survival under injurious conditions. To assess the impact of Pol κ deficiency on cisplatin-exposed DRG neurons, Pol κ levels were reduced using siRNA. Pol κ targeting siRNA diminished the cisplatin-induced nuclear Pol κ immunoreactivity in DRG neurons and decreased the extent of cisplatin-induced DNA repair synthesis, as reflected in reduced incorporation of thymidine analog into nuclear DNA. Moreover, Pol κ depletion exacerbated global transcriptional suppression induced by cisplatin in DRG neurons. Collectively, these findings provide the first evidence for critical role of Pol κ in DNA damage response in the nervous system and call attention to implications of polymorphisms that modify Pol κ activity, on maintenance of genomic integrity and neuronal function in exogenously challenged PNS.

Neurotoxicity of cytarabine (Ara-C) in dorsal root ganglion neurons originates from impediment of mtDNA synthesis and compromise of mitochondrial function

“…The University of Texas Medical Branch Institutional Animal Care and Use Committee approved all mouse-handling procedures. Dorsal root ganglion neurons were isolated from 3–4 months old C57BL/6 male mice (Envigo Laboratories, USA) according to established protocols [25–27] and as we described [28–30]. Briefly, ganglia were collected from all spinal levels, placed in cold dissecting solution (130 mM NaCl, 5 mM KCl, 2 mM KH2PO4, 1.5 mM CaCl2, 6 mM MgCl2, 10 mM glucose and 10 mM Hepes, pH 7.2), incubated with collagenase A (Roche) and trypsin (1 h/37°C) followed by DNase I (Roche), dissociated by 20 gentle triturations and spun (175 g/3 min).…”

“…DRG neurons seeded on coverslips were treated as indicated and fixed in 4% paraformaldehyde as we described [28–30]. Cells were permeabilized with 0.1% Triton X-100/0.1% sodium citrate in PBS and incubated with 3% BSA (w/v)/1% Donkey serum (v/v) in PBS.…”

Mammalian DNA Polymerase Kappa Activity and Specificity

“…By using plasmids carrying a specific single-site lesion and knocking down TLS polymerases, pol κ activity in combination with pol ζ resulted in error-prone bypass past CP adducts. Research focused on the regulation of pol κ function in eukaryotic cells demonstrated the involvement of pol κ in DNA repair pathways induced by CP and MMC treatment [108,109,110]. TLS across ICLs formed on oligodeoxynucleotides with acrolein-derived crosslinks and on plasmids with trimethylene crosslinks demonstrated that human pol κ carried out accurate incorporation opposite the crosslinked guanine and extended the primer beyond the lesion [111].…”

One, No One, and One Hundred Thousand: The Many Forms of Ribonucleotides in DNA

“…It should be emphasized that these polymerases are often active outside of the S phase [53][54][55][56] when the concentration of dNTPs is even lower than in the S phase [13], which may contribute to more significant incorporation of rNMPs into DNA. We can then speculate that "non-replicative" ribonucleotides may become particularly relevant in post-mitotic cells, such as neurons, where TLS has been recently found to take place [57].…”