Translesion DNA synthesis polymerase κ is essential to a carcinogen-induced nucleolar stress response

Abstract

DNA polymerase kappa (Polk) has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences and nucleotide excision repair. Since multiple polymerases are present at the same DNA lesion, by utilizing the selective Polk substrate, N2 (4 ethynylbenyl) 2 deoxyguanosine (EBndG) as bait, isolation of proteins on Pol kappa synthesized DNA (iPoKD) followed by mass spectrometry captured proteins associated specifically with nascent DNA synthesized with Polk. We show that, Polk is active in the nucleolus and Polk synthesized DNA are enriched with nucleolar proteins. Exposure of cells to benzo a pyrene diol epoxide (BPDE) increases Polk stability and induces nucleophosmin 1 translocation to the nucleoplasm, nucleolar cap formation, and reduction in rDNA transcription, hallmarks of nucleolar stress. The expression and activity of Polk is regulated by the polycomb complex protein Ring Finger Protein 2 (RNF2) and by the PARylation by poly(ADP) ribose polymerase 1 (PARP1). This study provides insight into the novel role of Polk in the nucleolus and in maintaining ribosomal DNA integrity after BPDE damage.

An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging neurons

“…To confirm the biochemical result, we used an immunofluorescence (IF) assay to visualize the spatial-temporal changes of POLK with age in a healthy wild-type mouse brain. We validated the POLK antibody, previously reported in immunofluorescence assays in human cell lines (Paul et al 2022), using mouse primary neuronal culture and observed a significant decrease in POLK signal upon two independent siRNA knock-downs but not in scrambled siRNA (Figure S1Ci). qPCR analysis showed a 35% reduction in Polk mRNA level upon siRNA knock-down (Figure S1Cii).…”

“…A progressive age-associated shiJ in subcellular localiza;on of Polk First, we validated the an4-POLK an4body for immunofluorescence assays by knocking out Polk in the U2OS cell line 33 and recovering endogenous POLK by immunoprecipita4on from cor4cal 4ssue. Compara4ve semiquan4ta4ve western blot of nuclear frac4ons from cor4cal 4ssue showed that POLK levels decreased with increasing age 1 month, versus 10 months and 18 months.…”

“…Since neurons endure an onslaught of a lifetime of DNA damage due to activity, genotoxic agents, and physiological age, we investigated if the levels of endogenous POLK in the mice brain also vary as a function of age. In addition, since POLK showed subcellular relocalization in cancer cells undergoing stress (Temprine et al 2020;Paul et al 2022), we further explored whether POLK in the brain shows similar changes during normative aging in C57BL/6J (RRID:IMSR_JAX:000664) wild-type mice. We performed immunoblots using two independently sourced antibodies against POLK on nuclear and cytoplasmic fractions from cortical tissue of young 1 month, middle age 10 months, and early-old age 18 months mice (Figure 1A).…”

“…We conducted proteomics of the POLK complex from human cell lines 33 and tested if one of the iden4fied DNA damage response proteins PRKDC can be observed in vivo. Upon coimmunostaining in 1-month-old brains, we observed that PRKDC expression was in the nucleus as dis4nct puncta that co-localizes with POLK puncta, which is lost in 10-month and 18-monthold brains (Fig 3).…”

“…There are hints that TLS polymerases including POLK play a role in NHEJ and BER in dividing cells, for example, POLK inserts the correct base opposite the 8oxo-dG lesion and extends from dC:8oxo-dG base pairs (Maddukuri et al 2014). We previously identified DNA repair pathway proteins by performing iPoKD -MS (isolation of proteins on Pol kappa synthesized DNA followed by mass spectrometry), where proteins were captured and bound to the nascent DNA synthesized by Polk in human cell lines (Paul et al 2022). Hence, we tested whether neuronal nuclear POLK is associated with BER and NHEJ pathway proteins identified in our iPoKD-MS data sets.…”