Spratt, T. E. | bioRxiv Science

bioRxiv2024

Activity of DNA polymerase κ across the genome in human fibroblasts

DNA polymerase {kappa} (Pol {kappa}) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replicating repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Pol {kappa} utilizing a Pol {kappa} - specific substrate, N2-(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200-500bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)- catalyzed alkyne-azide cycloaddition reaction, and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing (NGS) to generate genome-wide repair maps. We have termed this method polymerase {kappa} sequencing (polK-seq). Here we present the human genome maps for pol {kappa} activity in an undamaged cell line. We found that pol {kappa} activity was enhanced in euchromatin regions, the promoter of genes, and in DNA that is replicated early in S-phase.

cell biology↗

bioRxiv2022

Translesion DNA synthesis polymerase kappa functions in the nucleolar stress response

DNA polymerase kappa (Pol{kappa}) has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences and nucleotide excision repair. However, the mechanisms regulating Pol{kappa}s cellular activities are unknown. Since all polymerases insert the canonical deoxynucleotide triphosphates, it is difficult to determine which polymerase is active at a particular genomic site. To counter this difficulty, we utilized the selective Pol{kappa} substrate, N2-(4-ethynylbenyl)-2-deoxyguanosine (EBndG), as bait to capture proteins associated with Pol{kappa} synthesized DNA. Here we show that, Pol{kappa} is active in the nucleolus and Pol{kappa} synthesized DNA are enriched with nucleolar proteins. Exposure of cells to benzo[a]pyrene diol epoxide (BPDE) induced hallmarks of nucleolar stress, increased Pol{kappa} stability and nucleolar activity. Other agents that induce nucleolar stress such as mitomycin C, cisplatin and actinomycin D also increased Pol{kappa}s nucleolar activity. The Pol{kappa} activity was cell-cycle independent and dependent on PCNA ubiquitination. In addition, we identified that the expression and activity of Pol{kappa} is regulated by the polycomb complex protein Ring Finger Protein 2 (RNF2) and by poly(ADP)-ribose polymerase 1 (PARP1) catalyzed PARylation. This study provides insight into the novel role of Pol{kappa} in ribosomal RNA synthesis, in maintaining ribosomal DNA integrity after DNA damage thus protecting the cells from nucleolar stress.

molecular biology↗