Activity of DNA polymerase kappa across the genome in human fibroblasts
DNA polymerase kappa (PolK) 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 PolK utilizing a PolK - 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 k sequencing (polK-seq). Here we present the human genome maps for pol k activity in an undamaged cell line. We found that pol k activity was enhanced in euchromatin regions, the promoter of genes, and in DNA that is replicated early in S-phase Overall design: Cells treated with EBndG for 4 h, harvested and genomic DNA isolated. EBndG was attached to biotin via Click reaction. DNAwas sheared, EBndG containing DNA isolated with streptavidin beads and NGS libraries prepared