Activity of DNA polymerase κ across the genome in human fibroblasts

Abstract

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.