Activity of DNA polymerase κ across the genome in human fibroblasts

• 成果类型: Article
• 署名作者: Torres, Mariela C.; Rebok, Abbey; Sun, Dongxiao; Spratt, Thomas E.
• 署名单位: Pennsylvania Commonwealth System of Higher Education (PCSHE); Pennsylvania State University; Penn State Health; Pennsylvania Commonwealth System of Higher Education (PCSHE); Pennsylvania State University; Penn State Health; University of North Carolina; University of North Carolina Chapel Hill
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-11606
• DOI: 10.1073/pnas.2403130121
• 发表日期: 2024-07-09
• 关键词: repair; eta

摘要

DNA polymerase kappa (Pol kappa ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Pol kappa utilizing a Pol kappa -specific substrate, N 2- (4- ethynylbenzyl)- 2 '- deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp 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 to generate genome- wide repair maps. We have termed this method polymerase kappa sequencing. 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 GC- rich regions, euchromatin regions, the promoter of genes, and in DNA that is replicated early in the S phase.