Using a Human Papillomavirus Model to Study DNA Replication and Repair of Wild Type and Damaged DNA Templates in Mammalian Cells

Abstract

Human papillomaviruses have 8kbp DNA episomal genomes that replicate autonomously from host DNA. During initial infection, the virus increases its copy number to 20–50 copies per cell, causing torsional stress on the replicating DNA. This activates the DNA damage response (DDR) and HPV replicates its genome, at least in part, using homologous recombination. An active DDR is on throughout the HPV life cycle. Two viral proteins are required for replication of the viral genome; E2 binds to 12bp palindromic sequences around the A/T rich origin of replication and recruits the viral helicase E1 via a protein–protein interaction. E1 forms a di-hexameric complex that replicates the viral genome in association with host factors. Transient replication assays following transfection with E1–E2 expression plasmids, along with an origin containing plasmid, allow monitoring of E1-E2 replication activity. Incorporating a bacterial lacZ gene into the origin plasmid allows for the determination of replication fidelity. Here we describe how we exploited this system to investigate replication and repair in mammalian cells, including using damaged DNA templates. We propose that this system has the potential to enhance the understanding of cellular components involved in DNA replication and repair.

DNA Damage Repair-Related Genes Signature for Immune Infiltration and Outcome in Cervical Cancer

“…When HPV infects epithelial cells, a variety of pathways or mechanisms related to DNA damage repair are encouraged to ensure the completion of the virus life cycle. E6 and E7 are the most important proteins in HPV, which can bind and inhibit p53 and pRb respectively ( Spriggs and Laimins, 2017 ; Das et al, 2020 ). The result is that HPV can activate DNA damage repair mechanism for HPV self-preservation, thus hijacking the unit, which will lead to the secondary inhibition of DNA damage repair.…”

Recognition of DNA Lesions

“…DNA damage recognition is a very complex cellular process that consists of detecting rare modifications to DNA in a large pool of intact genomic material, and the efficiency of this first step is critical for downstream repair processes and their consequences. This Special Issue, which includes both original research [8][9][10][11][12][13] and review articles [14][15][16][17][18][19][20], focuses on the various strategies used by cells to recognize DNA damage, but also identifies new causative agents of DNA damage, explores novel mechanisms involved in regulating DNA repair pathways, and investigates the consequences of deficient repair pathways on human health and the potential of repair factors as drug targets.…”

“…Finally, this Special Issue also includes two articles that present important developments and approaches in the study of DNA repair processes [13,16]. Inagawa et al report the development of a novel Förster resonance energy transfer (FRET)-based assay to probe the DNA binding and release kinetics of the Ku70/Ku80 heterodimer to DNA ends [13].…”

“…This new assay allows DNA binding affinities to Ku to be measured in the context of a DNA-dependent kinase (DNA-PK) holocomplex, which paves the way for the future study of larger, physiologically more relevant NHEJ complexes to DNA ends. Das et al developed a human papillomavirus-based model to determine both DNA replication and repair in mammalian cells and discuss the potential of this system to decipher key DNA repair mechanisms [16].…”