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.
Highlights
Human papillomaviruses (HPV) are causative agents in around 5% of all cancers, including the majority of cervical and oropharyngeal cancers [1]
We demonstrated that SIRT1 regulates E1–E2 replication; as well as binding to TopBP1, it binds to E1 and E2 proteins and is part of the E1–E2 replication complex (as defined by chromatin immunoprecipitation (ChIP)) [47]
In support of the switch from reverse branch migration and homologous recombination (HR) to break-induced replication (BIR) in the absence of Werner helicase (WRN), more recently we demonstrated a significant increase of MUS81 recruitment to E1–E2-replicating DNA in the absence of WRN, as determined by ChIP; the FEN1 endonuclease has enhanced recruitment in the absence of WRN (Das, Morgan, and Morgan, unpublished)
Summary
Human papillomaviruses (HPV) are causative agents in around 5% of all cancers, including the majority of cervical and oropharyngeal cancers [1]. The paused forks trigger the DDR via increased stretches of ssDNA that activate ATR and the recruitment of the MRE11-RAD50-NBS1 (MRN) complex that mediates HR and activates ATM; HPV-replicating DNA recruits MRN components [24,40,41,42,48,49,50] This activation of the DDR is prevented from inducing a cell cycle arrest in the infected cells by the action of the E6 and E7 viral oncogenes, which disrupt. In HPV16 genomes that lack E6 and E7 expression (via introduction of stop codons), we demonstrated that the DDR is no different from that observed in wild-type HPV16 genome cells, but that the cells grow more slowly [52] This result demonstrated the potential of the process of viral replication to activate the DDR in the absence of E6 and E7, and that E6 and E7 are important for promoting cell proliferation in the presence of the DDR induced by active replication. The purpose of this review is to stimulate the idea that this system can be more widely used to study DNA lesions and DDR proteins during replication
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