DNA Fiber Assay upon Treatment with Ultraviolet Radiations

Abstract

Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV type C (UVC) exposure, the DNA fiber assay can be used to analyse the new origin firing and DNA synthesis rate ( Jackson et al., 1998 ; Merrick et al., 2004 ; Alfano et al., 2016 ). The DNA fiber assay technique was conceived in the 90s and then further developed through the use of thymidine analogues (such as CldU and IdU), which are incorporated into the nascent DNA strands. By treating the cells in sequential mode with these analogues, which can be visualized through specific antibodies carrying different fluorophores, it is possible to monitor the replication fork activity and assess how this is influenced by UV radiations or others agents.

Keywords: Cell cycle checkpoints; DNA damage; DNA fiber assay; Halogenated pyrimidines; Ultra Violet radiation.

Figure 1.. Schematic overview of the protocol

Figure 2.. Representative kinds of DNA fibers identified by using this protocol.

The sequential labelling provides a snapshot of different DNA replication steps: 1) Ongoing forks can appear as in a (in the case of unidirectional progression through the replication fork) or as in b (in the case of bidirectional progression through the replication fork). 2) Termination events can appear as in a (as an only-green line in the case of a replication origin in which DNA synthesis terminated while the cells were still undergoing incubation with the first label), or as in b (as adjoined red-green-red signals with divergent trend). 3) New fired origins appear as shown in the lower panel of the figure. These origins were fired during incubation with the second label (consisting of an only-red line, according to the protocol herein described).