Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

Abstract

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Beta Human Papillomavirus 8E6 Attenuates Non-Homologous End Joining by Hindering DNA-PKcs Activity

“…An alternative explanation consistent with the data shown in Figure 1 is that β-HPV 8E6 reduced the frequency of NHEJ. When repair complexes are not resolved, the repair proteins spread along nearby chromatin producing larger/brighter foci [ 58 , 59 , 60 ]. As a result, foci intensity was used as an indicator of repair efficiency (brighter foci indicate more persistent lesions).…”

Beta human papillomavirus 8 E6 allows colocalization of non-homologous end joining and homologous recombination repair factors

“…After washing, the cells were stained with 10 μM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described [72]. Cyclin E intensity was measured for each cell.…”

High Risk α-HPV E6 Impairs Translesion Synthesis by Blocking POLη Induction

“…The nucleus was stained using DAPI (300 nM). Foci analysis was conducted using ImageJ as described in [70].…”