In Situ Analysis of DNA-Protein Complex Formation upon Radiation-Induced DNA Damage

Abstract

The importance of determining at the cellular level the formation of DNA–protein complexes after radiation-induced lesions to DNA is outlined by the evidence that such interactions represent one of the first steps of the cellular response to DNA damage. These complexes are formed through recruitment at the sites of the lesion, of proteins deputed to signal the presence of DNA damage, and of DNA repair factors necessary to remove it. Investigating the formation of such complexes has provided, and will probably continue to, relevant information about molecular mechanisms and spatiotemporal dynamics of the processes that constitute the first barrier of cell defense against genome instability and related diseases. In this review, we will summarize and discuss the use of in situ procedures to detect the formation of DNA-protein complexes after radiation-induced DNA damage. This type of analysis provides important information on the spatial localization and temporal resolution of the formation of such complexes, at the single-cell level, allowing the study of heterogeneous cell populations.

Methods to Study Intracellular Movement and Localization of the Nucleotide Excision Repair Proteins at the DNA Lesions in Mammalian Cells

“…Similar protocols for selective depletion of unrelated protein molecules using a mild treatment with DNase or RNase have been used to improve the detection of the XPG, DDB2, XPC ( Dutto et al, 2017 ), and Ku80 ( Britton et al, 2013 ) at the lesion sites. Along with this, the live-cell imaging revealed that NER is highly dynamic process with a continuous exchange of the repair factors during the repair reaction ( Vermeulen, 2011 ; Ticli and Prosperi, 2019 ).…”

“…The progress in understanding mammalian NER was relatively slower than other DNA repair pathways, largely because of a conceptual and some technical hurdles. In the early stage, the specific biochemical reactions carried out by a majority of the mammalian NER proteins were identified using the in vitro DNA repair assays with purified proteins and from their equivalent proteins in bacteria and yeast ( Aboussekhra et al, 1995 ; Sugasawa et al, 1998 ; Ticli and Prosperi, 2019 ). However, these techniques lack the spatio-temporal properties ( Ticli and Prosperi, 2019 ).…”

“…In the early stage, the specific biochemical reactions carried out by a majority of the mammalian NER proteins were identified using the in vitro DNA repair assays with purified proteins and from their equivalent proteins in bacteria and yeast ( Aboussekhra et al, 1995 ; Sugasawa et al, 1998 ; Ticli and Prosperi, 2019 ). However, these techniques lack the spatio-temporal properties ( Ticli and Prosperi, 2019 ). Hence, the bigger challenge was to understand the dynamics of their rapid recruitment to the lesion site and coordination of sequential steps of NER along with other proteins in mammalian cells.…”

“…Despite its numerous advantages, including the low cost and use of basic equipment, the local irradiation technique needed optimization for the visualization of different repair proteins at damage site in different cell lines ( Ticli and Prosperi, 2019 ). For instance, we observed that the uniform distribution of signal for PARP1 throughout the nucleus before irradiation did not change after local UVC irradiation.…”

“…To stimulate more studies in NER, there is a need for widespread accessibility to techniques that will allow many more laboratories to explore this repair pathway in different models. In this context, some of the leading techniques in the field, such as live cell microscopy imaging using fluorescent tagged proteins ( Vermeulen, 2011 ; Ticli and Prosperi, 2019 ) or UVC laser with quartz optics to cause damage in defined sub-nuclear zones ( Dinant et al, 2007 ) have produced excellent data and will continue to be useful in future. However, these techniques need specialized reagents, engineered cell lines and expensive instruments which may not be available in most laboratories.…”