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.
Highlights
Cells in the human body are continuously exposed to a multitude of endogenous and exogenous agents, which can produce a broad range of DNA lesions, compromising the cell functionality
The analysis of DNA damage response (DDR) factors binding to DNA, and in particular to DNA lesions, may be performed in situ, i.e., in intact cells, either: (i) after appropriate cell/tissue fixation that is necessary to retain these proteins at their activity site, or (ii) directly on living cells, providing that DNA and proteins of interest may be visualized under the microscope
The techniques above described are characterized by limited spatial resolution, or do not allow the kinetic analysis of the protein binding to DNA, because they provide a freeze-frame; they are unsuitable for studying the dynamic binding of repair factors to DNA in living cells
Summary
Cells in the human body are continuously exposed to a multitude of endogenous and exogenous agents, which can produce a broad range of DNA lesions, compromising the cell functionality. From early studies using in vitro approaches based on purified proteins in reconstituted systems, components of DDR pathways were identified These methodologies were later considered incomplete and insufficient to address specific questions regarding the spatiotemporal characterization of these processes, taking into account the physiology and ultrastructure complexity of the cell [13]. For this reason, it has been necessary to develop new strategies to probe the formation of DNA-protein complexes in situ, to investigate the recruitment kinetics of DDR proteins at DNA damage sites, and to evaluate where they interact with DNA. A comparison of these techniques in terms of the advantages and limitations of each approach will be discussed
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