DPCs represent one of the most common and least-researched types of DNA damage. Their research intensified only after the discovery of the DNA-dependent proteases responsible for their repair, namely wss1 in yeast and SPRTN in mammals. It has been proven that DPCs are repaired by three mechanisms: direct hydrolysis of the covalent bond, excision of the damaged part of DNA, and proteolytic degradation of the protein part of the DPC lesion. However, the interdependence of these mechanisms is unknown. This dissertation presents the development of a new assay for the direct isolation of DPCs, called the STAR assay, which successfully isolates DPCs of different origins, under physiologically relevant conditions, while being sensitive enough to enable the visualization of peptides covalently bound to DNA resulting from proteolytic degradation. Using the assay, we determined the steps in the repair mechanism of DPCs, which are first proteolytically degraded by the action of SPRTN, during which covalently bound peptides remain on the DNA. Peptides are then removed by other repair mechanisms. The demonstrated dynamics of repair correlates with DNA damage signaling through the ATM/H2Ax axis, which is a newly described role for both proteins and is independent of DNA breaks. γH2Ax is crucial for binding DNA repair factors. The key evidence that proteolytic degradation is the first step in the repair of DPCs comes from the lack of activation of this signaling pathway in the absence of proteolysis, and it has been shown that DPCs can inhibit the activation of DNA strand break signaling if they are located near the break site. The STAR essay proved to be an extremely useful tool for studying the dynamics of the formation and repair of DPCs, which will enable progress in their study.