| Summary: | <p><strong><em>Introduction:</em></strong> DNA-Protein Crosslinks (DPCs) are a form of DNA damage caused by covalent attachment between proteins and DNA, which can lead to replication fork stalling, disruption of cell division, and ultimately cell death. DPCs repair is critical for maintaining genome stability, however, the knowledge of the DPC repair pathways is still limited. SPRTN, a DNA-dependent metalloprotease, has been identified as the major protease in repairing DPCs. The function of SPRTN in DPC repair is regulated by ubiquitination. The regulation of SPRTN activity, in particular the N-terminal SPRTN catalytic region (SprT) core, remains largely unknown.</p>
<p><strong><em>Aim:</em></strong> The fluorescently labeled histone H1 is used as the model substrate for SPRTN proteolysis. This project is designed to study the effect of ubiquitin and ubiquitin-like proteins on the activation of SPRTN SprT core towards the model H1-DPC substrate. We are also aiming to explore the effect of SPRTN proteolysis on H1 with different post-translational modifications.</p>
<p><strong><em>Results:</em></strong> SPRTN activity towards the model H1-DPC substrate is enhanced by the addition of ubiquitin chains on substrate, but not the ubiquitin-like protein ISG15. To better understand SPRTN activation by modified substrates, H1 constructs fused with mono-ubiquitin, linear tetra-ubiquitin or 2xISG15 were designed and purified. Surprisingly, the complete cleavage of tetra-ubiquitin-modified H1, but not the 2xISG15-H1 fusion, can be observed within 5 min by the SprT core, indicating the much more efficient activating effect of Ub chains on SPRTN proteolysis.</p>
<p><strong><em>Conclusion:</em></strong> Our study has demonstrated that ubiquitination of the DPC substrates is a key pathway in regulating SPRTN activity. These findings will not only contribute to the understanding of the mechanisms by which SPRTN proteolysis in DPCs repair, but also provide a powerful <em>in vitro</em> approach for drug screening to develop new cancer treatment strategies.</p>
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