The dual ubiquitin binding mode of SPRTN secures rapid spatiotemporal proteolysis of DNA-protein crosslinks

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DNA-protein crosslinks (DPCs) are toxic DNA lesions arising from endogenous or exogenous sources.
• Unrepaired DPCs are linked to severe health issues including cancer, neurodegeneration, and premature aging.
• The SPRTN protease and 26S proteasome are key players in DPC repair, with proteasome recognition dependent on ubiquitination.

Purpose of the Study:

• To elucidate the mechanism by which the SPRTN protease achieves substrate specificity for DPCs.
• To identify the molecular determinants of SPRTN's interaction with DPCs.

Main Methods:

• Biochemical assays
• Biophysical techniques
• Structural biology approaches
• Ubiquitin-binding assays

Main Results:

• A novel ubiquitin-binding domain, the Ubiquitin interface of SprT Domain (USD), was identified in the N-terminal catalytic region of SPRTN.
• The USD domain directly binds to ubiquitin chains.
• SPRTN exhibits a 67-fold increase in proteolysis activity towards polyubiquitinated DPCs compared to unmodified DPCs.

Conclusions:

• Ubiquitination serves as the critical signal for SPRTN's substrate specificity and efficient proteolysis of DPCs.
• The USD domain is essential for SPRTN's recognition and processing of ubiquitinated DPCs.
• This discovery provides a molecular basis for understanding DPC repair pathways and their implications in disease.