RNA-splicing factor SART3 regulates translesion DNA synthesis

Area of Science:

• Molecular Biology
• DNA Repair Mechanisms
• Cancer Biology

Background:

• Translesion DNA synthesis (TLS) is crucial for replicating damaged DNA using specialized polymerases.
• DNA polymerase eta (Polη) plays a key role in TLS across UV-induced DNA lesions, and its dysfunction is linked to skin cancer.
• The precise mechanism for recruiting Polη to stalled replication forks remains incompletely understood.

Purpose of the Study:

• To identify novel regulators of Polη recruitment to stalled replication forks.
• To elucidate the role of SART3 in DNA damage tolerance and UV response.
• To investigate the functional consequences of SART3 mutations in cancer.

Main Methods:

• Affinity purification to isolate Polη-containing complexes.
• Co-immunoprecipitation assays to study protein-protein interactions.
• Western blotting to assess PCNA monoubiquitination and RPA focus formation.
• Cellular assays to evaluate UV sensitivity and mutation frequency.

Main Results:

• SART3 was identified as a novel interactor of Polη and RAD18, crucial for Polη recruitment after UV exposure.
• SART3 homodimerization promotes Polη/RAD18 interaction and PCNA monoubiquitination, a key TLS event.
• SART3 depletion impairs UV-induced ssDNA generation, RPA focus formation, and Polη recruitment, leading to increased UV sensitivity and mutation frequency.
• Cancer-associated SART3 mutations were found to reduce its stimulatory effect on PCNA monoubiquitination.

Conclusions:

• SART3 acts as a critical regulator of Polη/RAD18 association, independent of its RNA-binding function.
• SART3 plays a significant role in protecting cells against UV-induced DNA damage.
• SART3 is a potential therapeutic target for enhancing cancer treatment efficacy.