REV1 promotes PCNA monoubiquitylation through interacting with ubiquitylated RAD18

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Translesion DNA synthesis (TLS) is crucial for DNA damage tolerance, impacting genome mutagenesis and chromatin integrity.
• Proliferating cell nuclear antigen (PCNA) monoubiquitylation is a key determinant in selecting TLS pathways.
• The precise regulatory mechanisms governing TLS pathway choice remain incompletely understood.

Purpose of the Study:

• To elucidate the regulatory role of TLS polymerase REV1 in PCNA monoubiquitylation.
• To investigate the molecular mechanism by which REV1 influences PCNA monoubiquitylation and subsequent TLS.
• To determine the specific DNA damaging agents that trigger REV1-mediated PCNA monoubiquitylation.

Main Methods:

• UV radiation, hydroxyurea, mitomycin C, and methyl methanesulfonate (MMS) treatments in cellular models.
• Analysis of PCNA monoubiquitylation status.
• Investigation of protein-protein interactions between REV1 and RAD18 using co-immunoprecipitation assays.
• Assessment of RAD18 recruitment to chromatin.

Main Results:

• TLS polymerase REV1 was found to promote PCNA monoubiquitylation following UV radiation.
• REV1 enhances the interaction with ubiquitylated RAD18, facilitating the release of nonubiquitylated RAD18 for chromatin recruitment.
• This stimulatory effect was observed after exposure to hydroxyurea and mitomycin C, but not MMS.
• The absence of ubiquitylated RAD18 after MMS treatment correlated with the lack of REV1-mediated stimulation.

Conclusions:

• REV1 plays a significant role in promoting PCNA monoubiquitylation, a critical step in TLS pathway regulation.
• The interaction between REV1 and ubiquitylated RAD18 is a key mechanism for facilitating RAD18's function in TLS.
• The findings highlight a pathway-specific regulation of TLS, dependent on the type of DNA damage and the resulting ubiquitylation status of RAD18.