ena-STUDY-NOVO NORDISK FOUNDATION CENTER FOR PROTEIN RESEARCH-06-07-2020-11:43:47:523-1151
Here we identify a critical role of the E3 ubiquitin ligase RFWD3 in promoting DNA synthesis across polymerase-stalling lesions. We show that in the absence of RFWD3, ubiquitin signalling and protein accumulation at damaged chromatin are severely compromised.
Lesions on DNA can uncouple DNA synthesis from helicase unwinding, generating stretches of unreplicated single stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication and thereby avoid the generation of DNA double-stranded breaks (DSBs), a major source of genomic instability. Gap filling repair involves either translesion DNA synthesis (TLS) or template switching (TS) to bypass the lesion. At the heart of these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes non-specific ubiquitylation of proteins on ssDNA, to enhance protein accumulation and stimulate gap filling repair. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin, including ubiquitin and proteins known to ubiquitylate and interact with ubiquitylated PCNA. As a result, PCNA ubiquitylation is severely inhibited without RFWD3, and TLS across different DNA lesions drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it triggers widespread ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
Lesions on DNA can uncouple DNA synthesis from helicase unwinding, generating stretches of unreplicated single stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication and thereby avoid the generation of DNA double-stranded breaks (DSBs), a major source of genomic instability. Gap filling repair involves either translesion DNA synthesis (TLS) or template switching (TS) to bypass the lesion. At the heart of these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes non-specific ubiquitylation of proteins on ssDNA, to enhance protein accumulation and stimulate gap filling repair. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin, including ubiquitin and proteins known to ubiquitylate and interact with ubiquitylated PCNA. As a result, PCNA ubiquitylation is severely inhibited without RFWD3, and TLS across different DNA lesions drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it triggers widespread ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.