RESUMO
Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling 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 ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
Assuntos
Cromatina/metabolismo , Quebras de DNA de Cadeia Simples , Reparo do DNA , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Linhagem Celular Tumoral , Cromatina/genética , DNA Polimerase Dirigida por DNA/metabolismo , Feminino , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Especificidade por Substrato , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Xenopus laevisRESUMO
DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.
Assuntos
Reparo do DNA , Replicação do DNA , DNA/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , DNA/química , DNA/genética , Feminino , Masculino , Conformação de Ácido Nucleico , Complexo de Endopeptidases do Proteassoma/genética , Domínios e Motivos de Interação entre Proteínas , Proteólise , Células Sf9 , Relação Estrutura-Atividade , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.