Plant-specific and conserved mechanisms of the polymerase-associated factor 1 complex in replication stress responses

Abstract DNA replication stress threatens genome stability in eukaryotes. The evolutionarily conserved kinase WEE1 is essential for the activation of the replication stress response. Although the polymerase-associated factor 1 complex (PAF1C) is highly conserved in eukaryotes, its role in the DNA replication stress response remains unclear. Here, we show that Arabidopsis PAF1C is essential for replication stress response. PAF1C-deficient mutants exhibit hypersensitivity to hydroxyurea (HU)-induced replication stress. Mechanistically, we uncover a plant-specific regulatory pathway in which WEE1 interacts with and phosphorylates the PAF1 subunit within its unique N-terminal domain, thereby preventing PAF1 polyubiquitination and subsequent proteasomal degradation to ensure PAF1 accumulation under stress. Genetically, overexpression of a phospho-mimetic PAF1 variant suppresses the HU hypersensitivity of wee1 , revealing that PAF1 acts downstream of WEE1. However, the WEE1-PAF1 regulatory axis is absent in yeast, indicating its lineage-specific innovation. Further studies reveal that the replication factor C (RFC) complex interacts with and recruits PAF1 to the stalled replication forks. PAF1 then sequentially recruits the E2 ubiquitin-conjugating enzymes UBC1/2 and the E3 ubiquitin ligases HUB1/2 to promote histone H2B monoubiquitination (H2Bub), thereby facilitating replication fork stability. This RFC-dependent recruitment mechanism is conserved in yeast. Collectively, this study suggests that PAF1 regulates replication stress responses by integrating a plant-specific protein stability control mechanism (WEE1-PAF1) with a conserved recruitment mechanism (RFC-PAF1-UBC1/2-HUB1/2), uncovering a novel function of PAF1C and revealing new mechanisms of WEE1 and the RFC complex.