Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

• Published in: Nucleic acids research 2018-11, Vol.46 (21), p.11340-11356
• Main Authors: Masuda, Yuji, Mitsuyuki, Satoshi, Kanao, Rie, Hishiki, Asami, Hashimoto, Hiroshi, Masutani, Chikahide
• Format: Article
• Language: English
• Subjects: Genome Integrity, Repair and
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gky943

Abstract DNA-damage tolerance protects cells via at least two sub-pathways regulated by proliferating cell nuclear antigen (PCNA) ubiquitination in eukaryotes: translesion DNA synthesis (TLS) and template switching (TS), which are stimulated by mono- and polyubiquitination, respectively. However, how cells choose between the two pathways remains unclear. The regulation of ubiquitin ligases catalyzing polyubiquitination, such as helicase-like transcription factor (HLTF), could play a role in the choice of pathway. Here, we demonstrate that the ligase activity of HLTF is stimulated by double-stranded DNA via HIRAN domain-dependent recruitment to stalled primer ends. Replication factor C (RFC) and PCNA located at primer ends, however, suppress en bloc polyubiquitination in the complex, redirecting toward sequential chain elongation. When PCNA in the complex is monoubiquitinated by RAD6-RAD18, the resulting ubiquitin moiety is immediately polyubiquitinated by coexisting HLTF, indicating a coupling reaction between mono- and polyubiquitination. By contrast, when PCNA was monoubiquitinated in the absence of HLTF, it was not polyubiquitinated by subsequently recruited HLTF unless all three-subunits of PCNA were monoubiquitinated, indicating that the uncoupling reaction specifically occurs on three-subunit-monoubiquitinated PCNA. We discuss the physiological relevance of the different modes of the polyubiquitination to the choice of cells between TLS and TS under different conditions.