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.
Highlights
DNA-damage tolerance (DDT) pathways protect cells from a wide variety of endogenous and exogenous genotoxic agents by recovering stalled DNA replication caused by insult to DNA
The total amount of ubiquitin in the chains generated by helicase-like transcription factor (HLTF) from dimer to gel top was determined by western blotting with an anti-ubiquitin antibody using commercially available Lys63-linked tetra-ubiquitin as the standard
We found that the ubiquitin ligase activity of HLTF is intrinsically stimulated by dsDNA, and the HIRAN domain prevents direct loading of the catalytic domain on dsDNA by restricting its recruitment to the 3 -OH of primer termini
Summary
DNA-damage tolerance (DDT) pathways protect cells from a wide variety of endogenous and exogenous genotoxic agents by recovering stalled DNA replication caused by insult to DNA. TS is promoted by K63-linked polyubiquitination of PCNA catalyzed by the combined actions of the RAD6-RAD18 complex and another E3–E2 pair, such as helicase-like transcription factor (HLTF) and MMS2UBC13 [1,6,7]. HIRAN is a 3 -OH-binding-module, and its biochemical activity is required for replication fork reversal together with the SWI/SNF helicase domain [9,10,11,12,13,14,15]. The RING domain is required for the polyubiquitination of PCNA [6,7,16,17,18], and is involved in the monoubiquitination of PCNA [19]. HLTF controls many genes involved in a variety of cellular processes through its capacity to bind to DNA sequences [21]
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