Abstract
Translesion synthesis is a fundamental biological process that enables DNA replication across lesion sites to ensure timely duplication of genetic information at the cost of replication fidelity, and it is implicated in development of cancer drug resistance after chemotherapy. The eukaryotic Y-family polymerase Rev1 is an essential scaffolding protein in translesion synthesis. Its C-terminal domain (CTD), which interacts with translesion polymerase ζ through the Rev7 subunit and with polymerases κ, ι, and η in vertebrates through the Rev1-interacting region (RIR), is absolutely required for function. We report the first solution structures of the mouse Rev1 CTD and its complex with the Pol κ RIR, revealing an atypical four-helix bundle. Using yeast two-hybrid assays, we have identified a Rev7-binding surface centered at the α2-α3 loop and N-terminal half of α3 of the Rev1 CTD. Binding of the mouse Pol κ RIR to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn α-helix, with the helix stabilized by an N-terminal cap. RIR binding also induces folding of a disordered N-terminal loop of the Rev1 CTD into a β-hairpin that projects over the shallow α1-α2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev1 CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis.
Highlights
Translesion synthesis requires the scaffolding function of the Rev1 C-terminal domain (CTD)
Binding of the mouse Pol Rev1-interacting region (RIR) to the Rev1 CTD induces folding of the disordered RIR peptide into a three-turn ␣-helix, with the helix stabilized by an N-terminal cap
Conserved and Acquired Roles of Rev1 CTD in Translesion Synthesis—Rev1 is a unique member of the eukaryotic Y-family polymerases and is conserved from yeast to human
Summary
Results: We determined the structures of the Rev CTD and its complex with Pol and mapped its Rev7-binding surface. Conclusion: Distinct surfaces of the Rev CTD separately mediate the assembly of extension and insertion translesion polymerase complexes. RIR binding induces folding of a disordered N-terminal loop of the Rev CTD into a -hairpin that projects over the shallow ␣1-␣2 surface and creates a deep hydrophobic cavity to interact with the essential FF residues juxtaposed on the same side of the RIR helix. Our combined structural and biochemical studies reveal two distinct surfaces of the Rev CTD that separately mediate the assembly of extension and insertion translesion polymerase complexes and provide a molecular framework for developing novel cancer therapeutics to inhibit translesion synthesis. Translesion synthesis has been shown to function in replication-coupled DNA interstrand cross-link repair in a Pol -dependent manner [9], and monoubiquitinated Rev has been found to interact with the Fanconi anemia core complex, providing a critical link between the Fanconi anemia pathway and
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