Selective Binding Of Human Rev1 With G-Quadruplex Dna Is Determined By A Region Unique To Higher Eukaryotes

Rev1 is a special translesion synthesis (TLS) DNA polymerase that uses a unique protein-template mechanism to bypass DNA lesions like abasic sites and guanine adducts. Earlier work from our laboratory revealed that human Rev1 (hRev1) was capable of disrupting G-quadruplex (G4) structures and preventing their refolding, and that this was independent of its nucleotidyl transfer activity. In the present study, we investigated the G4 binding specificity of hRev1 further, and demonstrated that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Site-directed mutagenesis helped identify specific amino acids in the insert-2 region of the protein to be important for high G4 DNA binding affinity. Using a forward mutagenesis assay with plasmids containing the supF gene with an engineered G4 insert, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence lacking the G4 insert. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >800-fold over control and deletions upstream of the G4 site more than doubled. Complementation experiments further showed that these mutagenic effects could be partially rescued by wild-type and E466K hRev1, while the E466A and Y470A mutants failed to suppress the PDS-induced increase in G4 mutation frequency. These findings have implications for the role of insert-2, a motif conserved in animals but not yeast, trypanosomes, or plants, in Rev1-mediated suppression of mutagenesis during G4 replication.