Article ; Online: Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs.
2021 Volume 49, Issue 4, Page(s) 2065–2084
Abstract: We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for ... ...
| Abstract | We previously reported that human Rev1 (hRev1) bound to a parallel-stranded G-quadruplex (G4) from the c-MYC promoter with high affinity. We have extended those results to include other G4 motifs, finding that hRev1 exhibited stronger affinity for parallel-stranded G4 than either anti-parallel or hybrid folds. Amino acids in the αE helix of insert-2 were identified as being important for G4 binding. Mutating E466 and Y470 to alanine selectively perturbed G4 binding affinity. The E466K mutant restored wild-type G4 binding properties. Using a forward mutagenesis assay, we discovered that loss of hRev1 increased G4 mutation frequency >200-fold compared to the control sequence. Base substitutions and deletions occurred around and within the G4 motif. Pyridostatin (PDS) exacerbated this effect, as the mutation frequency increased >700-fold over control and deletions upstream of the G4 site more than doubled. Mutagenic replication of G4 DNA (±PDS) was partially rescued by wild-type and E466K hRev1. The E466A or 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 vertebrates but not yeast or plants, in Rev1-mediated suppression of mutagenesis during G4 replication. |
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| MeSH term(s) | Cell Line ; DNA/chemistry ; DNA/metabolism ; DNA Replication ; DNA-Directed DNA Polymerase/metabolism ; G-Quadruplexes ; Genes, myc ; Humans ; Models, Molecular ; Mutation ; Nucleotide Motifs ; Nucleotidyltransferases/chemistry ; Nucleotidyltransferases/genetics ; Nucleotidyltransferases/metabolism ; Protein Binding | |||||
| Chemical Substances | DNA (9007-49-2) ; Nucleotidyltransferases (EC 2.7.7.-) ; REV1 protein, human (EC 2.7.7.-) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) | |||||
| Language | English | |||||
| Publishing date | 2021-02-09 | |||||
| Publishing country | England | |||||
| Document type | Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S. | |||||
| ZDB-ID | 186809-3 | |||||
| ISSN | 1362-4962 ; 1362-4954 ; 0301-5610 ; 0305-1048 | |||||
| ISSN (online) | 1362-4962 ; 1362-4954 | |||||
| ISSN | 0301-5610 ; 0305-1048 | |||||
| DOI | 10.1093/nar/gkab041 | |||||
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| Database | MEDical Literature Analysis and Retrieval System OnLINE |
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