Human Rev1 relies on insert-2 to promote selective binding and accurate replication of stabilized G-quadruplex motifs

Amit Ketkar, Lane Smith, Callie Johnson, Alyssa Richey, Makayla Berry, Jessica H. Hartman, Leena Maddukuri, Megan R. Reed, Julie E.C. Gunderson, Justin W.C. Leung, Robert L. Eoff

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)2065-2084
Number of pages20
JournalNucleic acids research
Volume49
Issue number4
DOIs
StatePublished - Feb 26 2021
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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