High Affinity binding of Yeast Nucleolin Nsr1 to Co-transcriptionally Formed G4 DNA Obstructs Replication and Elevates Genome Instability

ABSTRACTA significant increase in genome instability is associated with the conformational shift of a guanine-run-containing DNA strand into the four-stranded G-quadruplex (G4) DNA. The mechanism underlying the recombination and genome rearrangements following the formation of G4 DNA in vivo has been difficult to elucidate but has become better clarified by the identification and functional characterization of several key G4 DNA-binding proteins. Mammalian nucleolin NCL is a highly specific G4 DNA-binding protein with a well-defined role in the transcriptional regulation of genes with associated G4 DNA-forming sequence motifs at their promoters. The consequence of the in vivo interaction between G4 DNA and nucleolin in respect to the genome instability has not been previously investigated. We show here that G4 DNA-binding is a conserved function in the yeast nucleolin Nsr1. Furthermore, we demonstrate that the Nsr1-G4 DNA complex formation results in replication obstruction and is a major factor in inducing the genome instability associated with the co-transcriptionally formed G4 DNA in the yeast genome. The G4-associated genome instability and the G4 DNA-binding in vivo requires the arginine-glycine-glycine (RGG) repeats located at the C-terminus of the Nsr1 protein. Nsr1 with the deletion of RGG domain supports normal cell growth and is sufficient for its pre-rRNA processing function. However, the truncation of RGG domain of Nsr1 significantly weakens its interaction with G4 DNA in vitro and in vivo and restores unhindered replication, overall resulting in a sharp reduction in the G4-associated genome instability. Our data suggest that the interaction between Nsr1 with the intact RGG repeats and G4 DNA impairs genome stability by precluding the access of G4-resolving proteins and obstructing replication.AUTHOR SUMMARYGenome instability is uniquely elevated at sequences containing multiple runs of guanines, which can fold into the unusual, four-stranded G-quadruplex (G4) DNA. In this study, we report a novel finding that a highly conserved G4 DNA binding protein Nsr1 can elevate the rate of recombination and chromosomal rearrangement occurring at a G4 DNA-forming sequence in the genome of Saccharomyces cerevisiae. The elevated genome instability requires the C-terminally located RGG domain of Nsr1, which supports the high-affinity interaction between the protein and G4 DNA. The connection between G4-specific genome instability and the function of Nsr1 to form stable complex with G4 DNA led to the hypothesis that the high-affinity Nsr1-G4 DNA complexes can become a barrier to replication. We demonstrate here that the presence of Nsr1 in fact slows the replication past a G4 DNA-containing genomic site and that the RGG domain is required to facilitate such replication block.