Guanine-rich DNA can fold into highly stable non-canonical four-stranded DNA structures called G-quadruplexes. These structures present obstacles for the DNA replication machinery, and it has been hypothesized that both eukaryotic DNA helicases and polymerases have evolved to resolve G4 DNA in vivo. Since the discovery of G-quadruplex DNA in the early 1960's, a number of studies have emerged reporting G-quadruplex DNA unfolding by helicase enzymes and DNA synthesis past G4 by specialized translesion polymerase enzymes. Recently, the discovery of the primase-polymerase PrimPol and its role in G4 bypass has sparked even more excitement in the G-quadruplex and DNA replication fields. This review presents an overview of the molecular interactions of G-quadruplexes with DNA helicases and polymerases implicated in their resolution, with an emphasis on how the regulation and coordination of these enzymes is critical for genome homeostasis. Targeting G4-interacting DNA helicases and polymerases for therapeutic strategies is discussed.

As the powerhouses of the eukaryotic cell, mitochondria must maintain their genomes which encode proteins essential for energy production. Mitochondria are characterized by guanine-rich DNA sequences that spontaneously form unusual three-dimensional structures known as G-quadruplexes (G4). G4 structures can be problematic for the essential processes of DNA replication and transcription because they deter normal progression of the enzymatic-driven processes. In this study, we addressed the hypothesis that mitochondrial G4 is a source of mutagenesis leading to base-pair substitutions. Our computational analysis of 2757 individual genomes from two Italian population cohorts (SardiNIA and InCHIANTI) revealed a statistically significant enrichment of mitochondrial mutations within sequences corresponding to stable G4 DNA structures. Guided by the computational analysis results, we designed biochemical reconstitution experiments and demonstrated that DNA synthesis by two known mitochondrial DNA polymerases (Pol γ, PrimPol) in vitro was strongly blocked by representative stable G4 mitochondrial DNA structures, which could be overcome in a specific manner by the ATP-dependent G4-resolving helicase Pif1. However, error-prone DNA synthesis by PrimPol using the G4 template sequence persisted even in the presence of Pif1. Altogether, our results suggest that genetic variation is enriched in G-quadruplex regions that impede mitochondrial DNA replication.

Helicases are molecular motors that play central roles in nucleic acid metabolism. Mutations in genes encoding DNA helicases of the RecQ and iron–sulfur (Fe–S) helicase families are linked to hereditary disorders characterized by chromosomal instabilities, highlighting the importance of these enzymes. Moreover, mono-allelic RecQ and Fe–S helicase mutations are associated with a broad spectrum of cancers. This review will discuss and contrast the specialized molecular functions and biological roles of RecQ and Fe–S helicases in DNA repair, the replication stress response, and the regulation of gene expression, laying a foundation for continued research in these important areas of study.