Timeless couples G-quadruplex detection with processing by DDX11 helicase during DNA replication

Regions of the genome with the potential to form secondaryDNAstructures pose a frequent and significant impediment toDNAreplication and must be actively managed in order to preserve genetic and epigenetic integrity. How the replisome detects and responds to secondary structures is poorly understood. Here, we show that a core component of the fork protection complex in the eukaryotic replisome, Timeless, harbours in its C-terminal region a previously unappreciatedDNA-binding domain that exhibits specific binding to G-quadruplex (G4)DNAstructures. We show that this domain contributes to maintaining processive replication through G4-forming sequences, and exhibits partial redundancy with an adjacentPARP-binding domain. Further, this function of Timeless requires interaction with and activity of the helicaseDDX11. Loss of both Timeless andDDX11 causes epigenetic instability at G4-forming sequences andDNAdamage. Our findings indicate that Timeless contributes to the ability of the replisome to sense replication-hindering G4 formation and ensures the prompt resolution of these structures byDDX11 to maintain processiveDNAsynthesis.