Pif1 DNA helicase is a potent unwinder of G-quadruplex (G4) structures in vitro and functions to maintain genome stability at G4 sequences in Saccharomyces cerevisiae. Here, we developed and utilized a live-cell imaging approach to quantitatively measure the progression rates of single replication forks through different G4 containing sequences in individual yeast cells. We show that in the absence of Pif1, replication rates through specific lagging strand G4 sequences in vivo is significantly decreased. In contrast, we found that in the absence of Pif1, replication rates through the same G4s on the leading strand are not decreased relative to the respective WT strains, showing that Pif1 is essential only for efficient replication through lagging strand G4s. Additionally, we show that a canonical PIP sequence in Pif1 interacts with PCNA and that replication through G4 structures is significantly slower in the absence of this interaction in vitro and in vivo. Thus, Pif1–PCNA interaction is essential for optimal replisome progression through G4 sequences, highlighting the importance of coupling between Pif1 activity and replisome progression during yeast genome replication.

We describe a simple and direct approach to measure the progression of single DNA replication forks in living cells by monitoring two fluorescently labeled loci downstream of an origin of replication. We employ this approach to investigate the roles of several leading and lagging strand factors in overall replisome function and show that fork progression is strongly dependent on proper maturation of Okazaki fragments. We also demonstrate how related cellular phenotypes, such as cell-cycle progression and the dynamics of sister chromatid cohesion, may be simultaneously monitored and correlated to DNA replication at the single-cell level.

Dynamic switching of PCNA-partner interactions is essential for normal DNA replication and repair in yeast.

The homotrimeric sliding clamp proliferating cell nuclear antigen (PCNA) mediates Okazaki fragment maturation through tight coordination of the activities of DNA polymerase δ (Pol δ), flap endonuclease 1 (FEN1) and DNA ligase I (Lig1). Little is known regarding the mechanism of partner switching on PCNA and the involvement of PCNA's three binding sites in coordinating such processes. To shed new light on PCNA-mediated Okazaki fragment maturation, we developed a novel approach for the generation of PCNA heterotrimers containing one or two mutant monomers that are unable to bind and stimulate partners. These heterotrimers maintain the native oligomeric structure of PCNA and exhibit high stability under various conditions. Unexpectedly, we found that PCNA heterotrimers containing only one functional binding site enable Okazaki fragment maturation by efficiently coordinating the activities of Pol δ, FEN1, and Lig1. The efficiency of switching between partners on PCNA was not significantly impaired by limiting the number of available binding sites on the PCNA ring. Our results provide the first direct evidence, to our knowledge, that simultaneous binding of multiple partners to PCNA is unnecessary, and if it occurs, does not provide significant functional advantages for PCNA-mediated Okazaki fragment maturation in vitro. In contrast to the "toolbelt" model, which was demonstrated for bacterial and archaeal sliding clamps, our results suggest a mechanism of sequential switching of partners on the eukaryotic PCNA trimer during DNA replication and repair.

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