Ulrich Rass

PIF1 family helicases promote genome stability during DNA replication and repair, yet the biochemical activity of human PIF1 (hPIF1) remains poorly understood. Here, we directly compare full-length hPIF1 and budding yeast Pif1 (yPif1) and show that the two enzymes operate differently. In bulk assays, hPIF1 displays weak net DNA unwinding because its robust intrinsic single-stranded DNA (ssDNA) annealing activity rapidly reforms duplex DNA behind the helicase motor. Single-molecule magnetic tweezers experiments reveal that hPIF1 is only modestly slower than yPif1 and similarly processive, indicating that poor apparent unwinding primarily reflects rapid reannealing rather than impaired motor activity. Mutagenesis and structural modeling indicate that this functional divergence depends on a yeast-specific insertion within the catalytic domain. Unlike yPif1, hPIF1 fails to stimulate Polδ-dependent displacement-loop (D-loop) extension, arguing that it does not promote DNA unwinding ahead of the migrating bubble. However, similarly to yPif1, hPIF1 likely unwinds newly synthesized DNA coupled with DNA reannealing behind the migrating D-loop. hPIF1 efficiently remodels intramolecular G-quadruplex DNA into fully base-paired dsDNA, whereas yPif1 generates ssDNA products. Together, our data support a model in which hPIF1 acts primarily as a DNA translocase that couples local unwinding with rapid rewinding to clear secondary DNA structures without generating extensive ssDNA.

Break-induced replication (BIR) facilitates single-ended DNA double-strand break (DSB) repair. Upon homologous recombination-mediated strand invasion into a homologous repair template, BIR is catalysed by a minimal replisome comprising PCNA, DNA polymerase δ (Pol δ), and the Pif1 helicase. Here, we identify an interaction between Pol δ and single-stranded DNA (ssDNA)-binding protein RPA mediated by an RPA-binding module (RBM) within Pol δ subunit Pol32 and RPA subunit Rfa1. Pol32 RBM phosphorylation at Thr256 and Thr257 increases its affinity for Rfa1, while corresponding phospho-mimetic amino-acid substitutions promote BIR efficiency in vivo. This suggests that Pol32 functions as a rheostat whose phosphorylation enhances Pol δ’s affinity for RPA-bound BIR intermediates, thereby boosting BIR efficiency. Modelling indicates that Pol32 phospho-RBM-Rfa1 interactions mirror the binding mode of RBMs in Pif1 and the FANCM helicase and BIR antagonist Mph1. This implies a key role for RPA in the dynamic orchestration of the enzymes mediating BIR.

Nuclease–helicase DNA2 is a multifunctional genome caretaker that is essential for cell proliferation in a range of organisms, from yeast to human ^1–4 . Bi-allelic DNA2 mutations that reduce DNA2 concentrations cause a spectrum of primordial dwarfism disorders, including Seckel and Rothmund–Thomson-related syndromes ^5–7 . By contrast, cancer cells frequently express high concentrations of DNA2 (refs. ^8–11 ). The mechanism that precludes cell proliferation in the absence of DNA2 and the molecular aetiology of DNA2 -linked diseases remain elusive. Here we used yeast and human cells to demonstrate that DNA2 suppresses homologous recombination-restarted replication and checkpoint activation at stalled DNA replication forks. Loss of this control mechanism upon degradation of DNA2 in human cells causes recombination-dependent DNA synthesis and build-up of RPA-bound single-stranded DNA in the G2 phase of the cell cycle. Consequently, DNA2 deprivation triggers the DNA damage checkpoint and invariably leads to ATR–p21-dependent cell-cycle exit before mitosis. These findings explain why DNA2 is essential for cell proliferation and reveal that replication fork processing to restrict recombination is indispensable for avoiding cellular senescence. Stochastic entry into senescence stifles the proliferative potential of cells following the expression of a Seckel syndrome patient-derived DNA2 hypomorph or partial degradation of DNA2, providing a conceptual framework to explain global growth failure in DNA2 -linked primordial dwarfism disorders.

The conserved nuclease-helicase DNA2 has been linked to mitochondrial myopathy, Seckel syndrome, and cancer. Across species, the protein is indispensable for cell proliferation. On the molecular level, DNA2 has been implicated in DNA double-strand break (DSB) repair, checkpoint activation, Okazaki fragment processing (OFP), and telomere homeostasis. More recently, a critical contribution of DNA2 to the replication stress response and recovery of stalled DNA replication forks (RFs) has emerged. Here, we review the available functional and phenotypic data and propose that the major cellular defects associated with DNA2 dysfunction, and the links that exist with human disease, can be rationalized through the fundamental importance of DNA2-dependent RF recovery to genome duplication. Being a crucial player at stalled RFs, DNA2 is a promising target for anti-cancer therapy aimed at eliminating cancer cells by replication-stress overload.

The disease-associated nuclease–helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.

DNA2 is an essential nuclease–helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5′-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5′-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2’s role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1’s ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2’s role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.

DNA double-strand breaks (DSBs) disrupt the structural integrity of chromosomes. Proper DSB repair pathway choice is critical to avoid the type of gross chromosomal rearrangements that characterize cancer cells. Recent findings reveal S-fatty acylation and membrane anchorage of Rap1-interacting factor 1 (Rif1) as a mechanism providing spatial control over DSB repair pathway choice.

Rif1 is involved in telomere homeostasis, DNA replication timing, and DNA double-strand break (DSB) repair pathway choice from yeast to human. The molecular mechanisms that enable Rif1 to fulfill its diverse roles remain to be determined. Here, we demonstrate that Rif1 is S-acylated within its conserved N-terminal domain at cysteine residues C466 and C473 by the DHHC family palmitoyl acyltransferase Pfa4. Rif1 S-acylation facilitates the accumulation of Rif1 at DSBs, the attenuation of DNA end-resection, and DSB repair by non-homologous end-joining (NHEJ). These findings identify S-acylation as a posttranslational modification regulating DNA repair. S-acylated Rif1 mounts a localized DNA-damage response proximal to the inner nuclear membrane, revealing a mechanism of compartmentalized DSB repair pathway choice by sequestration of a fatty acylated repair factor at the inner nuclear membrane.

Complete genome duplication in every cell cycle is fundamental for genome stability and cell survival. However, chromosome replication is frequently challenged by obstacles that impede DNA replication fork (RF) progression, which subsequently causes replication stress (RS). Cells have evolved pathways of RF protection and restart that mitigate the consequences of RS and promote the completion of DNA synthesis prior to mitotic chromosome segregation. If there is entry into mitosis with underreplicated chromosomes, this results in sister-chromatid entanglements, chromosome breakage and rearrangements and aneuploidy in daughter cells. Here, we focus on the resolution of persistent replication intermediates by the structure-specific endonucleases (SSEs) MUS81, SLX1-SLX4 and GEN1. Their actions and a recently discovered pathway of mitotic DNA repair synthesis have emerged as important facilitators of replication completion and sister chromatid detachment in mitosis. As RS is induced by oncogene activation and is a common feature of cancer cells, any advances in our understanding of the molecular mechanisms related to chromosome underreplication have important biomedical implications.