Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes

Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81-Mms4/MUS81-EME1, Yen1/GEN1, and Slx1-Slx4/SLX1-SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication process

Freedom of movement

Holliday junction resolvases lock dynamic DNA four-way junctions into specific structural conformations for symmetric DNA cleavage. Single-molecule studies now reveal that resolvases can relax their grip, enabling Holliday junction conformer transitions and branch migration in the enzyme-bound form

Nachweis und Charakterisierung von Crp1p, einem neuen Holliday-Struktur bindenden Protein der Hefe Saccharomyces cerevisiae

Bei genetischen Rekombinationsvorgängen tritt eine vierarmige DNA-Struktur, die sog. Holliday-Struktur auf. Für in vitro Experimente wird ein Strukturanalog, die sog. X-DNA verwendet. Eine Reihe sehr unterschiedlicher Proteine ist in der Lage, diese charakteristische DNA-Struktur spezifisch zu erkennen. In einigen Fällen lässt sich ein klarer Bezug zur Rekombination herstellen. In der vorliegenden Arbeit wurde ein neues X-DNA bindendes Protein der Hefe Saccharomyces cerevisiae entdeckt. Durch eine Serie von Flüssigkeitschromatographien wurde es aus Rohextrakten soweit angereinigt, dass seine Identität geklärt werden konnte. Die Identifikation gelang mit Hilfe der Southwestern Technik. Eine anschließende Proteinsequenzierung ergab, dass es sich um das bisher uncharakterisierte Produkt des offenen Leserasters YHR146W handelte. Dem 465 Aminosäuren umfassenden Protein wurde der funktionelle Name Cruciform DNA-Recognising Protein 1 (Crp1p) gegeben. Das Gen CRP1 wurde kloniert und in E. coli exprimiert. Die Aktivität des rekombinanten Proteins in EMSAs bestätigte Crp1p als Urheber der mit Hefe-Extrakten beobachteten Aktivität. Das rekombinante Protein erwies sich allerdings als anfällig für eine effiziente posttranslationale Spaltung in der direkten Umgebung von Aminosäure 160. Dabei ergibt sich ein X-DNA bindendes N-terminales- und ein unter bestimmten Bedingungen X-DNA bindendes, aber in Anwesenheit von Kompetitor-DNA inaktives, C-terminales Subpeptid. Die auf der Architektur des DNA-Substrates beruhende DNA-Bindung des N-terminalen Peptids wird durch eine neuartige X-DNA-Bindedomäne vermittelt. Diese besitzt nach derzeitigem Stand eine 22 Aminosäuren umfassende, positiv geladene Minimalsequenz, die als autonomes X-DNA bindendes Subpeptid fungieren kann. Phänotypische Untersuchungen an crp1-Mutanten ergaben bislang keinen Hinweis auf eine Beteiligung an Rekombinationsprozessen. So ließ sich keine erhöhte Sensitivität gegenüber UV- oder Röntgenstrahlung und kein meiotischer Defekt nachweisen. Um weiter zu klären, an welchem molekularen Ablauf Crp1p beteiligt ist, wurde mit Hilfe der Hefe-2-Hybrid-Analyse nach Proteinen gesucht, die mit Crp1p in Wechselwirkung treten. Dabei wurden zwei RNA bindende Proteine, Drs1p und Gno1p gefunden. Beide sind an der rRNA-Reifung beteiligt, so dass die Möglichkeit aufgeworfen wurde, dass Crp1p in vivo nicht strukturierte DNA, sondern RNA bindet

A new role for Holliday junction resolvase Yen1 in processing DNA replication intermediates exposes Dna2 as an accessory replicative helicase

DNA replication is mediated by a multiprotein complex known as the replisome. With the hexameric MCM (minichromosome maintenance) replicative helicase at its core, the replisome splits the parental DNA strands, forming replication forks (RFs), where it catalyses coupled leading and lagging strand DNA synthesis. While replication is a highly effective process, intrinsic and oncogene-induced replication stress impedes the progression of replisomes along chromosomes. As a consequence, RFs stall, arrest, and collapse, jeopardiz- ing genome stability. In these instances, accessory fork progression and repair factors, orchestrated by the replication checkpoint, promote RF recovery, ensuring the chromosomes are fully replicated and can be safely segregated at cell division. Homologous recombination (HR) proteins play key roles in negotiating replication stress, binding at stalled RFs and shielding them from inappropriate processing. In addition, HR-mediated strand exchange reactions restart stalled or collapsed RFs and mediate error-free post-replicative repair. DNA transactions at stalled RFs further involve various DNA editing factors, notably helicases and nucleases. A study by Ölmezer et al. (2016) has recently identified a role for the structure-specific nuclease Yen1 (GEN1 in human) in the resolution of dead-end DNA replication intermediates after RF arrest. This new function of Yen1 is distinct from its previously known role as a Holliday junction resolvase, mediating the removal of branched HR intermediates, and it becomes essential for viable chromosome segregation in cells with a defective Dna2 helicase. These findings have revealed greater complexity in the tasks mediated by Yen1 and expose a replicative role for the elusive helicase activity of the conserved Dna2 nuclease-helicase

Structure-specific endonucleases and the resolution of chromosome underreplication

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

Compartmentalized DNA repair: Rif1 S-acylation links DNA double-strand break repair to the nuclear membrane

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

Resolving branched DNA intermediates with structure-specific nucleases during replication in eukaryotes

Genome duplication requires that replication forks track the entire length of every chromosome. When complications occur, homologous recombination-mediated repair supports replication fork movement and recovery. This leads to physical connections between the nascent sister chromatids in the form of Holliday junctions and other branched DNA intermediates. A key role in the removal of these recombination intermediates falls to structure-specific nucleases such as the Holliday junction resolvase RuvC in Escherichia coli. RuvC is also known to cut branched DNA intermediates that originate directly from blocked replication forks, targeting them for origin-independent replication restart. In eukaryotes, multiple structure-specific nucleases, including Mus81-Mms4/MUS81-EME1, Yen1/GEN1, and Slx1-Slx4/SLX1-SLX4 (FANCP) have been implicated in the resolution of branched DNA intermediates. It is becoming increasingly clear that, as a group, they reflect the dual function of RuvC in cleaving recombination intermediates and failing replication forks to assist the DNA replication proces

Limiting homologous recombination at stalled replication forks is essential for cell viability: DNA2 to the rescue

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 in chromosome stability and cell survival—is it all about replication forks?

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

Shepherding DNA ends: Rif1 protects telomeres and chromosome breaks

Cells have evolved conserved mechanisms to protect DNA ends, such as those at the termini of linear chromosomes, or those at DNA double-strand breaks (DSBs). In eukaryotes, DNA ends at chromosomal termini are packaged into proteinaceous structures called telomeres. Telomeres protect chromosome ends from erosion, inadvertent activation of the cellular DNA damage response (DDR), and telomere fusion. In contrast, cells must respond to damage-induced DNA ends at DSBs by harnessing the DDR to restore chromosome integrity, avoiding genome instability and disease. Intriguingly, Rif1 (Rap1-interacting factor 1) has been implicated in telomere homeostasis as well as DSB repair. The protein was first identified in as being part of the proteinaceous telosome. In mammals, RIF1 is not associated with intact telomeres, but was found at chromosome breaks, where RIF1 has emerged as a key mediator of pathway choice between the two evolutionary conserved DSB repair pathways of non-homologous end-joining (NHEJ) and homologous recombination (HR). While this functional dichotomy has long been a puzzle, recent findings link yeast Rif1 not only to telomeres, but also to DSB repair, and mechanistic parallels likely exist. In this review, we will provide an overview of the actions of Rif1 at DNA ends and explore how exclusion of end-processing factors might be the underlying principle allowing Rif1 to fulfill diverse biological roles at telomeres and chromosome breaks