A common mechanism for recruiting the Rrm3 and RTEL1 accessory helicases to the eukaryotic replisome.

The eukaryotic replisome is assembled around the CMG (CDC45-MCM-GINS) replicative helicase, which encircles the leading-strand DNA template at replication forks. When CMG stalls during DNA replication termination, or at barriers such as DNA-protein crosslinks on the leading strand template, a second helicase is deployed on the lagging strand template to support replisome progression. How these 'accessory' helicases are targeted to the replisome to mediate barrier bypass and replication termination remains unknown.

CMG helicase disassembly is essential and driven by two pathways in budding yeast.

The CMG helicase is the stable core of the eukaryotic replisome and is ubiquitylated and disassembled during DNA replication termination. Fungi and animals use different enzymes to ubiquitylate the Mcm7 subunit of CMG, suggesting that CMG ubiquitylation arose repeatedly during eukaryotic evolution. Until now, it was unclear whether cells also have ubiquitin-independent pathways for helicase disassembly and whether CMG disassembly is essential for cell viability.

An acidic loop in the forkhead-associated domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, Saccharomyces cerevisiae. MEK1 limits resection at double-strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, MEK1 promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover-specific pathway that enables chromosome synapsis.

Mechanism of human PINK1 activation at the TOM complex in a reconstituted system.

Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) are a frequent cause of early-onset Parkinson's disease (PD). Stabilization of PINK1 at the translocase of outer membrane (TOM) complex of damaged mitochondria is critical for its activation. The mechanism of how PINK1 is activated in the TOM complex is unclear.

An acidic loop in the FHA domain of the yeast meiosis-specific kinase Mek1 interacts with a specific motif in a subset of Mek1 substrates.

The meiosis-specific kinase Mek1 regulates key steps in meiotic recombination in the budding yeast, limits resection at the double strand break (DSB) ends and is required for preferential strand invasion into homologs, a process known as interhomolog bias. After strand invasion, promotes phosphorylation of the synaptonemal complex protein Zip1 that is necessary for DSB repair mediated by a crossover specific pathway that enables chromosome synapsis. In addition, Mek1 phosphorylation of the meiosis-specific transcription factor, Ndt80, regulates the meiotic recombination checkpoint that prevents exit from pachytene when DSBs are present.

Replicon-seq: seeing is believing.

Claussin et al. introduce Replicon-seq, a new genome-wide DNA sequencing technology that monitors the progression of individual replisomes at high resolution in vivo.

A conserved mechanism for regulating replisome disassembly in eukaryotes.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF in budding yeast, CUL2 in metazoa), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA.

TIMELESS-TIPIN and UBXN-3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans.

The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 "unfoldase". Here, we combine in vitro reconstitution with in vivo studies in Caenorhabditis elegans embryos, to show that the replisome-associated TIMELESS-TIPIN complex is required for CUL-2 recruitment and efficient CMG helicase ubiquitylation.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp14/nsp10 exoribonuclease.

SARS-CoV-2 is a coronavirus that emerged in 2019 and rapidly spread across the world causing a deadly pandemic with tremendous social and economic costs. Healthcare systems worldwide are under great pressure, and there is an urgent need for effective antiviral treatments. The only currently approved antiviral treatment for COVID-19 is remdesivir, an inhibitor of viral genome replication.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp15 endoribonuclease.

SARS-CoV-2 is responsible for COVID-19, a human disease that has caused over 2 million deaths, stretched health systems to near-breaking point and endangered economies of countries and families around the world. Antiviral treatments to combat COVID-19 are currently lacking. Remdesivir, the only antiviral drug approved for the treatment of COVID-19, can affect disease severity, but better treatments are needed.

CMG helicase disassembly is controlled by replication fork DNA, replisome components and a ubiquitin threshold.

The eukaryotic replisome assembles around the CMG helicase, which stably associates with DNA replication forks throughout elongation. When replication terminates, CMG is ubiquitylated on its Mcm7 subunit and disassembled by the Cdc48/p97 ATPase. Until now, the regulation that restricts CMG ubiquitylation to termination was unknown, as was the mechanism of disassembly.

Pif1-Family Helicases Support Fork Convergence during DNA Replication Termination in Eukaryotes.

The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown.

Phosphopeptide binding by Sld3 links Dbf4-dependent kinase to MCM replicative helicase activation.

The initiation of eukaryotic DNA replication requires the assembly of active CMG (Cdc45-MCM-GINS) helicases at replication origins by a set of conserved and essential firing factors. This process is controlled during the cell cycle by cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), and in response to DNA damage by the checkpoint kinase Rad53/Chk1. Here we show that Sld3, previously shown to be an essential CDK and Rad53 substrate, is recruited to the inactive MCM double hexamer in a DDK-dependent manner.

MCM: one ring to rule them all.

Precise replication of the eukaryotic genome is achieved primarily through strict regulation of the enzyme responsible for DNA unwinding, the replicative helicase. The motor of this helicase is a hexameric AAA+ ATPase called MCM. The loading of MCM onto DNA and its subsequent activation and disassembly are each restricted to separate cell cycle phases; this ensures that a functional replisome is only built once at any replication origin.

Regulated eukaryotic DNA replication origin firing with purified proteins.

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides.

Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen.

Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12].