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. Using reconstituted assays with budding yeast CMG, we generated the mcm7-10R allele that compromises ubiquitylation by SCFDia2. mcm7-10R delays helicase disassembly in vivo, driving genome instability in the next cell cycle. These data indicate that defective CMG ubiquitylation explains the major phenotypes of cells lacking Dia2. Notably, the viability of mcm7-10R and dia2∆ is dependent upon the related Rrm3 and Pif1 DNA helicases that have orthologues in all eukaryotes. We show that Rrm3 acts during S-phase to disassemble old CMG complexes from the previous cell cycle. These findings indicate that CMG disassembly is essential in yeast cells and suggest that Pif1-family helicases might have mediated CMG disassembly in ancestral eukaryotes.

Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase.

The p97/Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here, we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1, or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin-binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

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.

Reconstitution of human CMG helicase ubiquitylation by CUL2LRR1 and multiple E2 enzymes.

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.

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. By reconstituting these processes with purified budding yeast proteins, we show that ubiquitylation is tightly repressed throughout elongation by the Y-shaped DNA structure of replication forks. Termination removes the repressive DNA structure, whereupon long K48-linked ubiquitin chains are conjugated to CMG-Mcm7, dependent on multiple replisome components that bind to the ubiquitin ligase SCFDia2. This mechanism pushes CMG beyond a '5-ubiquitin threshold' that is inherent to Cdc48, which specifically unfolds ubiquitylated Mcm7 and thereby disassembles CMG. These findings explain the exquisite regulation of CMG disassembly and provide a general model for the disassembly of ubiquitylated protein complexes by Cdc48.