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  1. Author Response

    Reviewer #1 (Public Review):

    This paper describes a systematic biochemical analysis of UBX proteins in facilitating protein unfolding by the p97-UFD1-NPL4 (referred to here is the p97 complex). The p97 complex binds Ub and unfolds it to allow the ubiquitylated protein to be translocated into the p97 ATPase pore for unfolding. This paper demonstrates that UBX proteins are able to reduce the necessary ubiquitin chain length in order to support unfolding by p97. They explore this using ubiquitylated CMG helicase as a substrate. Removal of CMG helicase from replicated DNA is required for completion of DNA synthesis.

    First the authors demonstrate that the p97 complex only only unfolds CMG with very long Ub chains. The then show that the high threshold for Ub is reduced when UBXN7, FAF1 or FAF2 are added. These proteins bind to both the p97 complex and Ub in substrates. This is then followed up in cells by demonstrating that removal of UBXN7 and FAF1 reduces CMG disassembly and is synthetic with reduced CMG ubiquitin ligase activity.

    The conclusion that human p97 requires UBX proteins to support unfolding/segregase activity when Ub chains are short would be strengthened by more precise characterization of the length of ubiquitin chains being studied, as the methods do not precisely determine the chain lengths and how this is overlapping with the number and location of primary ubiquitylation sites on Mcm7.

    Please see our reply above to essential revision point 2 (data in Figure 1-figure supplement 1 and Figure 2-figure supplement 3)

    The in cellulo results, while consistent with a contributing role for FAF1 and UBXN7 in disassembly of the CMG by p97, indicate that either other factors are required in cells or that p97 can disassemble CMG with relative short chains in cells without the need for the UBX proteins. This needs to be reconciled with the proposed model.

    We now discuss on lines 444-450 that CMG disassembly in the absence of UBXN7 and FAF1 might be promoted by additional UBX proteins not characterised in this study, or else be due to extensive CMG-MCM7 ubiquitylation that bypasses the requirement for UBX proteins (as predicted by our data in Figure 1). Note that short ubiquitin chains on CMG-MCM7 in cells treated with p97 inhibitor need to be interpreted with caution, as it is likely that p97 inhibition lowers the pool of free ubiquitin in cells. This point is discussed on lines 444-445 of the revised manuscript.

    Reviewer #3 (Public Review):

    The ATPase p97 (Cdc48 in yeast) unfolds ubiquitinated substrates with the help of its heterodimeric cofactor UFD1-NPL4 (U-N). Using the previously established CMG helicase complex as model substrate in a fully reconstituted biochemical assay, Fujisawa and Labib show that p97-U-N can efficiently disassemble the helicase complex only when it is modified with multiple, long ubiquitin chains. This is in contrast to the yeast Cdc48-U-N complex, which disassembles helicase complexes carrying long or short (6-10 ubiquitin moieties) chains with similar efficiency. The authors demonstrate that the requirement of p97-U-N for long chains can be overcome by the presence of p97 cofactors of the UBA-UBX type, including UBXN7, FAF1, FAF2 and (much less so) UBXN1. They show that this reduction in the 'ubiquitin threshold' of p97-U-N by UBXN7, FAF1 and FAF2 requires their UBX domain mediating p97 binding. They further show that the UBA and UIM domains of UBXN7 contribute to its activity in the assay, whereas the UBA domain of FAF1 and FAF2 is dispensable. Instead, a coiled-coil domain preceding the UBX domain of FAF1 and FAF2 is required for their activity, and both the coiled-coil-UBX domain organization and its activity are conserved in the worm homologue UBXN-3. Using UBXN7 and FAF1 knockout cells, Fujisawa and Labib then demonstrate that UBXN7 is required for efficient CMG helicase disassembly during S phase, with a minor contribution of FAF1, whereas both cofactors possess redundant roles in mitotic CMG helicase disassembly. Finally, the authors show that UBXN7 and FAF1 double knockout cells are hypersensitive to the NEDDylation inhibitor MLN4924 and suggest that this reflects their importance for p97-U-N unfoldase activity under conditions of restricted ubiquitination activity.

    This manuscript describes the intriguing observation that the yeast and mammalian Cdc48/p97-U-N complexes have distinct requirements, at least in the in vitro assay used, with respect to the substrate´s ubiquitination state and to the presence of additional cofactors. While the concept of UBA-UBX cofactors assisting/stimulating Cdc48/p97-U-N activity is well-established, their link to ubiquitin chain length is novel and unexpected. The experiments are performed to a high technical standard, and the conclusions are mostly supported by the data. However, a shortcoming of the paper is that it remains entirely descriptive regarding the effect of the UBX proteins on the ubiquitin threshold, without providing mechanistic insights into their function or the molecular basis underlying the distinct thresholds.

    1. It remains unclear if the failure of p97-U-N to disassemble the helicase complex carrying short ubiquitin chains reflects impaired binding, priming or translocation of the substrate. It should be straightforward to test if the UBA-UBX cofactors simply stabilize the p97-U-N-substrate complex.

    As shown in previous studies, human UFD1-NPL4 bind stably to p97 in the absence of UBX proteins (our new data in Figure 3-figure supplement 2D illustrate this).

    The distinct domain requirements for UBXN7 (UBA, UIM, UBX) and FAF1/FAF2 (coiled-coil-UBX) suggest different mechanisms of stimulation, which should be discussed in more detail.

    We discuss further the roles of UBXN7 and FAF1/FAF2 on lines 533-548.

    The additive defects of the UBXN7 and FAF1 double knockout cells could indicate either redundant functions (as the authors propose) or synergistic function of both cofactors. To that end, the authors could test if UBXN7 and FAF1 can bind simultaneously to the same p97-U-N-substrate complex and if they act synergistically in helicase disassembly, e.g. at limiting cofactor concentrations.

    Previous studies have found that UBXN7 binds to p97 and UFD1-NPL4 with a 1:6:1 ratio and the same is true for FAF1, without any evidence of both UBXN7 and FAF1 binding to the same p97-UFD1-NPL4 complexes (Hanzelmann et al., 2011). Correspondingly, we did not observe any synergistic effect of FAF1 with UBXN7 upon the disassembly of ubiquitylated CMG by p97-UFD1-NPL4, when comparing reactions with a single UBX protein or reactions with both (our unpublished data).

    1. Having all purified proteins at hand, the authors should test which component of the system causes the elevated ubiquitin threshold of mammalian p97-U-N, by combining yeast Cdc48 with mammalian U-N and vice versa, etc.

    We thank the reviewer for this very interesting suggestion. The data are presented in Figure 3, showing that human UFD1-NPL4 and yeast Ufd1-Npl4 set the ubiquitin threshold for their cognate unfoldase enzymes.

    Can yeast Ubx5, which is a clear homologue of UBXN7, substitute for the mammalian UBA-UBX cofactors?

    This was also an interesting suggesting – we tested Ubx5 and didn’t see any stimulation. We didn’t include the data as we lack a positive control for Ubx5 activity.

    1. The authors emphasize that mammalian p97-U-N in the absence of UBA-UBX cofactors requires long ubiquitin chains for activity. However, they should consider the possibility that the critical property is chain topology, rather than chain length. There is evidence that p97-U-N prefers substrates with branched chains (see PMIDs 28512218, 29033132), and multiple ubiquitin chains on the helicase substrate may mimic those.

    We thank the reviewer for raising this important point and we now cite the two papers mentioned above, on lines 171 and 177.

    In the revised version of the manuscript, we characterise carefully the ubiquitin chains that are formed under the various conditions used (Figure 1-figure supplement 1). Importantly, we also show that human p97-UFD1-NPL4 can disassemble highly ubiquitylated CMG, regardless of whether there are several or just one ubiquitin chains attached to CMG-Mcm7 (Figure 1-figure supplement A+C; Figure 2-figure supplement 3A).

    Moreover, we also show that human p97-UFD1-NPL4 is comparable to yeast Cdc48-Ufd1-Npl4 in being able to disassemble CMG that is highly ubiquitylated with ‘K48-only’ ubiquitin that cannot form mixed chain linkages (Figure 2-figure supplement 3B).

    These data indicate that p97-UFD1-NPL4 can disassemble heavily ubiquitylated CMG complexes with long K48-linked ubiquitin chains on CMG-Mcm7, regardless of the number of chains and regardless of the presence of other chain linkages (in addition to K48-linked chains).

    It appears that worm CDC48-U-N in the absence of UBXN-3 cannot efficiently disassemble substrate carrying even long chains (Fig. 3 - supplement 2). The authors should discuss this finding in the context of their ubiquitin threshold model.

    This is an interesting point, suggesting that the threshold of C. elegans CDC-48_UFD-1_NPL-4 is even higher than human p97-UFD1-NPL4, in the absence of UBX proteins. However, we think that this issue is beyond the scope of our manuscript and likely requires structural biology to provide a definitive explanation. Our manuscript just uses the C. elegans enzymes to make one simple and clear point – namely that the essential role of the coiled coil domain of human FAF1 is conserved in its worm orthologue UBXN-3.

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  2. Evaluation Summary:

    This paper describes a biochemical analysis of the roles of Ub chain length on Ub-dependent segregase activity of yeast and human p97 and the role of UBX proteins on the disassembly of the CMG replicative helicase complex. The human p97 complex does not segregate substrates with shorter ubiquitin chains as efficiently as does the yeast complex but the human complex can be enhanced in vitro by 3 UBX proteins - FAF1, FAF2, and UBXN7. Cellular studies indicate a partial role for FAF1 and UBXN7 in cells. The paper would be strengthened by additional mechanistic understanding of how the UBX domain functions in activation of segregase activity and the contribution of this pathway in cells.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

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  3. Reviewer #1 (Public Review):

    This paper describes a systematic biochemical analysis of UBX proteins in facilitating protein unfolding by the p97-UFD1-NPL4 (referred to here is the p97 complex). The p97 complex binds Ub and unfolds it to allow the ubiquitylated protein to be translocated into the p97 ATPase pore for unfolding. This paper demonstrates that UBX proteins are able to reduce the necessary ubiquitin chain length in order to support unfolding by p97. They explore this using ubiquitylated CMG helicase as a substrate. Removal of CMG helicase from replicated DNA is required for completion of DNA synthesis.

    First the authors demonstrate that the p97 complex only only unfolds CMG with very long Ub chains. The then show that the high threshold for Ub is reduced when UBXN7, FAF1 or FAF2 are added. These proteins bind to both the p97 complex and Ub in substrates. This is then followed up in cells by demonstrating that removal of UBXN7 and FAF1 reduces CMG disassembly and is synthetic with reduced CMG ubiquitin ligase activity.

    The conclusion that human p97 requires UBX proteins to support unfolding/segregase activity when Ub chains are short would be strengthened by more precise characterization of the length of ubiquitin chains being studied, as the methods do not precisely determine the chain lengths and how this is overlapping with the number and location of primary ubiquitylation sites on Mcm7. The in cellulo results, while consistent with a contributing role for FAF1 and UBXN7 in disassembly of the CMG by p97, indicate that either other factors are required in cells or that p97 can disassemble CMG with relative short chains in cells without the need for the UBX proteins. This needs to be reconciled with the proposed model.

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  4. Reviewer #2 (Public Review):

    The authors employ a biochemical assay in which ubiquitylated MCM7 is dissociated by p97-Ufd1-Npl4 from the helicase complex immobilized on beads. They optimized the assay so that they can generate either shorter or very long chains. They convincingly demonstrate that mobilization by human p97-Ufd1-Npl4 in the presence of short chains is inefficient and can be stimulated by either FAF1, FAF2, or UBXN7. They map the required sequence in these proteins to the p97-binding UBX domain and an adjacent coiled-coil region. Surprisingly, established ubiquitin binding domains are not required for stimulatory activity. They reiterate these experiments with C. elegans proteins. Moreover, they validate significance of the accessory factors for the mobilization of the helicase component SLD5 in mouse ES cells. The latter was analyzed in mitosis, reflecting a backup pathway for S-phase events, which allows convenient time-course analysis.

    The findings are important for the understanding of replication termination. They are likely also more generally significant for other p97/Cdc48 mediated unfolding and segregation processes. While involvement of accessory factors FAF1, FAF2 or UBXN7 has been demonstrated in various processes before, and the recruitment function been anticipated, this manuscript directly demonstrates this function and links it to ubiquitin chain length. Therefore, it is an important step forward. The positive assessment is somewhat dampened by the fact that the authors do not provide kinetic data and, more significantly, that they fail to define the mechanistic function of the common coiled-coil region that they show is critical in FAF1 and FAF2.

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  5. Reviewer #3 (Public Review):

    The ATPase p97 (Cdc48 in yeast) unfolds ubiquitinated substrates with the help of its heterodimeric cofactor UFD1-NPL4 (U-N). Using the previously established CMG helicase complex as model substrate in a fully reconstituted biochemical assay, Fujisawa and Labib show that p97-U-N can efficiently disassemble the helicase complex only when it is modified with multiple, long ubiquitin chains. This is in contrast to the yeast Cdc48-U-N complex, which disassembles helicase complexes carrying long or short (6-10 ubiquitin moieties) chains with similar efficiency. The authors demonstrate that the requirement of p97-U-N for long chains can be overcome by the presence of p97 cofactors of the UBA-UBX type, including UBXN7, FAF1, FAF2 and (much less so) UBXN1. They show that this reduction in the 'ubiquitin threshold' of p97-U-N by UBXN7, FAF1 and FAF2 requires their UBX domain mediating p97 binding. They further show that the UBA and UIM domains of UBXN7 contribute to its activity in the assay, whereas the UBA domain of FAF1 and FAF2 is dispensable. Instead, a coiled-coil domain preceding the UBX domain of FAF1 and FAF2 is required for their activity, and both the coiled-coil-UBX domain organization and its activity are conserved in the worm homologue UBXN-3. Using UBXN7 and FAF1 knockout cells, Fujisawa and Labib then demonstrate that UBXN7 is required for efficient CMG helicase disassembly during S phase, with a minor contribution of FAF1, whereas both cofactors possess redundant roles in mitotic CMG helicase disassembly. Finally, the authors show that UBXN7 and FAF1 double knockout cells are hypersensitive to the NEDDylation inhibitor MLN4924 and suggest that this reflects their importance for p97-U-N unfoldase activity under conditions of restricted ubiquitination activity.

    This manuscript describes the intriguing observation that the yeast and mammalian Cdc48/p97-U-N complexes have distinct requirements, at least in the in vitro assay used, with respect to the substrate´s ubiquitination state and to the presence of additional cofactors. While the concept of UBA-UBX cofactors assisting/stimulating Cdc48/p97-U-N activity is well-established, their link to ubiquitin chain length is novel and unexpected. The experiments are performed to a high technical standard, and the conclusions are mostly supported by the data. However, a shortcoming of the paper is that it remains entirely descriptive regarding the effect of the UBX proteins on the ubiquitin threshold, without providing mechanistic insights into their function or the molecular basis underlying the distinct thresholds.

    1. It remains unclear if the failure of p97-U-N to disassemble the helicase complex carrying short ubiquitin chains reflects impaired binding, priming or translocation of the substrate. It should be straightforward to test if the UBA-UBX cofactors simply stabilize the p97-U-N-substrate complex.
    The distinct domain requirements for UBXN7 (UBA, UIM, UBX) and FAF1/FAF2 (coiled-coil-UBX) suggest different mechanisms of stimulation, which should be discussed in more detail. The additive defects of the UBXN7 and FAF1 double knockout cells could indicate either redundant functions (as the authors propose) or synergistic function of both cofactors. To that end, the authors could test if UBXN7 and FAF1 can bind simultaneously to the same p97-U-N-substrate complex and if they act synergistically in helicase disassembly, e.g. at limiting cofactor concentrations.

    2. Having all purified proteins at hand, the authors should test which component of the system causes the elevated ubiquitin threshold of mammalian p97-U-N, by combining yeast Cdc48 with mammalian U-N and vice versa, etc. Can yeast Ubx5, which is a clear homologue of UBXN7, substitute for the mammalian UBA-UBX cofactors?

    3. The authors emphasize that mammalian p97-U-N in the absence of UBA-UBX cofactors requires long ubiquitin chains for activity. However, they should consider the possibility that the critical property is chain topology, rather than chain length. There is evidence that p97-U-N prefers substrates with branched chains (see PMIDs 28512218, 29033132), and multiple ubiquitin chains on the helicase substrate may mimic those.
    It appears that worm CDC48-U-N in the absence of UBXN-3 cannot efficiently disassemble substrate carrying even long chains (Fig. 3 - supplement 2). The authors should discuss this finding in the context of their ubiquitin threshold model.

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