Peer review of: Structural mechanism for replication origin binding and remodeling by a metazoan origin (2020)

REVIEWERS' COMMENTS: 

Reviewer #1 (Remarks to the Author): 

JM Schmidt and F Bleichert report a structural mechanism for replication origin binding and remodeling 
by the Drosophyla melanogaster’s Origin Recognition Complex (ORC) and its co-loader Cdc6. While 
ORC recognizes consensus DNA sequences in S. cerevisiae, metazoan ORC (such as D. melanogaster) 
associates with chromosomal DNA independently of specific sequence elements. This work aims to 
decipher the structural basis for DNA engagement by ORC in the absence of specific consensus DNA 
sequences. 
The authors report near atomic resolution cryo-EM structures of D. melanogaster ORC bound to 
different DNA substrates in the absence and presence of its co-loader Cdc6. These structures show 
how the ATPase modules of subunits Orc1 and Orc4 constitute the primary DNA binding site in the 
center of the ORC ring and cooperate with the winged-helix domains to stabilize DNA bending. 
Correlating structural observations with biochemical data further shows that DNA sequence modulates 
DNA binding and remodeling, and that DNA bending in turn promotes Mcm2-7 loading. To explain the 
distinct DNA sequence-dependencies of S. cerevisiae and D. melanogaster (metazoan), the DNA 
contacts between S. cerevisiae and metazoan ORC are extensively compared. These findings support a 
model in which DNA geometry and DNA bendability contribute to the selection of chromosomal Mcm2-
7 helicase loading sites in metazoans. 
This work answers fundamental questions about the initiation of DNA replication in eukaryotes. The 
manuscript is well-written, the figures are nice and informative. The conclusions are sound and based 
on an impressive amount of functional and structural data. Please find below a few points that the 
authors should consider when preparing the revised version of their manuscript. 

Major points: 

The DNA contacts are extensively described. My main concern is that non-specialist readers may find 
it difficult to capture some of the article's important conclusions. 
- Figure 7 summaries the main conclusions of the article. The figure is very nice and informative. 
However, the figure may be extended by detailing and numbering the different conformational 
transitions in metazoan ORC and DNA remodeling. 
- A movie may be useful to compare the DNA contacts between S. cerevisiae and metazoan ORC, and 
explain the distinct DNA sequence-dependencies of S. cerevisiae and D. melanogaster. 

Minor points: 
- P.2: … the eukaryotic kingdom… should be … the eukaryotic domain of life … (The taxonomic rank of 
Eukarya is a domain not a kingdom). 
- P.9: “backbone phosphates” may be “phosphate backbones”. 
- P.11: You may consider splitting the following sentence: “Two notable exceptions include the WH 
domain of Orc2, which is detached from the AAA+ layer and flexible in the absence of Cdc6 (Extended 
Data Figures 6E and 6G), and the arginine finger helix in Orc1, a region that interacts with the Cdc6 
AAA+ module in the ternary complex and is less ordered in the DNA-free DmORC and the 
DmORC·DNA assembly, suggesting it undergoes a disorder-to-order transition upon Cdc6 binding.” 
- “van der Waals contacts” or “van der Waals interactions” is used in many occasions in the 
manuscript to describe the interactions within the different protein complexes. Hydrophobic 
interactions may be more appropriate ? 
- The interactions within the different protein complexes are extensively described. However several 
sentences may be confusing for the readers. The following sentences may be revised: 
P.7: In agreement with this evolutionary kinship, Orc1 and Cdc6 are predicated on the same protein 
folds and adopt similar 3D structures in the ternary initiator·co-loader complex. 
P.9: The ISMs of Orc1, Orc4, and Cdc6 in particular form several hydrogen bonds and van der Waals 
contacts with the sugar-phosphate backbone of one of the DNA strands that together extend for half a 

turn of the duplex 
- P31-32 (mat & meth): imidazole p31 … or … Imidazole p32 : please homogenize. 

Reviewer #2 (Remarks to the Author): 

Schmidt and Bleichert report the reconstitution of the MCM helicase loading reaction in vitro, using 
purified Drosophila proteins, and the cryo-EM structure of the Drosophila ORC-DNA and ORC-DNA-
Cdc6 complex. They find that, in both complexes, DNA is bent as it traverses the central pore of the 
ORC ring. The ORC-Cdc6 ring forms a flat ring configuration, structurally distinct from previous 
helicase loading intermediates observed in yeast. The ORC-Cdc6-DNA complex also contains a 
conformation distinct from the auto-inhibited state previously observed for the isolated ORC complex. 
Using structural and biochemical tools, the authors describe a new B-loop element in Orc1 that is 
responsible for the high-affinity interaction of Drosophila ORC with DNA. The authors also describe the 
cryo-EM structures of DmORC-Cdc6 bound to DNA of different sequence compositions. Interestingly, 
they establish, that duplex DNA segments differing in sequence can be bent to different degrees, and 
that DNA bendability affects MCM loading efficiency. 

This work is simply outstanding. The in vitro reconstitution of the MCM loading with metazoan proteins 
alone would grant in my opinion publication in a high impact journal. In addition to this however, the 
authors report an extremely high quality structural and biochemical study, describing important 
physical aspects of helicase loading by ORC. 

I only have a few minor comments: 

1. The manuscript in general is very clearly written, although I must say in certain passages the 
language appears unnecessarily complicated. For example, in the abstract, “a nucleotidyl hydrolase 
that uses adenosine triphosphate (ATP) binding and turnover to control its association with 
chromosomal regions” could be summarized with “a DNA binding ATPase”. I would suggest that the 
authors critically read their manuscript to eliminate such passages, to improve an otherwise 
exquisitely written manuscript. 

2. I would also recommend that the authors put a bigger emphasis on their achievement of the 
reconstitution of MCM loading using purified Drosophila proteins. Again, this is probably the most 
significant aspect of the work presented, and at the moment it feels like something appended to the 
study, rather than its central piece. 

3. Negative stain EM micrographs should be added as supplementary material for the MCM loading. 

4. The authors describe an ORC-Cdc6 complex that exists in a flat configuration that is different from 
the spiral form described for the yeast OCCM, and they speculate that MCM engagement could 
reconfigure the ORC-Cdc6. Could the authors clarify to what extent the ATPase sites in the Orc-Cdc6 
complex would be altered?