. 2016 Jan 21;61(2):287-96.
Epub 2015 Dec 24.
Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins
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Mechanism of Archaeal MCM Helicase Recruitment to DNA Replication Origins
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Cellular DNA replication origins direct the recruitment of replicative helicases via the action of initiator proteins belonging to the AAA+ superfamily of ATPases. Archaea have a simplified subset of the eukaryotic DNA replication machinery proteins and possess initiators that appear ancestral to both eukaryotic Orc1 and Cdc6. We have reconstituted origin-dependent recruitment of the homohexameric archaeal MCM in vitro with purified recombinant proteins. Using this system, we reveal that archaeal Orc1-1 fulfills both Orc1 and Cdc6 functions by binding to a replication origin and directly recruiting MCM helicase. We identify the interaction interface between these proteins and reveal how ATP binding by Orc1-1 modulates recruitment of MCM. Additionally, we provide evidence that an open-ring form of the archaeal MCM homohexamer is loaded at origins.
Copyright © 2016 Elsevier Inc. All rights reserved.
Figure 1. A Defined System for Origin-Dependent Recruitment of MCM
A) Coomassie-stained 10% SDS-PAGE gel of 2 μg of purified recombinant Orc1-1 (E147A) and MCM. ( B) Association of MCM with immobilized origin DNA in reactions with and without Orc1-1 following low and high salt washes. Proteins were detected by immuno-blotting after SDS-PAGE. Input shows the input MCM only. ( C) Cartoon of the organization of oriC1. The grey arrows indicate the ORB consensus elements, the initiation sites mapped in vivo are indicated by black arrows. MCM loading assays were performed with oriC1 derivatives containing point mutations in the ORB elements ( 8). The remaining intact ORB elements are named. Proteins were detected by immuno-blotting after SDS-PAGE of high salt-stable recruited material. See also Figure S1 and S5.
Figure 2. The Molecular Basis of MCM’s Interaction with Orc1-1
A) Cartoon of the MCM proteins used in the experiment shown in the panel to the right. Dotted lines indicate the site of amino acid substitutions. WT= wild-type, WA = Walker A mutation (K346A), WB = Walker B mutation (E448A), ΔC = truncation of C-terminal wH-domain. ( B) Association of indicated MCM proteins with immobilized origin DNA in reactions with and without Orc1-1 following low and high salt washes. Proteins were detected by immuno-blotting after SDS-PAGE. Input shows the input WT MCM only See also Figure S5.
Figure 3. The wH Domain of MCM is a Conserved Protein-Protein Interaction Module
A) Sequence alignment of the wH domains of Sulfolobus MCM and human RPA32. Structures of the indicated regions of human RPA32 (PDB 1DPU) and Sulfolobus MCM (PDB 2M45) are shown below. The left-hand and middle images show cartoon and surface representations of the isolated wH-domains with identical amino acids colored in yellow. The C-terminal residue of MCM is shown in cyan. The right-hand images show the complex of RPA32 with a peptide from human UNG2; the lower image is a model of Sulfolobus MCM interacting with UNG2 generated by superimposing the wH domains of the two species. ( B) Association of the indicated MCM proteins with immobilized origin DNA in reactions with and without Orc1-1 after low and high salt washes. Proteins were detected by immuno-blotting after SDS-PAGE. Input shows the input WT MCM only. See also Supplemental Experimental Procedures and Figure S2 and S5.
Figure 4. Identification of the MCM-Interacting Interface of Orc1-1
A) Alignment of Orc1/Cdc6 proteins from a diverse range of archaeal species. Proteins are indicated by species abbreviations followed by annotated ORF number. Crenarchaea : SiRe – Sulfolobus islandicus, SSO – Sulfolobus solfataricus, SacRon12I – Sulfolobus acidiocaldarius, DKAM – Desulfurococcus kamchatkensis, Igni – Ingicoccus hospitalis, Msed – Metallosphaera sedula. Euryarchaea : PAB - Pyrococcus abyssi, MTH – Methanothermobacter thermautotrophicus. Thaumarchaea : CENSY – Cenarchaeum symbiosum. UNG2 is the sequence of the peptide derived from human ING2 that forms a complex with the RPA32 wH domain. The representatives of clades related to Sulfolobus Orc1-1, Orc1-2 and Orc1-3 are indicated and key conserved residues highlighted, including the Sensor 2 residue, R250 ( S. islandicus Orc1-1 numbering). ( B) Association of the MCM with immobilized origin DNA in reactions with and without the indicated Orc1-1 proteins following low and high salt washes. Proteins were detected by immuno-blotting after SDS-PAGE. ( C) The upper panels show the results of flow cytometry of Δ orc1-1 cells expressing the indicated Orc1-1 proteins from a plasmid (see ref. 5). The lower panels show the results of 2D gels to interrogate firing of oriC1 in vivo in the complemented strains. See also Figure S3, S5 and S6 and Table S1.
Figure 5. Basis of ATP-Dependence of the Orc1-1 MCM Interaction
A) Structure of Orc1-1 ADP on DNA. The alpha-helix highlighted in red corresponds to the highlighted region of Orc1-1 in Figure S2A [Figure generated using Pymol (Schrödinger, LLC) from PDB file 2QBY]. The ADP moiety is shown in blue stick form and indicated. The Sensor 2 residue (R250) is in purple. Residues A240 and R244 are shown with atoms depicted as red spheres. The α/β “base” subdomain of the AAA+ domain is shown pale blue, the α-helical “lid” domain is in pink. The wH DNA binding domain of Orc1-1 is in gray. DNA is in wheat. Views from two perspectives are given, differing by a 90° rotation. ( B) The same view as the left hand panel of A but with the wH domain removed for clarity. ( C) A magnified view of the nucleotide binding pocket of Orc1-1 showing the incumbent ADP and the position of the Sensor 2 R250 residue. ( D) Association of the MCM with immobilized origin DNA in reactions with and without the indicated Orc1-1 proteins following low and high salt washes. Proteins were detected by immuno-blotting after SDS-PAGE. Input shows the input WT MCM. ( E) The upper panels show the results of flow cytometry of Δ orc1-1 cells expressing the indicated Orc1-1 proteins from a plasmid. The lower panels show the results of 2D gels to interrogate firing of oriC1 in vivo in the complemented strains. See also Figure S3, S5 and S6.
Figure 6. An Open-Ring Form of MCM is Preferentially Recruited to
oriC1 by Orc1-1
A) MCM was treated at the indicated temperatures prior to addition to the loading assay. Following low and high salt washes, origin-associated proteins were detected by immuno-blotting after SDS-PAGE. Input shows the input WT MCM. ( B) MCM was fractionated over a Superose 6 gel filtration column. Elution was monitored at 280 nm (lower panel) and 0.4 ml fractions collected. Fractions starting at the indicated volume were diluted to equal concentrations (see “input” panel) and used in recruitment assays. As previously, proteins were detected by immuno-blotting after SDS-PAGE. ( C) Representative electron micrographs of negative stained MCM recovered from early eluting fractions (left panel) and later eluting (right panel) fractions following gel filtration; the scale bar indicates 100 nm. ( D) 2D class averages of the particles in the early eluting fractions (upper panel) and later eluting fractions (lower panel).
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Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Adenosine Triphosphate / metabolism
Archaeal Proteins / chemistry
Archaeal Proteins / metabolism*
DNA Helicases / chemistry
DNA Helicases / metabolism*
Protein Interaction Mapping
Protein Structure, Tertiary