Du W , Josephrajan A , Adhikary S , Bowles T , Bielinsky AK , Eichman BF .

R01 GM074917 NIGMS NIH HHS , R01 GM080570 NIGMS NIH HHS

	Figure 2. Trimerization of MBP-CC.(A,B) Crystal structure of the MBP-CC asymmetric unit, with each protomer colored differently. Maltose-binding protein is shown as a Cα-trace, and the xMcm10 coiled-coil is depicted as a cartoon ribbon. (C,D) Sedimentation velocity profiles of MBP-CC95–132 (C) and free MBP (D) at pH 4.7. Molecular masses (kDa) calculated from the sedimentation data are shown above each peak. The molecular mass of a single polypeptide calculated from the amino acid composition are 45.1 kDa (MBP-CC) and 40.4 kDa (MBP).
	Figure 3. Crystal structure of the Mcm10 coiled-coil.(A) Sequence alignment of the coiled-coil region from Xenopus laevis (x), Homo sapiens (h), Mus musculus (m), and Saccharomyces cerevisiae (sc) Mcm10. Red boxes indicate identical amino acids. The predicted heptad repeat pattern is labeled by letters a-g at the bottom. The position of the helix is shown schematically at the top (brown, MBP-CC95–124; grey, MBP-CC95–132). (B) Composite 2Fo−Fc omit electron density map (contoured at 1σ) with carbon atoms colored according to protomer. (C) Crystal structure of xMcm10-CC95–124. Residues at the interface are shown in ball and stick, and the amino acid numbers labeled in black. (D) View down the helical axis, rotated 90° from the view in B.
	Figure 4. A model of the dimeric Mcm10 CC.(A) Structure-based sequence alignment between Mcm10 CC and the trimeric and dimeric forms of GCN4 CC. (B) Stereoview of the Mcm10 CC trimer (green) superimposed onto the isoleucine GCN4 trimer (grey, PDB ID 1 GCM). Side chains at the CC interface are shown, with the exception of the N-terminal methionine in GCN4. Two Mcm10 residues are labeled for orientation. The view is rotated 45° clockwise with respect to Figure 3C. (C) Stereoview of the Mcm10 CC dimer model (green) superimposed on the CGN4 CC (grey, PDB ID 2ZTA). (D) The relationship between trimer (left) and dimer (right) forms of the CC. Schematics are oriented with respect to the green helix. The conformation of the dimer can be constructed from the trimer by a 60° rotation and 8 à translation of the magenta helix. The interhelical distances (dashed line) are 15.5 à (trimer) and 10 à (dimer).
	Figure 5. Coiled-coil mutations disrupt CC and NTD dimerization.Sedimentation velocity data for MBP-CC95–124 (A) and NTD (B) constructs as wild-type (WT) or containing L104D/L108D (2D) or L104A/L108A/M115A/L118A (4A) mutations. Molecular masses corresponding to each peak are reported in Table S1 in the Supporting Information.
	Figure 6. Coiled-coil mutations disrupt Mcm10 self-association.(A, B) Three individual strains (1, 2, 3) harboring yeast two-hybrid plasmids that express the indicated proteins either as a fusion with the Gal4-binding domain (BD) or -activation domain (AD) were spotted onto drop-out plates lacking tryptophan and leucine (-Trp -Leu) or quadruple drop-out plates lacking tryptophan, leucine, adenine and histidine (-Trp -Leu -Ade -His). Cells were spotted at a number of 2×107 (1) or a 10-fold dilution (1∶10) for full-length Xenopus Mcm10, the L104D/L108D mutant (Mcm102D), the L104A/L108A/M115A/L118A mutant (Mcm104A), and the NTD deletion mutant spanning residues 230–860 (Mcm10ΔN). p53 and large T-antigen (LTag) served as a positive control, EV indicates empty vector controls. (C) Western blot showing wild-type and mutant xMcm10 protein expression in representative strains. Gal4-AD fusions were detected by a HA-specific antibody. Strains carrying empty vector controls, or Gal4-AD fusion genes and Gal4-BD empty vectors are shown on the left (Empty vector controls). Strains expressing pair-wise combinations of the Gal4-AD and Gal4-BD fusion genes as indicated in panels A and B are shown on the right (Two-hybrid strains). Full-length xMcm10 and the 2D and 4A mutants ran at an approximate size of 140 kDa, whereas the truncated form of xMcm10 ran at an approximate size of 94 kDa. Tubulin served as a loading control. (D) Gal4-BD fusions were detected by a Myc-specific antibody. Extracts from the identical two-hybrid strains shown in (C) were loaded in the same order. The asterisk denotes a non-specific band.
	Figure 1. The NTD is necessary for Mcm10 self-association.(A) Schematic of Xenopus laevis Mcm10 constructs used to study self-association. Theoretical molecular masses are 95.4 kDa (Mcm10) and 70.4 kDa (Mcm10ΔN). (B) Sedimentation velocity data for full-length Mcm10 (black dotted line) and Mcm10ΔN (blue) as described in Materials and Methods. The molecular mass of the Mcm10ΔN major peak was calculated to be 68.8 kDa. Molecular masses could not be accurately determined from the full-length data. (C,D) SEC-MALS analysis of Mcm10 (C) and Mcm10ΔN (D). The UV trace is shown as a black dotted line and the light scattering trace is red. (C) Estimated molecular masses were calculated from the three shaded regions to be 90.4 kDa (I), 189.3 kDa (II), and 322.7 kDa (III). The peak at 18 min corresponds to the void volume. (D) The molecular mass of Mcm10ΔN was calculated to be 75.1±0.8 kDa.

Adachi, A globular complex formation by Nda1 and the other five members of the MCM protein family in fission yeast. 1997, Pubmed

Adachi, A globular complex formation by Nda1 and the other five members of the MCM protein family in fission yeast. 1997, Pubmed
Adams, PHENIX: building new software for automated crystallographic structure determination. 2002, Pubmed
Apger, Multiple functions for Drosophila Mcm10 suggested through analysis of two Mcm10 mutant alleles. 2010, Pubmed
Bell, DNA replication in eukaryotic cells. 2002, Pubmed
Burkhard, Coiled coils: a highly versatile protein folding motif. 2001, Pubmed
Carr, A spring-loaded mechanism for the conformational change of influenza hemagglutinin. 1993, Pubmed
Center, Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein. 1998, Pubmed
Chan, Core structure of gp41 from the HIV envelope glycoprotein. 1997, Pubmed
Chattopadhyay, Human Mcm10 regulates the catalytic subunit of DNA polymerase-alpha and prevents DNA damage during replication. 2007, Pubmed
Ciani, Molecular basis of coiled-coil oligomerization-state specificity. 2010, Pubmed
Cook, A novel zinc finger is required for Mcm10 homocomplex assembly. 2003, Pubmed
Costa, The structural basis for MCM2-7 helicase activation by GINS and Cdc45. 2011, Pubmed
Das-Bradoo, Interaction between PCNA and diubiquitinated Mcm10 is essential for cell growth in budding yeast. 2006, Pubmed
Du, Structural biology of replication initiation factor Mcm10. 2012, Pubmed
Dutta, pH-induced folding of an apoptotic coiled coil. 2001, Pubmed
Dutta, Stabilization of a pH-sensitive apoptosis-linked coiled coil through single point mutations. 2003, Pubmed
Eisenberg, Novel DNA binding properties of the Mcm10 protein from Saccharomyces cerevisiae. 2009, Pubmed
Emsley, Coot: model-building tools for molecular graphics. 2004, Pubmed
Fatoba, Human SIRT1 regulates DNA binding and stability of the Mcm10 DNA replication factor via deacetylation. 2013, Pubmed
Fien, Fission yeast Mcm10p contains primase activity. 2006, Pubmed
Fien, Primer utilization by DNA polymerase alpha-primase is influenced by its interaction with Mcm10p. 2004, Pubmed , Xenbase
Gambus, A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome. 2009, Pubmed
Gambus, GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. 2006, Pubmed
Gibbons, Formation and characterization of the trimeric form of the fusion protein of Semliki Forest Virus. 2000, Pubmed
Harbury, A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. 1993, Pubmed
Harbury, Crystal structure of an isoleucine-zipper trimer. 1994, Pubmed
Hart, Fission yeast Cdc23 interactions with DNA replication initiation proteins. 2002, Pubmed
Haworth, Ubc4 and Not4 regulate steady-state levels of DNA polymerase-α to promote efficient and accurate DNA replication. 2010, Pubmed
Heller, Eukaryotic origin-dependent DNA replication in vitro reveals sequential action of DDK and S-CDK kinases. 2011, Pubmed
Homesley, Mcm10 and the MCM2-7 complex interact to initiate DNA synthesis and to release replication factors from origins. 2000, Pubmed
Ilves, Activation of the MCM2-7 helicase by association with Cdc45 and GINS proteins. 2010, Pubmed
Im, Assembly of the Cdc45-Mcm2-7-GINS complex in human cells requires the Ctf4/And-1, RecQL4, and Mcm10 proteins. 2009, Pubmed
Izumi, The human homolog of Saccharomyces cerevisiae Mcm10 interacts with replication factors and dissociates from nuclease-resistant nuclear structures in G(2) phase. 2000, Pubmed
Kanke, Mcm10 plays an essential role in origin DNA unwinding after loading of the CMG components. 2012, Pubmed
Kobe, Crystal structure of human T cell leukemia virus type 1 gp21 ectodomain crystallized as a maltose-binding protein chimera reveals structural evolution of retroviral transmembrane proteins. 1999, Pubmed
Laskowski, AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. 1996, Pubmed
Lee, Alternative mechanisms for coordinating polymerase alpha and MCM helicase. 2010, Pubmed
Lee, The Cdc23 (Mcm10) protein is required for the phosphorylation of minichromosome maintenance complex by the Dfp1-Hsk1 kinase. 2003, Pubmed
Lee, Structural basis for inhibition of the replication licensing factor Cdt1 by geminin. 2004, Pubmed , Xenbase
Lei, Mcm2 is a target of regulation by Cdc7-Dbf4 during the initiation of DNA synthesis. 1997, Pubmed
LOWEY, COMPARATIVE STUDY OF THE ALPHA-HELICAL MUSCLE PROTEINS. TYROSYL TITRATION AND EFFECT OF PH ON CONFORMATION. 1965, Pubmed
Marvin, The rational design of allosteric interactions in a monomeric protein and its applications to the construction of biosensors. 1997, Pubmed
Merchant, A lesion in the DNA replication initiation factor Mcm10 induces pausing of elongation forks through chromosomal replication origins in Saccharomyces cerevisiae. 1997, Pubmed
Moon, A synergistic approach to protein crystallization: combination of a fixed-arm carrier with surface entropy reduction. 2010, Pubmed
Moutevelis, A periodic table of coiled-coil protein structures. 2009, Pubmed
Moyer, Isolation of the Cdc45/Mcm2-7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. 2006, Pubmed
Mueller, The structure of the dust mite allergen Der p 7 reveals similarities to innate immune proteins. 2010, Pubmed
Okorokov, Hexameric ring structure of human MCM10 DNA replication factor. 2007, Pubmed
O'Shea, Peptide 'Velcro': design of a heterodimeric coiled coil. 1993, Pubmed
O'Shea, Mechanism of specificity in the Fos-Jun oncoprotein heterodimer. 1992, Pubmed
O'Shea, X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. 1991, Pubmed
Otwinowski, Processing of X-ray diffraction data collected in oscillation mode. 1997, Pubmed
Pacek, Localization of MCM2-7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. 2006, Pubmed , Xenbase
Remus, Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. 2009, Pubmed
Ricke, A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-alpha in budding yeast. 2006, Pubmed
Ricke, Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. 2004, Pubmed
Robertson, Domain architecture and biochemical characterization of vertebrate Mcm10. 2008, Pubmed , Xenbase
Saxena, A dimerized coiled-coil domain and an adjoining part of geminin interact with two sites on Cdt1 for replication inhibition. 2004, Pubmed , Xenbase
Schuck, Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. 2000, Pubmed
Schuck, Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems. 2002, Pubmed
Sheu, Cdc7-Dbf4 phosphorylates MCM proteins via a docking site-mediated mechanism to promote S phase progression. 2006, Pubmed
Spínola-Amilibia, BRMS151-98 and BRMS151-84 are crystal oligomeric coiled coils with different oligomerization states, which behave as disordered protein fragments in solution. 2013, Pubmed
Spínola-Amilibia, The structure of BRMS1 nuclear export signal and SNX6 interacting region reveals a hexamer formed by antiparallel coiled coils. 2011, Pubmed
Tanaka, CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast. 2007, Pubmed
Thépaut, Crystal structure of the coiled-coil dimerization motif of geminin: structural and functional insights on DNA replication regulation. 2004, Pubmed
Thu, Enigmatic roles of Mcm10 in DNA replication. 2013, Pubmed
van Deursen, Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation. 2012, Pubmed
Warren, Physical interactions between Mcm10, DNA, and DNA polymerase alpha. 2009, Pubmed
Watase, Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, Cdc45-Mcm-GINS. 2012, Pubmed
Wigge, Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. 1998, Pubmed
Wohlschlegel, Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45. 2002, Pubmed , Xenbase
Woolfson, The design of coiled-coil structures and assemblies. 2005, Pubmed
Xu, MCM10 mediates RECQ4 association with MCM2-7 helicase complex during DNA replication. 2009, Pubmed , Xenbase
Yang, Nuclear distribution and chromatin association of DNA polymerase alpha-primase is affected by TEV protease cleavage of Cdc23 (Mcm10) in fission yeast. 2005, Pubmed
Zegerman, Phosphorylation of Sld2 and Sld3 by cyclin-dependent kinases promotes DNA replication in budding yeast. 2007, Pubmed
Zhu, Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. 2007, Pubmed , Xenbase