1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression
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Antagonistic control of DDK binding to licensed replication origins by Mcm2 and Rad53

  1. Syafiq Abd Wahab
  2. Dirk Remus  Is a corresponding author
  1. Molecular Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, United States
  2. Weill-Cornell Graduate School of Medical Sciences, United States
Research Article
Cite this article as: eLife 2020;9:e58571 doi: 10.7554/eLife.58571
7 figures, 2 tables and 1 additional file

Figures

Figure 1 with 1 supplement
Residues 1–127 of the Mcm2 NTE are dispensable for MCM DH stability.

(A) Schematic of Mcm2 domain structure. Numbers indicate amino acid positions. The position of the TEV cleavage site is highlighted in red. NLS: Nuclear localization sequence; HBD: Histone binding domain; NTD: N-terminal domain; AAA+: ATPase domain. (B) Cdt1·MCM2-TEV was mock-treated or digested with TEV protease for 1 hr at 30°C, as indicated. Reactions were fractionated on SDS-PAGE and stained with Coomassie blue. (C) MCM loading reactions were performed on 3 kbp ARS305-containing DNA in the presence of ATPγS (γS) or ATP as indicated. DNA-bound material was washed with high-salt buffer, mock-treated or digested with TEV protease as indicated, washed again with high-salt buffer, and analyzed by SDS-PAGE and silver staining. * denotes Orc1 protein. (D) Gel-filtration analysis of purified Cdt1·MCM2-TEV following digestion with TEV protease. The digestion reaction was fractionated on a Superdex 200 column and fractions analyzed by SDS-PAGE and Coomassie stain. (E) Mcm2-7 loading reactions with either wildtype Cdt1·MCM (lanes 1+2) or Cdt1·MCM2-Δ127 (lanes 3+4). Reactions were performed either in the presence of ATPγS or ATP as indicated and DNA-beads subsequently washed with high-salt buffer. DNA-bound fractions were analyzed by SDS-PAGE and silver stain.

Figure 1—figure supplement 1
Time course analysis of Cdt1·MCM2-TEV and Cdt1·MCM2-WT cleavage by TEV protease.

Fractions of the reactions were analyzed by SDS-PAGE and Coomassie stain.

Figure 2 with 2 supplements
The Mcm2 NTE is important for DNA replication.

(A) Experimental outline. (B) In vitro DNA replication reactions were performed on naked (lanes 1–3) or chromatinized (lanes 4–6) circular plasmid DNA (p1017, 4.8 kbp). TEV protease was added to each reaction following MCM loading for 1 hr at 30°C, before addition of DDK and standard initiation/replisome factors. Chromatin replication reactions additionally contained FACT and Nhp6. Products were analyzed by 0.8% denaturing agarose gel-electrophoresis and autoradiography. Lead: Leading strand product; lag: Lagging strand product. (C) Purified Cdt1·MCM complexes containing either wildtype Mcm2 (Cdt1·MCM2-WT), Mcm2-TEV (Cdt1·MCM2-TEV), or Mcm2-2A (Cdt1·MCM2-2A).

Figure 2—figure supplement 1
FACT/Nhp6-dependent chromatin replication.

(A) Purified FACT and Nhp6. Samples were analyzed by SDS-PAGE and Coomassie stain. (B) In vitro DNA replication reaction was performed on chromatinized p470 (10 kbp) in the absence or presence of FACT and Nhp6 as indicated. Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography.

Figure 2—figure supplement 2
Attenuation of DNA synthesis in the presence of Mcm2-TEV is dependent on TEV protease cleavage.

Standard DNA replication reactions using naked or chromatinized p1017 (4.8 kbp) as template were performed with Cdt1·MCM2-TEV or Cdt1·MCM2-WT as indicated. TEV protease or mock buffer was added to the reaction following MCM loading and preceding origin activation as indicated. Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography.

The Mcm2 NTE promotes DDK function during origin activation.

(A) Experimental outline for experiment in B. Variable addition points for TEV protease are highlighted in red. (B) Standard in vitro DNA replication reactions were performed using p1017 (4.8 kb) as a template. TEV protease or mock buffer was added for 1 hr at 30°C as indicated. Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography (top). A fraction of each reaction was analyzed by SDS-PAGE and western blot using antibodies against Mcm2 and Mcm5 (bottom); note that the N-terminal epitope recognized by the Mcm2 antibody is lost after TEV protease cleavage. (C) MCM DHs assembled with Cdt1·MCM2-TEV were either mock-treated (lanes 1–4) or digested with TEV protease (lanes 5–8). DDK was subsequently added to the reactions at the indicated concentrations and reactions analyzed by SDS-PAGE and silver stain or western blot using antibodies against Mcm4 and Mcm5. (D) Plasmid unwinding assay. CMGs were assembled with Cdt1·MCM2-WT (lanes 1–4) or Cdt1·MCM2-TEV (lanes 5–8) using p79 (3 kbp) as substrate. TEV protease was added to the reactions after the MCM loading step, prior to the addition of DDK, CDK, Sld2, Sld3⋅7, Dpb11, GINS, Cdc45, Pol ε, RPA, and Mcm10 as indicated. DNA was repurified from the reaction and analyzed by native agarose gel-electrophoresis and EtBr stain. U: U-form DNA.

The Mcm2 NTE promotes binding of DDK to MCM DHs.

(A) MCM DHs were assembled on bead-immobilized DNA, washed with high-salt buffer, and subsequently incubated with ATPγS and DDK at the indicated concentrations. As a control, Cdt1·MCM was omitted from the MCM loading reaction in lane 6. After incubation with DDK, DNA-bound material was isolated and analyzed by SDS-PAGE and silver stain (top) or western blot using antibodies against Mcm7, Dbf4, or Cdc7 (bottom). (B) MCM loading reactions were carried out either in the presence of ATP (lane 1) or ATPγS (lanes 2–8). DNA beads were subsequently washed with low-salt buffer and incubated with DDK in the presence of ATPγS. DNA-bound material was analyzed as in A. (C) MCM DHs were assembled from Cdt1·MCM2-TEV, mock-treated or digested with TEV protease as indicated and incubated with purified DDK at the indicated concentrations. DNA-bound material was analyzed as in A. (D) MCM DHs were assembled from Cdt1·MCM2-TEV and mock-treated or digested with TEV protease as indicated. In lane 3, DDK was added after TEV protease, in lane 4 DDK was added before TEV protease. DDK was included at 150 nM. DNA-bound material was analyzed as in A. (E) DNA-bound DDK-MCM DH complexes were washed with buffer containing the indicated concentration of KOAc, and where indicated followed by a wash with buffer containing 500 mM NaCl. (F) MCM DHs were assembled on bead-immobilized DNA, washed to remove free ATP, and subsequently incubated with DDK in the presence of ATP or ATP analogues, as indicated. DNA-bound material was analyzed as in A.

Figure 4—source data 1

Figure 4A, silver stain.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data1-v2.pdf
Figure 4—source data 2

Figure 4 A+E, immunoblot: Mcm7, Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data2-v2.pdf
Figure 4—source data 3

Figure 4 A+E, immunoblot: Dbf4.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data3-v2.pdf
Figure 4—source data 4

Figure 4B, silver stain.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data4-v2.pdf
Figure 4—source data 5

Figure 4B, immunoblot: Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data5-v2.pdf
Figure 4—source data 6

Figure 4B, immunoblot: Dbf4.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data6-v2.pdf
Figure 4—source data 7

Figure 4C, silver stain.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data7-v2.pdf
Figure 4—source data 8

Figure 4C, immunoblot: Mcm7, Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data8-v2.pdf
Figure 4—source data 9

Figure 4C, immunoblot: Dbf4.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data9-v2.pdf
Figure 4—source data 10

Figure 4D, silver stain.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data10-v2.pdf
Figure 4—source data 11

Figure 4D, immunoblot: Mcm7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data11-v2.pdf
Figure 4—source data 12

Figure 4D, immunoblot: Dbf4.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data12-v2.pdf
Figure 4—source data 13

Figure 4D, immunoblot: Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data13-v2.pdf
Figure 4—source data 14

Figure 4E, silver stain.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data14-v2.pdf
Figure 4—source data 15

Figure 4F, silver stain.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data15-v2.pdf
Figure 4—source data 16

Figure 4F, immunoblot: Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data16-v2.pdf
Figure 4—source data 17

Figure 4F, immunoblot: Mcm7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data17-v2.pdf
Figure 4—source data 18

Figure 4F, immunoblot: Dbf4.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig4-data18-v2.pdf
Figure 5 with 1 supplement
Mcm2-WT does not rescue the Mcm2Δ127 replication defect.

(A) Standard DNA replication reaction using p1017 (4.8 kb) as template. Cdt1·MCM2-Δ127 and Cdt1·MCM2-WT were included at the MCM loading step at the indicated ratios; the total concentration of Cdt1·MCM was 80 nM in the Mcm2-7 loading reaction. (B) Quantification of total relative DNA synthesis in reactions of experiment in C. Bars represent the average of two independent experiments. (C) Lane traces of experiment in C.

Figure 5—figure supplement 1
The effect of Cdt1·MCM and DDK concentrations on DNA replication in vitro.

(A) Cdt1·MCM titration experiment using standard DNA replication conditions. Template: p1017 (4.8 kbp). Left: Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography. Right: Plot of total normalized DNA synthesis. (B) DDK titration experiment using standard DNA replication conditions, but Cdt1·MCM2-Δ127 in place of Cdt1·MCM2-WT. Template: p1017 (4.8 kbp). Left: Reaction products were analyzed by denaturing agarose gel-electrophoresis and autoradiography. Right: Plot of total normalized DNA synthesis.

Steric inhibition of DDK binding to MCM DHs by Rad53.

(A) DDK binding to purified MCM DHs was monitored in the presence of ATP and in the absence or presence of Rad53-WT or Rad53-kd, as indicated. In lanes 5+6 DDK and Rad53 were co-incubated in the presence of ATP prior to addition to DNA-bound MCM-7 DHs; in lanes 7+8 DDK was incubated with purified MCM DHs before addition of Rad53. DNA-bound material was analyzed SDS-PAGE and silver stain or western blot as indicated. (B) Standard DNA replication reaction using p1017 (4.8 kb) as template. Rad53 and DDK were either co-incubated prior to simultaneous addition after the MCM loading step (lanes 3+4) or Rad53 was added after DDK prior to the addition of activation factors (lanes 5+6). Replication products were analyzed by denaturing agarose gel-electrophoresis and autoradiography. The results of two experiment repeats are plotted in the graph on the right. (C) DDK binding to DNA-bound MCM DHs was monitored in the presence of AMP-PNP. DDK and Rad53 were either co-incubated in the presence of AMP-PNP prior to addition to purified DNA-bound MCM DHs (lane 4), or added sequentially to MCM DHs (lane 5) as indicated.

Rad53-WT, but not Rad53-kd, can form a stable complex with DDK.

(A) Gel-filtration analysis of purified Rad53-WT (top) or Rad53-kd (bottom), as indicated. Samples were analyzed by SDS-PAGE and Coomassie stain. (B) Gel-filtration analysis of Rad53-WT + DDK (top), DDK alone (center), or Rad53-kd + DDK (bottom). Samples were analyzed by SDS-PAGE and Coomassie stain or western blot, as indicated. (C) Model illustrating the inhibition of DDK-MCM DH complex formation by competitive binding of activated Rad53 to DDK.

Figure 7—source data 1

Figure 7A, Rad53-WT.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data1-v2.pdf
Figure 7—source data 2

Figure 7A, Rad53-kd.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data2-v2.pdf
Figure 7—source data 3

Figure 7B, Rad53-WT + DDK.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data3-v2.pdf
Figure 7—source data 4

Figure 7B, Rad53-WT + DDK, immunoblot: Dbf4.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data4-v2.pdf
Figure 7—source data 5

Figure 7B, Rad53-WT + DDK, immunoblot: Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data5-v2.pdf
Figure 7—source data 6

Figure 7B, DDK.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data6-v2.pdf
Figure 7—source data 7

Figure 7B, DDK, immunoblot: Dbf4, Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data7-v2.pdf
Figure 7—source data 8

Figure 7B, Rad53-kd + DDK.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data8-v2.pdf
Figure 7—source data 9

Figure 7B, Rad53-kd + DDK, immunoblot: Dbf4, Cdc7.

https://cdn.elifesciences.org/articles/58571/elife-58571-fig7-data9-v2.pdf

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional
information
Strain, strain background (Saccharomyces cerevisiae)YDR125This paperOverexpression and
purification
of FACT (see Table 1)
Strain, strain background (Saccharomyces cerevisiae)YJF38PMID:23474987Overexpression and purification of Cdt1·Mcm2-7WT
Strain, strain background (Saccharomyces cerevisiae)YMC5PMID:24566988Overexpression and purification of DDK
Strain, strain background (Saccharomyces cerevisiae)YSA11This paperOverexpression and purification of Cdt1·Mcm2-72-TEV (see Table 1)
Strain, strain background (Saccharomyces cerevisiae)YSA27This paperOverexpression and purification of Cdt1·Mcm2-72-2A (see Table 1)
Strain, strain background (Saccharomyces cerevisiae)YSA35This paperOverexpression and purification of DDK
(see Table 1)
AntibodyAnti-Cdc7 (yN-18) (goat polyclonal)Santa Cruz BiotechnologyCat. #: sc-11964
RRID:AB_638349
(1:2000)
AntibodyAnti-Dbf4 (yA-16) (goat polyclonal)Santa Cruz BiotechnologyCat. #: sc-5706
RRID:AB_637654
(1:2000)
AntibodyAnti-Mcm2 (yN-19) (goat polyclonal)Santa Cruz BiotechnologyCat. #: sc-6680
RRID:AB_648843
(1:2000)
AntibodyAnti-Mcm4 (yC-19) (goat polyclonal)Santa Cruz BiotechnologyCat. #: sc-6685
RRID:AB_648862
(1:2000)
AntibodyAnti-Mcm5 (yN-19) (goat polyclonal)Santa Cruz BiotechnologyCat. #: sc-6687
RRID:AB_648872
(1:2000)
AntibodyAnti-Mcm7 (yN-19) (goat polyclonal)Santa Cruz BiotechnologyCat. #: sc-6688
RRID:AB_647936
(1:2000)
AntibodyAnti-goat IgG-HRP (mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-6688
RRID:AB_628490
(1:5000)
Recombinant DNA reagentp79 (pARS1.4.1)PMID:3281162Plasmid unwinding assay
Recombinant DNA reagentp470 (pARS305)PMID:27989437Template for MCM loading, phosphorylation, DDK binding, and replication assays
Recombinant DNA reagentp779 (pRS306G-MCM2/FLAG-MCM3)PMID:23474987Yeast overexpression of Mcm2 and FLAG-Mcm3, template for Mcm2 modifications
Recombinant DNA reagentp993 (pRS305G-CBP-POB3++)This paperYeast overexpression of CBP-Pob3
Recombinant DNA reagentp1000 (pRS306G-SPT16++)This paperYeast overexpression of Spt16
Recombinant DNA reagentp1017 (pARS1)PMID:27989437Template for replication assay
Recombinant DNA reagentp1034 (pRS306G-MCM2-TEV/FLAG-MCM3)This paperYeast overexpression of Mcm2-TEV and FLAG-Mcm3
Recombinant DNA reagentp1035 (pet15b-NHP6)This paperBacterial overexpression of His-Nhp6
Recombinant DNA reagentp1162 (pRS306G-MCM2-2A/FLAG-MCM3)This paperYeast overexpression of Mcm2-2A and FLAG-Mcm3
Recombinant DNA reagentp1220 (pRS305G-CDC7-myc/DBF4-ybbR-FLAG)This paperYeast overexpression of DDK with Cdc7-myc and Dbf4-ybbR-FLAG
Sequence-based reagentDR772IDTPCR primer/5PCBio/CCATTATCGAAGGCA
Sequence-based reagentDR2417BioSynthesisPCR primerTACTGAAATGGTATAC[5-Fluoro-2'-dC]GGTAGATGCATAACGAATTCGCTGCGTAGCATTTGGAG
Peptide, recombinant proteinNap1 (6xHis-Nap1)PMID:27989437
Peptide, recombinant proteinISW1a (Isw1-3xFLAG)PMID:27989437
Peptide, recombinant proteinORC (CBP-Orc1)PMID:23474987
Peptide, recombinant proteinCdc6PMID:24566988
Peptide, recombinant proteinCdt1·Mcm2-7WT(3xFLAG-Mcm3)PMID:23474987
Peptide, recombinant proteinCdt1·Mcm2-72-TEV(3xFLAG-Mcm3)This paperPurified from Saccharomyces cerevisiae cells
Peptide, recombinant proteinCdt1·Mcm2-72-2A(3xFLAG-Mcm3)This paperPurified from Saccharomyces cerevisiae cells
Peptide, recombinant proteinDDK (Cdc7-myc)PMID:24566988
Peptide, recombinant proteinDDK (Dbf4-ybbR-3xFLAG/Cdc7 myc)This paperPurified from Saccharomyces cerevisiae cells (see Materials and methods)
Peptide, recombinant proteinSld3·7 (10xHis-Smt3-Sld3)PMID:27989437
Peptide, recombinant proteinCdc45 (Cdc45-3xFLAGint)PMID:27989437
Peptide, recombinant proteinCDK (Clb5-CBP)PMID:27989437
Peptide, recombinant proteinGINS (Psf1-CBP)PMID:27989437
Peptide, recombinant proteinPol ε (CBP-Pol2)PMID:27989437
Peptide, recombinant proteinDpb11 (Dpb11-CBP)PMID:32341532
Peptide, recombinant proteinSld2 (Sld2-3xFLAG)PMID:32341532
Peptide, recombinant proteinRPAPMID:27989437
Peptide, recombinant proteinPol α (CBP-Pri1)PMID:27989437
Peptide, recombinant proteinCtf4 (6xHis-Ctf4)PMID:27989437
Peptide, recombinant proteinRFC (Rfc1-FLAG-HAT)PMID:27989437
Peptide, recombinant proteinPCNA (6xHis-PCNA)PMID:27989437
Peptide, recombinant proteinPol δ (GST-Pol3)PMID:27989437
Peptide, recombinant proteinCsm3·Tof1 (CBP-Csm3)PMID:32341532
Peptide, recombinant proteinMrc1 (Mrc1-3xFLAG)PMID:32341532
Peptide, recombinant proteinMcm10 (6xHis-Mcm10)PMID:24566988
Peptide, recombinant proteinTop1 (Top1-CBP)PMID:27989437
Peptide, recombinant proteinTop2 (CBP-Top2)PMID:27989437
Peptide, recombinant proteinNhp6 (6xHis-Nhp6)This paperPurified from
E. coli BL21-CodonPlus (DE3)-RIL cells (see Materials and methods)
Peptide, recombinant proteinFACT (CBP-Pob3)This paperPurified from Saccharomyces cerevisiae cells (see Materials and methods)
Peptide, recombinant proteinRad53 (6xHis-Rad53)PMID:32341532
Peptide, recombinant proteinRad53D339A(6xHis-Rad53D339A)PMID:32341532
Peptide, recombinant proteinHpaII methyltransferaseNEBCat. #: M0214S
Commercial assay or kitSilverQuest Silver Staining KitInvitrogen (ThermoFisher)Cat. #: LC6070
Chemical compound, drugATPThermo Scientific (Thermo Fisher)Cat. #: R1441
Chemical compound, drugATPγSRoche (MilliporeSigma)Cat. #: 11162306001
Chemical compound, drugAMP-PNPRoche (MilliporeSigma)Cat. #: 10102547001
Software, algorithmImageJ softwareImageJ (http://imagej.nih.gov/ij/)RRID:SCR_003070
Software, algorithmGraphPad Prism softwareGraphPad Prism (https://graphpad.com)RRID:SCR_015807
Table 1
Yeast strains.
Strain nameGenotypePurpose
YDR125W303-1a MATa ade2-1 trp1-1 can1-100 pep4::kanMX bar::hphNAT1 (hygromycinB) his3-11,15::P/Gal 1,10-GAL4 (HIS3) leu2-3,112::P/Gal 1,10-CBP-POB3++ (LEU2) ura3-1::P/Gal 1,10-SPT16++ (URA3)Overexpression and purification of FACT
YSA11W303-1a MATa ade2-1 can1-100 pep4::kanMX bar1::hphNAT1 (hygromycinB) his3-11,15::GAL4-P/Gal1,10-CDT1 (HIS3) trp1-1::MCM5-P/Gal1,10-MCM4 (TRP1) leu2-3,112::MCM7-P/Gal1,10-MCM6 (LEU2) ura3-1::MCM2-TEV-P/Gal1,10-FLAG-MCM3 (URA3)Overexpression and purification of Cdt1·Mcm2-72-TEV
YSA27W303-1a MATa ade2-1 can1-100 pep4::kanMX bar1::hphNAT1 (hygromycinB) his3-11,15::GAL4-P/Gal1,10-CDT1 (HIS3) trp1-1::MCM5-P/Gal1,10-MCM4 (TRP1) leu2-3,112::MCM7-P/Gal1,10-MCM6 (LEU2) ura3-1::MCM2-2A-P/Gal1,10-FLAG-MCM3 (URA3)Overexpression and purification of Cdt1·Mcm2-72-2A
YSA35W303-1a MATa ade2-1 ura3-1 trp1-1 can1-100 pep4::kanMX bar::hphNAT1 (hygromycinB) his3-11,15::P/Gal 1,10-GAL4 (HIS3) leu2-3,112::CDC7-myc-P/Gal 1,10-DBF4-ybbR-FLAG (LEU2)Overexpression and purification of DDK

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