| REAGENT or RESOURCE | SOURCE | IDENTIFIERr |
|---|---|---|
| Antibodies | ||
| rabbit anti-CDT1 mAb [EPR17891] | Abcam | Cat# ab202067, RRID:AB_2651122 |
| rabbit anti-Geminin pAb | Atlas Antibodies | Cat# HPA049977, RRID:AB_2680978 |
| mouse anti-Cyclin A mAb [B-8] | Santa Cruz Biotech | Cat# sc-271682, RRID:AB_10709300 |
| mouse anti-PCNA mAb [PC10] | Santa Cruz Biotech | Cat# sc-56, RRID:AB_628110 |
| rabbit anti-MCM2 mAb [D7G11] XP | Cell Signaling Technology | Cat# 3619, RRID:AB_2142137 |
| rabbit anti-p21 mAb [12D1] | Cell Signaling Technology | Cat# 2947, RRID:AB_823586 |
| rabbit anti-HA tag mAb [C29F4] | Cell Signaling Technology | Cat# 3724, RRID:AB_1549585 |
| rabbit anti-CDC45 mAb [D7G6] | Cell Signaling Technology | Cat# 11881, RRID:AB_2715569 |
| rabbit anti-POLA2 pAb | Atlas Antibodies | Cat# HPA037570, RRID:AB_10672280 |
| rabbit anti-POLD2 pAb | Atlas Antibodies | Cat# HPA026745, RRID:AB_1855520 |
| rabbit anti-POLE2 pAb | Atlas Antibodies | Cat# HPA027555, RRID:AB_10610282 |
| rabbit anti-Timeless mAb [EPR5275] | Abcam | Cat# ab109512, RRID:AB_10863023 |
| rabbit anti-phospho-Chk1(S317) mAb [D12H3] | Cell Signaling Technology | Cat# 12302, RRID:AB_2783865 |
| rabbit anti-phospho-Chk2(T68) pAb | Cell Signaling Technology | Cat# 2661, RRID:AB_331479 |
| rabbit anti-phospho-histone H2A.X(S139) pAb | Cell Signaling Technology | Cat# 2577, RRID:AB_2118010 |
| rabbit anti-RPA70/RPA1 mAb [EPR3472] | Abcam | Cat# ab79398, RRID:AB_1603759 |
| mouse anti-CDC6 mAb [180.2] | Santa Cruz Biotech | Cat# sc-9964, RRID:AB_627236 |
| rabbit anti-GAPDH mAb [D16H11] XP | Cell Signaling Technology | Cat# 5174, RRID:AB_10622025 |
| mouse anti-GFP [9F9.F9] mAb | Abcam | Cat# ab1218, RRID:AB_298911 |
| rabbit anti-p27 mAb [D69C12] XP mAb | Cell Signaling Technology | Cat# 3686, RRID:AB_2077850 |
| rabbit anti-Geminin pAb | Proteintech | Cat# 10802-1-AP, RRID:AB_2110945 |
| mouse anti-GAPDH (6C5) mAb | Santa Cruz Biotech | Cat# sc-32233, RRID:AB_627679 |
| goat anti-rabbit IgG, HRP-linked Antibody | Cell Signaling Technology | Cat# 7074, RRID:AB_2099233 |
| horse anti-mouse IgG, HRP-linked Antibody | Cell Signaling Technology | Cat# 7076, RRID:AB_330924 |
| goat anti-rabbit IgG Alexa Fluor 647 | Thermo Fisher Scientific | Cat# A-21245, RRID:AB_2535813 |
| goat anti-rabbit IgG Alexa Fluor 514 | Thermo Fisher Scientific | Cat#A31558, RRID:AB_10375589 |
| goat anti-mouse IgG Alexa Fluor 647 | Thermo Fisher Scientific | Cat# A-21235, RRID:AB_2535804 |
| goat anti-mouse IgG Alexa Fluor 514 | Thermo Fisher Scientific | Cat# A-31555, RRID:AB_2536171 |
| goat anti-mouse IgG Alexa Fluor 488 | Thermo Fisher Scientific | Cat# A-11029, RRID:AB_2534088 |
| goat anti-rabbit IgG Alexa Fluor 568 | Thermo Fisher Scientific | Cat# A-11036, RRID:AB_10563566 |
| Chemicals, peptides, and recombinant proteins | ||
| DMSO | Sigma-Aldrich | Cat# D2650 |
| Hydroxyurea | Cayman Chemical | Cat# 23725 |
| AZ-20 (ATRi) | Cayman Chemical | Cat# 17589 |
| MK-1775 (WEE1i) | Cayman Chemical | Cat# 21266 |
| Doxycycline hyclate (Dox) | Sigma-Aldrich | Cat# D9891 |
| MLN-4924 | Abcam | Cat# ab216470 |
| indole-3 acetic acid (auxin) | MP Biomedicals | Cat# 0210203705 |
| BMS-650032 | Adooq Bioscience | Cat# A112955 |
| L-mimosine | Cayman Chemical | Cat# 14337 |
| Hoechst 33342 | Invitrogen | Cat# H3570 |
| 5-ethynyl-2’-deoxyuridine (EdU) | Cayman Chemical | Cat# 20518 |
| AFDye 488 picolyl azide | Click Chemistry Tools | Cat# 1276 |
| AFDye 647 picolyl azide | Click Chemistry Tools | Cat# 1300 |
| DAPI | Thermo Fisher Scientific | Cat# 62248 |
| thymidine | Sigma-Aldrich | Cat# T9250-1G |
| aphidicolin | Sigma-Aldrich | Cat# 5047440001 |
| Recombinant human CMG (CDC45, MCM2, MCM3, MCM4, MCM5, MCM6, MCM7, PSF1, PSF2, PSF3, SLD5) | This study | N/A |
| Recombinant human Pol ε (POLE1, POLE2, POLE3, POLE4) | This study | N/A |
| Recombinant human Pol α (POLA1, POLA2, PRIM1, PRIM2) | This study | N/A |
| Recombinant human Pol δ (POLD1, POLD2, POLD3, POLD4) | This study | N/A |
| Recombinant human CTF18-RFC (CTF18, CTF8, DCC1, RFC2, RFC3, RFC4, RFC5) | This study | N/A |
| Recombinant human TIMELESS-TIPIN (TIMELESS, TIPIN) | This study | N/A |
| Recombinant human AND1 | This study | N/A |
| Recominant human CLASPIN | This study | N/A |
| Recombinant human PCNA | This study | N/A |
| Recombinant human RPA (RPA1, RPA2, RPA3) | This study | N/A |
| Recombinant human CDT1 | This study | N/A |
| Recombinant human Geminin | Abcam | Cat# ab86447 |
| Deposited data | ||
| Data for plotting figures | Dryad (This study) | https://doi.org/10.5061/dryad.4xgxd2599 |
| Original images and uncropped gels | Mendeley Data (This study) | https://doi.org/10.17632/6c63g8jjg7.1 |
| Experimental models: Cell lines | ||
| Human: MCF-10A | ATCC | Cat# CRL-10317, RRID:CVCL_0598 |
| Human: MCF-10A(H2B-Turq +EYFP-PCNA) | This study | N/A |
| Human: MCF-10A(H2B-Turq + EYFP-PCNA + TetOn-CDT1-mCherry) | This study | N/A |
| Human: MCF-10A(H2B-Turq + EYFP-PCNA + TetOn-CDT1ΔCy-mCherry) | This study | N/A |
| Human: MCF-10A(H2B-Turq + EYFP-PCNA + TetOn-CDT1ΔPIP-mCherry) | This study | N/A |
| Human: MCF-10A(H2B-Turq + C-CRL4Cdt2 reporter + APC/C reporter) | This study | N/A |
| Human: MCF-10A(H2B-Turq + C-CRL4Cdt2 reporter + APC/C reporter + TetOn-GemininΔDbox) | This study | N/A |
| Human: MCF-10A(H2B-Turq + N-CRL4Cdt2 reporter + APC/C reporter) | This study | N/A |
| Human: MCF-10A(H2B-Turq + N-CRL4Cdt2 reporter + APC/C reporter + TetOn-ND-CDT1-HA) | This study | N/A |
| Human: MCF-10A(H2B-Turq + N-CRL4Cdt2 reporter + APC/C reporter + TetOn-ND-CDT1 Δ499-546-HA) | This study | N/A |
| Human: MCF-10A(H2B-Turq + N-CRL4Cdt2 reporter + APC/C reporter + TetOn-ND-CDT1R198A/R210A-HA) | This study | N/A |
| Human: MCF-10A(H2B-Turq + N-CRL4Cdt2 reporter + Cyclin E/A-CDK reporter + TetOn-ND-CDT1-HA) | This study | N/A |
| Human: MCF-10A(H2B-Turq + APC/C reporter + TetOn-CDT1-mCherry) | This study | N/A |
| Human: MCF-10A(H2B-Turq + APC/C reporter + TetOn-ND-CDT1-mCherry) | This study | N/A |
| Human: MCF-10A(H2B-Turq + APC/C reporter + Cyclin E/A-CDK reporter) | Meyer Laboratory; Cappell et al.22 | N/A |
| Human: MCF-10A(H2B-Turq + APC/C reporter + Cyclin E/A-CDK reporter + CDK4/6 reporter) | Meyer Laboratory; Yang et al.58 | N/A |
| Human: MCF-10A(H2B-Turq + N-CRL4Cdt2 reporter + APC/C reporter + Cyclin E/A-CDK reporter) | This study | N/A |
| Human: RPE-1 TP53-/- CDC6d/d | Laboratory of Arne Lindqvist ; Lemmens et al.43 | N/A |
| Human: RPE-1 TP53-/- CDC6d/d (H2B-Turq + APC/C reporter + TetOn-ND-CDT1-mCherry) | This study | N/A |
| Human: RPE-1 TP53-/- CDC6d/d (H2B-Turq + APC/C reporter + TetOn-NLS-mCherry) | This study | N/A |
| Human: U2OS | ATCC | Cat#HTB-96, RRID:CVCL_0042 |
| Human: U2OS (H2B-Turq + N-CRL4Cdt2 reporter + APC/C reporter + Cyclin E/A-CDK reporter) | This study | N/A |
| Human: U2OS GFP-CDC45 | Laboratory of Jiri Lukas; Sedlackova et al.59 | N/A |
| Human: U2OS GFP-CDC45(TetOn-ND-CDT1-mCherry) | This study | N/A |
| Human: U2OS GFP-CDC45(TetOn-ND-CDT1-HA) | This study | N/A |
| Human: HeLa | ATCC | Cat#CCL-2, RRID:CVCL_0030 |
| Human: HeLa(H2B-Turq + APC/C reporter) | This study | N/A |
| Oligonucleotides | ||
| siRNA | See Table S1 | N/A |
| Recombinant DNA | ||
| tFucci(CA)2/pCSII-EF (N-CRL4Cdt2 reporter + APC/C reporter) | RIKEN BRC: Laboratory of Atsushi Miyawaki; Sakaue-Sawano et al.15 | Cat# RDB15446 |
| pLV-hCDT1(1-100)ΔCy-mCherry-P2A-mVenus-hGeminin(1-110)-IRES-Blast (C-CRL4Cdt2 reporter + APC/C reporter) | This study | Addgene # 193139 |
| pLV-mCherry-hCDT1 (1-100)ΔCy (N-CRL4Cdt2 reporter) | This study | Addgene# 193759 |
| CSII-pEF1a-H2B-mTurquoise | Meyer Laboratory; Spencer et al.40 | N/A |
| CSII-pEF1a-hDHB(994-1087)-mVenus(Cyclin E/A-CDK reporter) | Meyer Laboratory; Spencer et al.40 | Addgene# 136461 |
| CSII-pEF1a-mVenus-hGeminin(1-110) (APC/C reporter) | Meyer Laboratory | N/A |
| pLV-mCherry-hGeminin(1-110)-IRES-Puro(APC/C reporter) | Meyer Laboratory | N/A |
| pLV-H2B-miRFP670 | Meyer Laboratory | N/A |
| pLV-hDHB(994-1087)-mTurquoise (Cyclin E/A-CDK reporter) | Meyer Laboratory | N/A |
| pLV-EYFP-PCNA | Meyer Laboratory; Hahn et al.18 | N/A |
| pLV-rtTA3-IRES-Puro | Meyer Laboratory | N/A |
| pLV-TetOn-CDT1-mCherry | This study | Addgene# 193760 |
| pLV-TetOn-ND-CDT1-mCherry | This study | Addgene# 193761 |
| pLV-TetOn-NLS-mCherry | This study | Addgene# 193762 |
| pCW-CDT1-mCherry-Puro | This study | Addgene# 193763 |
| pCW-CDT1 ΔCy-mCherry-Puro | This study | Addgene# 193764 |
| pCW-CDT1 ΔPIP-mCherry-Puro | This study | Addgene# 193765 |
| pCW-ND-CDT1-mCherry-Puro | This study | Addgene# 193766 |
| pCW-ND-CDT1-HA-Puro | This study | Addgene# 193767 |
| pCW-ND-CDT1 Δ499-546-HA-Puro | This study | Addgene# 193768 |
| pCW-ND-CDT1 ΔR198A/R210A-HA-Puro | This study | Addgene# 193769 |
| pCW-GemininΔDbox-HA-Puro | This study | Addgene# 193770 |
| pC1-ND-CDT1-mCitrine | This study | Addgene# 193771 |
| pMDLg/pRRE | Addgene: Laboratory of Didier Trono; Dull etal.60 | Addgene# 12251 |
| pRSV-rev | Addgene: Laboratory of Didier Trono; Dull etal.60 | Addgene# 12253 |
| pCMV-VSV-G | Addgene: Laboratory of Bob Weinberg;Stewart et al.61 | Addgene # 8454 |
| 2X FLAG-CDT1-pACEBac1 | This study | N/A |
| Software and algorithms | ||
| Image processing software (MATLAB) | This study (https://github.com/MeyerLab/image-analysis-ratnayeke-2022) | https://doi.org/10.5281/zenodo.7183750 |
| MATLAB R2020a | MathWorks | N/A |
| ImageJ v1.53c (Fiji distribution) | Schindelin et al.62 | N/A |
| QuickFigures (ImageJ plugin) | Mazo 2020 | N/A |
| Other | ||
| 96-well glass bottomed plates | Cellvis | Cat# P96-1.5H-N |
| Lipofectamine 2000 | Thermo Fisher Scientific | Cat# 11668019 |
| DharmaFECT 1 | Dharmacon | Cat# T-2001-03 |
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