Appendix 1—key resources table.
| Reagent type (species) or resource |
Designation | Source or reference |
Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | Rosetta (DE3) pLysS | Novagen | 70956 | N/A |
| Strain, strain background (Saccharomyces cerevisiae) | yJF1 | Frigola et al., 2013 | N/A | Background strain used for construction of yTDK5 |
| Strain, strain background (Saccharomyces cerevisiae) | ySDORC | Frigola et al., 2013 | N/A | ORC purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAM33 | Coster et al., 2014 | N/A | Cdt1-Mcm2-7 purification |
| Strain, strain background (Saccharomyces cerevisiae) | ySDK8 | On et al., 2014 | N/A | DDK purification |
| Strain, strain background (Saccharomyces cerevisiae) | yTD6 | Yeeles et al., 2015 | N/A | Sld3-7 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yTD8 | Yeeles et al., 2015 | N/A | Sld2 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yJY13 | Yeeles et al., 2015 | N/A | Cdc45 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yJY23 | Yeeles et al., 2015 | N/A | Pol α – primase purification |
| Strain, strain background (Saccharomyces cerevisiae) | yJY26 | Yeeles et al., 2015 | N/A | Dpb11 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAJ2 | Yeeles et al., 2015 | N/A | Polε purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAE31 | Yeeles et al., 2015 | N/A | RPA purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAE37 | Yeeles et al., 2015 | N/A | S-CDK purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAE40 | Yeeles et al., 2015 | N/A | Ctf4 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAE41 | Yeeles et al., 2015 | N/A | RFC purification |
| Strain, strain background (Saccharomyces cerevisiae) | yAE71 | John Diffley | N/A | Mrc1 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yTDK4 | Deegan et al., 2019 | N/A | Csm3-Tof1 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yTDK5 | This study | N/A | SCFDia2 purification MATa ade2-1 ura3-1 his3-11,15 trp1-1 leu2-3,112 can1-100 bar1∆::hphNT pep4∆::kanMX ura3::pRS306-SKP1+ProteinA-3TEV-DIA2 leu2::pRS305-HRT1+CDC53 |
| Strain, strain background (Saccharomyces cerevisiae) | yTDK6 | Deegan et al., 2019 | N/A | Top1 purification |
| Strain, strain background (Saccharomyces cerevisiae) | yTDK20 | This study | N/A | CMG purification MATa/MATα pep4∆::kanMX/pep4∆::kanMX bar1∆::hph-NT1/bar1∆::hph-NT1 ade2−1/ade2-1 ura3−1/ura3-1::pRS306-MCM2-GAL1,10-CBP-TEV-MCM3 his3-11::pRS303-CDC45iFLAG2-GAL1,10-GAL4/his3-11 trp1-1::pRS304-PSF1-GAL1,10-SLD5/trp1-1::pRS304-MCM5-GAL1,10-MCM4 leu2-3::pRS305-PSF2-GAL1,10-PSF3/leu2-3::pRS305-MCM7-GAL1,10-MCM6 ctf4-I901E/ctf4-I901E |
| Strain, strain background (Saccharomyces cerevisiae) | yPM224 | This study | N/A | CMG-Mcm7-K29A purification MATa/MATα pep4∆::kanMX/pep4∆::kanMX bar1∆::hph-NT1/bar1∆::hph-NT1 ade2−1/ade2-1 ura3−1/ura3-1::pRS306-MCM2-GAL1,10-CBP-TEV-MCM3 his3-11::pRS303-CDC45iFLAG2-GAL1,10-GAL4/his3-11 trp1-1::pRS304-PSF1-GAL1,10-SLD5/trp1-1::pRS304-MCM5-GAL1,10-MCM4 leu2-3::pRS305-PSF2-GAL1,10-PSF3/leu2-3::pRS305-MCM7-K29A-GAL1,10-MCM6 ctf4-I901E/ctf4-I901E |
| Antibody | Anti-yeast Mcm2 (sheep polyclonal) | Labib laboratory | 158 | (1:2000) |
| Antibody | Anti-yeast Mcm3 (sheep polyclonal) | Labib laboratory | 16 | (1:1000) |
| Antibody | Anti-yeast Mcm4 (sheep polyclonal) | Labib laboratory | 159 | (1:2000) |
| Antibody | Anti-yeast Mcm5 (sheep polyclonal) | Labib laboratory | 160 | (1:2000) |
| Antibody | Anti-yeast Mcm6 (sheep polyclonal) | Labib laboratory | 161 | (1:2000) |
| Antibody | Anti-yeast Mcm7 (sheep polyclonal) | Labib laboratory | 19 | (1:2000) |
| Antibody | Anti-yeast Psf1 (sheep polyclonal) | Labib laboratory | 58 | (1:2000) |
| Antibody | Anti-yeast Psf2 (sheep polyclonal) | Labib laboratory | 31 | (1:1000) |
| Antibody | Anti-yeast Psf3 (sheep polyclonal) | Labib laboratory | 33 | (1:1000) |
| Antibody | Anti-yeast Sld5 (sheep polyclonal) | Labib laboratory | 32 | (1:1000) |
| Antibody | Anti-yeast Cdc45 (sheep polyclonal) | Labib laboratory | 158 | (1:2000) |
| Antibody | Anti-yeast Ctf4 (sheep polyclonal) | Labib laboratory | 30 | (1:3000) |
| Antibody | Anti-yeast Mrc1 (sheep polyclonal) | Labib laboratory | 125 | (1:1000) |
| Antibody | Anti-yeast Pol2 (sheep polyclonal) | Labib laboratory | 11 | (1:2000) |
| Antibody | Anti-yeast Dpb2 (sheep polyclonal) | Labib laboratory | 122 | (1:2000) |
| Antibody | Anti-yeast Cdc48 (sheep polyclonal) | Labib laboratory | 90 | (1:2000) |
| Antibody | Anti-yeast Ufd1 (sheep polyclonal) | Labib laboratory | 99 | (1:2000) |
| Antibody | Anti-yeast Npl4 (sheep polyclonal) | Labib laboratory | 100 | (1:2000) |
| Antibody | Anti-yeast Cdc53 (rabbit polyclonal) | Santa Cruz Biotechnology | sc-50444 | (1:1000) |
| Antibody | Anti-yeast Skp1 (goat polyclonal) | Santa Cruz Biotechnology | sc-5328 | (1:500) |
| Antibody | Anti-sheep IgG HRP (from donkey) | Sigma-Aldrich | A3415 | (1:10000) |
| Antibody | Anti-rabbit IgG HRP (from donkey) | GE Healthcare | NA934 | (1:10000) |
| Antibody | Anti-goat IgG HRP (from rabbit) | Sigma-Aldrich | A5420 | (1:10000) |
| Recombinant DNA reagent | pAM3 | Frigola et al., 2013 | N/A | Cdc6 purification |
| Recombinant DNA reagent | pJY19 | Yeeles et al., 2017 | N/A | PCNA purification |
| Recombinant DNA reagent | pJFDJ5 | Yeeles et al., 2015 | N/A | GINS purification |
| Recombinant DNA reagent | pET28a-Mcm10 | Yeeles et al., 2015 | N/A | Mcm10 purification |
| Recombinant DNA reagent | pTF175 | Biswas et al., 2005 | N/A | FACT purification |
| Recombinant DNA reagent | pJW22 | Biswas et al., 2005 | N/A | FACT purification |
| Recombinant DNA reagent | pTDK10 | Deegan et al., 2019 | N/A | Pif1 purification |
| Recombinant DNA reagent | pTDK24 | Deegan et al., 2019 | N/A | Pif1-K264A purification |
| Recombinant DNA reagent | Ufd1 in K27SUMO | Stein et al., 2014 | N/A | Ufd1-Npl4 purification |
| Recombinant DNA reagent | Npl4 in pET21b | Stein et al., 2014 | N/A | Ufd1-Npl4 purification |
| Recombinant DNA reagent | Cdc48 in K27SUMO | Stein et al., 2014 | N/A | Cdc48 purification |
| Recombinant DNA reagent | Cdc48-FtsH in K27SUMO | Bodnar and Rapoport, 2017 | N/A | Cdc48-FtsH purification |
| Recombinant DNA reagent | FtsH in K27SUMO | Bodnar and Rapoport, 2017 | N/A | FtsH purification |
| Recombinant DNA reagent | pTDK3 | This study | N/A | SCFDia2 purification (pRS306-Skp1-Gal1-10-PrA-Dia2) |
| Recombinant DNA reagent | pTDK6 | This study | N/A | SCFDia2 purification (pRS305-Hrt1-Gal1-10-Cdc53) |
| Recombinant DNA reagent | pTDK7 | This study | N/A | Cdc34 purification (Cdc34 in pET28c vector) |
| Recombinant DNA reagent | pTDK35 | This study | N/A | Otu1 purification (Otu1 in K27SUMO vector) |
| Recombinant DNA reagent | pBS/ARS1 WTA | Marahrens and Stillman, 1992 | N/A | 3.2 kb template for in vitro DNA replication reactions |
| Recombinant DNA reagent | λ HindIII Digest | New England Biolabs | N3012S | Molecular weight marker for agarose gels |
| Sequence-based reagent | 6664 | This study | N/A |
CDC34 forward primer for construction of pTDK7 ATTCTAtctagaaataattttgtttaactttaagaaggagatataccATGAGTAGTCGCAAAAGCACCGCTTC |
| Sequence-based reagent | 6665 | This study | N/A |
CDC34 reverse primer for construction of pTDK7 atcgatCTCGAGtgatccgccctgaaaatacaggttttcTATTTTCTTTGAAACTCTTTCTACATCCTC |
| Sequence-based reagent | 8302 | This study | N/A |
OTU1 forward primer for construction of pTDK35 gaacagattggtggcATGAAACTGAAAGTTACTGGAGCAGG |
| Sequence-based reagent | 8303 | This study | N/A |
OTU1 reverse primer for construction of pTDK35 gtgcggccgcttattaTCTATTTTGGCCAAAATCAACG |
| Sequence-based reagent | Unblocked leading | This study | N/A | Leading strand template for construction of model replication fork DNA TAGAGTAGGAAGTGATGGTAAGTGATTAGAGAATTGGAGAGTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T [* denotes a phosphorothioate bond] |
| Sequence-based reagent | Biotinylated lagging (15 nt arm) | This study | N/A | Lagging strand template for construction of model replication fork DNA (15 nt 5’ flap) GGCAGGCAGGCAGGCACACACTCTCCAATTCTCTAATCACTTACCATCACTTCCTACTCTA-DesthioBiotin-TEG |
| Sequence-based reagent | Biotinylated lagging (five nt arm) | This study | N/A | Lagging strand template for construction of model replication fork DNA (5 nt 5’ flap) CAGGCACACACTCTCCAATTCTCTAATCACTTACCATCACTTCCTACTCTA-DesthioBiotin-TEG |
| Sequence-based reagent | Biotinylated lagging (no arm) | This study | N/A | Lagging strand template for construction of model replication fork DNA (no 5’ flap) ACACACTCTCCAATTCTCTAATCACTTACCATCACTTCCTACTCTA-DesthioBiotin-TEG |
| Sequence-based reagent | DBO2 | Joe Yeeles | N/A | Cy3-labelled leading strand template for construction of model replication fork DNA Cy3- TAGAGTAGGAAGTGATGGTAAGTGATTAGAGAATTGGAGAGTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT*T*T*T*T*T [* denotes a phosphorothioate bond, T is internally biotinylated] |
| Peptide, recombinant protein | ORC | Frigola et al., 2013 | N/A | N/A |
| Peptide, recombinant protein | Cdc6 | Frigola et al., 2013 | N/A | N/A |
| Peptide, recombinant protein | Cdt1- Mcm2-7 | Coster et al., 2014 | N/A | N/A |
| Peptide, recombinant protein | Mcm2-7 | Max Douglas | N/A | N/A |
| Peptide, recombinant protein | DDK | On et al., 2014 | N/A | N/A |
| Peptide, recombinant protein | S-CDK | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Sld3-7 | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Cdc45 | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Dpb11 | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Sld2 | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Pol ε | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | GINS | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Mcm10 | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Pol α - primase | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | RPA | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Ctf4 | Yeeles et al., 2015 | N/A | N/A |
| Peptide, recombinant protein | Mrc1 | Yeeles et al., 2017 | N/A | N/A |
| Peptide, recombinant protein | Csm3-Tof1 | Deegan et al., 2019 | N/A | N/A |
| Peptide, recombinant protein | RFC | Yeeles et al., 2017 | N/A | N/A |
| Peptide, recombinant protein | PCNA | Yeeles et al., 2017 | N/A | N/A |
| Peptide, recombinant protein | Top1 | Deegan et al., 2019 | N/A | N/A |
| Peptide, recombinant protein | FACT | Joe Yeeles | N/A | N/A |
| Peptide, recombinant protein | Pif1 | Deegan et al., 2019 | N/A | N/A |
| Peptide, recombinant protein | Pif1-K264A | Deegan et al., 2019 | N/A | N/A |
| Peptide, recombinant protein | CMG | This study | N/A | Details in Material and Methods |
| Peptide, recombinant protein | CMG-Mcm7-K29A | This study | N/A | Details in Material and Methods |
| Peptide, recombinant protein | Uba1 | This study | N/A | Details in Material and Methods |
| Peptide, recombinant protein | Cdc34 | This study | N/A | Details in Material and Methods |
| Peptide, recombinant protein | SCFDia2 | This study | N/A | Details in Material and Methods |
| Peptide, recombinant protein | Ubiquitin | Axel Knebel | N/A | N/A |
| Peptide, recombinant protein | USP2b | Axel Knebel | N/A | N/A |
| Peptide, recombinant protein | Ulp1 | Alexander Stein | N/A | N/A |
| Peptide, recombinant protein | Ufd1-Npl4 | Stein et al., 2014 | N/A | N/A |
| Peptide, recombinant protein | Cdc48 | Stein et al., 2014 | N/A | N/A |
| Peptide, recombinant protein | Cdc48-FtsH | Bodnar and Rapoport, 2017 | N/A | N/A |
| Peptide, recombinant protein | FtsH | Bodnar and Rapoport, 2017 | N/A | N/A |
| Software, algorithm | ImageJ | National Institute of Health | https://imagej.nih.gov/ij/ | N/A |