KEY RESOURCES TABLE
| REAGENT or RESOURCE | SOURCE | IDENTIFIER |
|---|---|---|
| Antibodies | ||
| Anti–FLAG (affinity gel agarose conjugate, mouse monoclonal, clone M2) | Millipore–SIGMA | Cat# A2220; RRID: AB_10063035 |
| anti–His6 antibody (Alexa fluor 488 conjugate) | Thermofisher | Cat# MA1–21315–A488; RRID: AB_2610645 |
| Bacterial and virus strains | ||
| E. coli Bl21 Rosetta2 (DE3) F–ompT hsdSB(rB– mB–) gal dcm (DE3) pRARE2 (CamR) | EMD Millipore | Cat# 714003 |
| E. coli NEB Turbo F proA+B+ lacIqD lacZ M15/ fhuA2 D(lac–proAB) glnV gal R(zgb– 210::Tn10)TetSendA1 thi–1 D(hsdS–mcrB)5 | NEB | Cat# C2984H |
| E. coli DH5alpha fhuA2 a(argF–lacZ)U169 phoA glnV44 a80a(lacZ)M15 gyrA96 recA1 relA1 endA1 thi–1 hsdR17 | NEB | Cat# C2987H |
| Chemicals, peptides, and recombinant proteins | ||
| Sc His–GFP–Rad55 | This Study | N/A |
| Sc FLAG–Rad57 | This Study | N/A |
| Sc Rad51 | Crickard et al., 2020 | N/A |
| Sc RPA–mCherry–6xHis | Crickard et al., 2020 | N/A |
| Sc RPA–6xHis | Crickard et al., 2020 | N/A |
| Sc Srs2(1–898) | Kaniecki et al., 2017 | N/A |
| Sc Srs2(1–898)–mCherry | Kaniecki et al., 2017 | N/A |
| Sc Srs2(1–898)–GFP | Kaniecki et al., 2017 | N/A |
| Sc Hed1–GFP | Crickard et al., 2018b | N/A |
| Sc Dmc1 | Crickard et al., 2018a | N/A |
| Streptavidin from Streptomyces | Millipore | Cat# S4762 |
| Albumin from bovine serum | Sigma–Aldrich | Cat# A7030 |
| Sc Ulp1–SUMO Protease | Sigma–Aldrich | Cat# SAE0067 |
| Talon Resin | Clontech | Cat# 635503 |
| HisTrap prepacked column | GE Healthcare | Cat# 17–5247–01 |
| GST–Resin | GE Healthcare | Cat# 17–0756–01 |
| 1,2–dioleoyl–sn–glycero–3–phosphocholine | Avanti Polar Lipids | Cat# 850375C |
| 1,2–dioleoyl–sn–glycero–3– phosphoethanolamine–N–(capbiotinyl)(sodium salt) | Avanti Polar Lipids | Cat# 870273P |
| 1,2–dioleoyl–sn–glycero–3–phosphoethanolamine–N–(methoxy(polyethylene glycol)–2000)(ammonium salt) | Avanti Polar Lipids | Cat# 880130P |
| Critical commercial assays | ||
| In–Fusion HD Cloning plus Kit | Clontech | Cat# 638909 |
| Deposited data | ||
| Kymographs used for all single molecule data analysis | Mendeley | https://doi.org/10.17632/wj38dgdgtp.1 |
| Experimental models: organisms/strains | ||
| S. cerevisiae strain for Rad55–Rad57 expression: yRP654 (MATα ura3–52 trp1Δ leu2Δ1 his3Δ200 pep4::HIS3 prb1Δ1.6R can1 GAL) | Gift from the L. Prakash laboratory and see Wilson et al. (2013) | N/A |
| S. cerevisiae strain for camptothecin sensitivity assays: LSY0697 (MATa met17–s, leu2–3,112, trp1–1, ura3–1, can1, his3–11,15) and LSY0848 (MATa rad55::LEU2 rad57::LEU2 met17–s leu2–3,112 trp1–1 ura3–1 can1 his3–11,15), transformed with the pESC–URA encoding His6–GFP–RAD55 and FLAG–RAD57 and containing the URA3 marker. | This Study | N/A |
| S. cerevisiae strain for live cell imaging: GFP–tagged Rad55, Rad57 and mutants thereof were expressed from the bi–directional GAL1–10 promoter on plasmids pESC–URA3–His–GFP– Rad55/FLAG–Rad57, pESC–URA3–His–GFP–Rad55–K49R/FLAG–Rad57 or pESC–URA3–His–GFP– Rad55/FLAG–Rad57–K131R in rad55Δ rad57Δ (ML1153–13B; MATa rad55Δ rad57::LEU2) or rad55Δ rad57Δ srs2Δ (ML1156–17B; MATa rad55Δ rad57::LEU2 srs2::HIS3) | This Study | ML1153–13B |
| Oligonucleotides | ||
| Primer for rolling circle replication: 5′–BIOTEG–TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT GTA AAA CGA CGG CCA GT –3′ | IDT | N/A |
| Forward PCR primer for Rad55 GFP insert: 5′– CAT CAC CAT CAC CAC CAT ATG AGC AAA GGA GAA GAA CTT TTC ACT GG –3′ | IDT | N/A |
| Reverse PCR primer for Rad55 GFP insert: 5′– GTA TAC CAA GCG ACA TGG ATC CTT TGT AGA GCT CAT CCA TGC CA –3′ | IDT | N/A |
| Forward PCR primer for Rad55–Rad57 vector: 5′– ATG TCG CTT GGT ATA CCA CTT TCC C –3′ | IDT | N/A |
| Reverse PCR primer for Rad55–Rad57 vector: 5′– GCT CAT ATG GTG GTG ATG GTG ATG CAT –3′ | IDT | N/A |
| Rad55 K49R mutation forward primer: 5′– GGG CAT TGG CAG GAC TAA TTT TGG C –3′ | IDT | N/A |
| Rad55 K49R mutation reverse primer: 5– TTA GTC CTG CCA ATG CCC GGA GG –3′ | IDT | N/A |
| Rad57 K131R mutation forward primer: 5′– GCTCTACTGGTA GATCTCAATTACTTATGCAG –3′ | IDT | N/A |
| Rad57 K131R mutation reverse primer: 5′– GTAATTGAGATCTA CCAGTAGAGCTCTCTCCG –3′ | IDT | N/A |
| Recombinant DNA | ||
| pESC(URA)–His–GFP–Rad55–FLAG–Rad57 | This study | N/A |
| pESC(URA)–His–GFP–Rad55(K49R)–FLAG–Rad57 | This study | N/A |
| pESC(URA)–His–GFP–Rad55–FLAG–Rad57(K131R) | This study | N/A |
| pET15b–GFP–Srs2(1–898) | This study | N/A |
| pET11C–SUMO–Rad51 | This study | N/A |
| p11D–sctRPA, sctRPA–GFP–70, sctRPA–mCherry | M. Wold Lab (University of Iowa) | N/A |
| Software and algorithms | ||
| Graphpad Prism 8.2 | Graphpad | https://www.graphpad.com/scientific-software/prism/ |
| Nikon Elements Ars 5.11 | Nikon | https://www.nikon.com/products/microscope-solutions/lineup/img_soft/nis-elements/table_of_features.htm |
| Other | ||
| Fisherfinest Premium Cover Glass | Fisher Scientific | 12–548–5P |
| Quartz Microscope Slides (1” × 3′’) | Finkenbeiner | N/A |
| TIRFM Microscope | This Study | N/A |