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Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases Α, Δ and Ε in Saccharomyces Cerevisiae

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Genetic Networks Required to Coordinate Chromosome Replication by DNA Polymerases Α, Δ and Ε in Saccharomyces Cerevisiae Genetic networks required to coordinate chromosome replication by DNA polymerases α, δ and ε in Saccharomyces cerevisiae Marion Dubarry1, Conor Lawless1, A. Peter Banks2, Simon Cockell3 and David Lydall1 1Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK 2High Throughput Screening Facility, Newcastle Biomedicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK 3Bioinformatics Support Unit, Faculty of Medical Sciences, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK Corresponding author: David Lydall [email protected] Faculty of Medical Sciences, Framlington Place, Institute for Cell and Molecular Biosciences, Cookson Building (M2.022) Newcastle University, Newcastle, NE2 4HH DOI: 10.1534/g3.115.021493 Figure S1 A R908G 1267 1463 1 376 719 794 T971A 1247 1468 exonuclease pol1-4 polymerase Zinc finger (no activity) G904D S1238G B 2182 1 123 442 638 1097 2103 2222 pol2-12 exonuclease polymerase Zinc finger non-essential N-terminal domain E2195* essential C-terminal domain C 1009 1081 P367H 1143 482 555 979 1097 cdc2-2 exonuclease polymerase N363D P603S Zinc finger Figure S1: DNA polymerase alleles used in this study A-C) Representation of the DNA polymerase defective alleles used (BY4741 background). Mutations were identified by sequencing and indicated in A) for Pol α, in B) for Pol ε and in C) for Pol δ. Conserved domains of DNA polymerases are shown (exonuclease (grey), polymerase (yellow) and zinc finger (brown)) and mutations are shown in orange. Pol α lacks exonuclease activity because the sequence of catalytic motifs in the Exo domains is destroyed (Pavlov and Shcherbakova 2010). M. Dubarry et al. 2 SI Figure S2 A a babB ab ab lyp1∆ his3∆ MDR=12.8 MDR=14.5 pol1-4 his3∆ MDR=8 MDR=5.41 MDP=6.89 MDP=6.98 MDP=6.87 MDP=6.95 a a 0.1 0.2 5e−3 b 5e−3 b 0 00.10.2 5e−4 5e-2 5e−4 5e-2 lyp1∆ rad9∆ MDR=14.1 MDR=11.9 pol1-4 rad9∆ MDR=0 MDR=0 MDP=6.61 MDP=7.02 MDP=0 MDP=0 a a 0.1 0.2 5e−3 b 5e−3 b 0 5e−4 5e-2 5e−4 5e-2 00.10.2 lyp1∆ ctf18∆ MDR=14.5 MDR=13.8 pol1-4 ctf18∆ MDR=0 MDR=0 MDP=6.81 MDP=6.84 MDP=0 MDP=0 a a 0.1 0.2 5e−3 b 5e−3 b Culture density (AU) Culture density (AU) 0 5e−4 5e-2 5e−4 5e-2 00.10.2 lyp1∆ elg1∆ MDR=12.9 MDR=12.8 pol1-4 elg1∆ MDR=4.88 MDR=4.93 MDP=7.09 MDP=7.11 MDP=6.89 MDP=6.95 a a 0.1 0.2 5e−3 b 5e−3 b 0 00.10.2 5e−4 5e-2 5e−4 5e-2 Photographs 30°C Photographs 33°C CDab a b ab ab pol2-12 his3∆ MDR=4.2 MDR=5.79 cdc2-2 his3∆ MDR=5.82 MDR=6.44 MDP=6.52 MDP=6.05 MDP=7.18 MDP=6.99 a a 0.1 0.2 5e−3 b b 5e−3 00.10.2 5e−4 5e-2 5e−4 5e-2 pol2-12 rad9∆ MDR=0 MDR=0 cdc2-2 rad9∆ MDR=0 MDR=0.183 MDP=0 MDP=0 MDP=0 MDP=4.51 a a 0.1 0.2 5e−3 b b 5e−3 0 00.10.2 5e−4 5e-2 5e−4 5e-2 MDR=0 MDR=0.372 MDR=8.93 MDR=3.66 pol2-12 ctf18∆ MDP=0 MDP=4.51 cdc2-2 ctf18∆ MDP=5.85 MDP=4.52 a a 0.1 0.2 5e−3 b b 5e−3 0 00.10.2 5e−4 5e-2 5e−4 5e-2 Culture density (AU) Culture density (AU) MDR=5.54 MDR=4.99 MDR=0 MDR=0 pol2-12 elg1∆ MDP=5.7 MDP=5.6 cdc2-2 elg1∆ MDP=0 MDP=0 a a 0.1 0.2 5e−3 b 5e−3 b 00.10.2 0 5e−4 5e-2 5e−4 5e-2 Photographs 36°C Photographs 30°C Figure S2: Quantitative fitness analysis A) First eleven photographs of a QFA time course showing two representative replicates (a and b) of his3Δ, rad9Δ, ctf18Δ and elg1Δ gene deletions combined with lyp1Δ (control) at 30°C (left panel). QFA growth curves (linear on the left and logarithmic on the right) for each of the double-mutants (a and b) are presented in the left panel at 30°C for 2.5 days (right panel). Cell density of individual cultures was determined after image-analysis. The generalized logistic growth model is fitted to each culture density time-series. Fitness is defined as the product of the MDR and MDP values. B) As in A) but gene deletions were combined with pol1-4 mutation at 33°C. C) As in A) but gene deletions were combined with pol2-12 mutation at 36°C. D) As in A) but gene deletions were combined with cdc2-2 mutation at 30°C. M. Dubarry et al. 3 SI Figure S3 A B Level of the Level of the GO hierarchy GO hierarchy 1 8 2 3 4 9 5 6 7 10 8 9 11 10 11 12 12 Level of the C GO hierarchy DNA strandstrand heteroduplexheteroduplex telomeretelomere 8 DNADNA replicationreplication DNADNA recombinationrecombination DNADNA repairrepair elongationelongation formation maintenancemaintenance DNADNA strandstrand elelongationongation recombinationalrecombinational ddouble-strandouble-strand base-excisionbase-excision non-recombinationalnon-recombinational mitotic recombination 9 involvedinvolved repair break repair repairrepair repair inin DNA replicationreplication double-stranddouble-strand telomeretelomere mitochondrialmitochondrial breakbreak repairrepair DNA damagedamage 10 maintenancemaintenance double-stranddouble-strand via homologoushomologous checkpointcheckpoint viavia recombinationrecombination breakbreak repairrepair recombinationrecombination mitochondrialmitochondrial double-stranddouble-strand mitoticmitotic G1/SG1/S double-stranddouble-strand breakbreak repairrepair G1G1 DNA damage mitoticmitotic DNA 11 transitiontransition breakbreak repairrepair viavia break-inducedbreak-induced checkpointcheckpoint damage checkpoint checkpointcheckpoint viavia homologoushomologous replicationreplication recombinationrecombination mitoticmitotic G1G1 intra-Sintra-S DNA DNA damagedamage 12 damage checkpoint checkpointcheckpoint Figure S3: Use of the GOrilla visualisation tool used to identify the enriched GO terms A) A screen shot of enriched GO terms identified by GOrilla for negative genetic interaction with Pol δ mutation. The lists of enriched GO terms with the three defective DNA polymerase mutants are shown in Tables S3-5. B-C) We filtered the results (Figure 1D-F) to include enriched GO terms that annotated more than one and less than 250 genes. Then, the list was filtered to include only the last terms (black boxes) of any branch ranked at least at the eighth level (dashed box) of the GO hierarchy. The GOrilla database version was updated on May 30, 2015. M. Dubarry et al. 4 SI Figure S4 Figure S4: DIXY illustrates rrm3Δ and sgs1Δ genetic interactions with Pol ε A-C) Snapshot of DIXY web page. Static scatter plots exported from DIXY shows the mean fitness of rrm3Δ and sgs1Δ mutants in pol2-12 screen versus lyp1Δ control screen. M. Dubarry et al. 5 SI Figure S5 A B 120 0˚C) 3 Fitness (28˚C) cdc13-1 HU 100mM Fitness ( 0 20406080100 0 100 200 300 0 20406080100120 0 100 200 300 Control Fitness (30˚C) Control Fitness (30˚C) CDMre11 complex ● mre11Δ rad50Δ Distance (D) to the target mutant: xrs2Δ RAD24 DRank Description RAD24 0 1 Checkpoint protein, 80 clamp loader of the Rad17-Mec3-Ddc1 RAD17 9.8 2 Checkpoint protein, forms a clamp with Ddc1 and Mec3 Mean fitness DDC1 17.4 3 Checkpoint protein, 40 forms a clamp with Rad17 and Mec3 ● RAD9 51.8 4 DNA damage checkpoint protein 20 ● ● ● ● ● 0 60 100 120 E bre1∆ ipp1-d ylr339c-d F rad57∆ ● ncb2-d ● shr3-d ● caf40∆ smi1∆ 120 120 rad52∆ pol32∆ ● htz1∆ lea1∆ ● 80 80 ● ● ●● Mean fitness Mean fitness 40 40 ● 20 20 ●● ● ● ● ● ● 0 60 100 0 60● ●● 100 ● α HU HU Pol Pol δ Pol ε Pol α Pol δ Pol ε Controlcdc13-1 Controlcdc13-1 Figure S5: Fitness profiles of regulators of DNA replication A) QFA fitness plot comparing cdc13-1 strains at 28°C with a lyp1Δ control strain at 30°C. Positive (blue upward triangles), negative (red downward triangles) and neutral (grey dot) genetic interactions are presented. The solid grey line is a linear regression through all points and the dashed line is the line of equal fitness. B) QFA Fitness plot comparing mutants from the deletion library growing on complete synthetic media (CSM) supplemented with 100mM HU with strains growing on CSM (Andrew et al. 2013). C) Fitness profiles of gene deletions affecting mre11Δ, rad50Δ and xrs2Δ (Mre11 complex). D) List of the top four gene deletions/DAmP mutations that show similar profiles to rad24Δ when combined with control (lyp1Δ), cdc13-1, pol1-4, pol2-12 and cdc2-2 mutations and in presence of 100mM HU. E-F) Fitness profiles of yfgΔ-d that negatively affect Pol α, Pol δ and Pol ε mutations (Table 1). E) Fitness profiles of yfgΔ-d that negatively affect the fitness of cdc13-1 mutants and the three DNA polymerase mutants. F) Fitness profiles of yfgΔ-d that do not affect the fitness of cdc13-1 mutants. M. Dubarry et al. 6 SI Figure S6 A B C rad55∆ pri1-d rsc8-d ● rad51∆ ● pri2-d ctf4∆ 120 rad54∆ 80 ● ● ● ● ● Mean fitness 40 ● ● ● 20 ● ● ● 0 60 100 HU HU HU Pol α Pol δ Pol ε Pol α Pol δ Pol ε Pol α Pol δ Pol ε Controlcdc13-1 Controlcdc13-1 Controlcdc13-1 Figure S6: Fitness profiles of regulators of Pol δ and Pol α defective strains A) Fitness profiles of rad51Δ, rad54Δ, rad55Δ mutations across the screens.
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