Smc5/6 functions with Sgs1-Top3-Rmi1 to complete chromosome replication at natural pause sites

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.


Statistics
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Data
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Sample size
No statistical methods were used to predetermine sample size, as this study did not include animal models or human participants. Sample size was determined based on the gold standards in the field and experiments to obtain statistical significance and reproducibility. We used 2000 million cells for each 2D gel point, 20 000 cells for each flow cytometry analysis point, 10 million cells for each ChIP-on-chip sample, over 100 molecules for EM analysis).
Data exclusions No data were excluded Replication All experimental findings were reliably reproduced as indicated in the figure legends. All experiments were repeated at least 2 times to ensure reproducibility. We have not experienced cases of non-reproducible data in this study. At least three biological replicates were included for ChIP-qPCR. The mean values of 3 experiments were calculated and shown with corresponding SEM. Statistical analyses were performed using a two-tailed unpaired t-test. 2D gel experiments were repeated at least 2 times and in some cases additionally different alleles, timepoints or regions were used to confirm the results. ChIP-on-chip experiments were repeated at least twice often with different components of the same complex for confirmation.
Randomization No randomization was done because this study did not involve animals or human participants. Samples were organized into groups based on treatment and genotype. Appropriate controls were included in all experiments.

Blinding
Before each experiment, the yeast strains that were used were given numbers instead of the genotype or condition and the numbers were then connected to the strains only after analysis.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. Validation All antibodies in this study were used for Western Blot analysis in S. cerevisiae yeast samples and the bands for the respective proteins corresponded with the expected size. The application (Western Blot) and species (S. cerevisiae) were indicated on the manufacturers websites.
We have validated anti-FLAG, anti-HA and anti-MYC antibodies in Western Blot by using an S. cerevisiae yeast strain without any tag to confirm specificity. The Pgk1 antibody is commonly used as a loading control in many publications (Gay et. al., 2018).

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April 2020 Software Tiling Array Suite software (TAS) from Affymetrix, details of the code as in Bermejo et al., 2009 Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
For flow cytometry analysis, approximately 1x107 cells for each timepoint were collected and fixed in 70% ethanol. Cells were suspended in 10 mM Tris pH 7.5 buffer, and RNA and proteins were removed by RNaseA (0.4 mg/ml) and proteinase K (1 mg/ml) treatment. Subsequently, cells were stained in SYTOX green solution (1μM) and analyzed using a FACSCalibur Flow Cytometer. Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.