Abstract
Smc5/6, a member of the conserved SMC family of complexes, is essential for growth in most organisms. Its exact functions in a mitotic cell cycle are controversial, as chronic Smc5/6 loss-of-function alleles produce varying phenotypes. To circumvent this issue, we acutely depleted Smc5/6 in budding yeast and determined the first cell cycle consequences of Smc5/6 removal. We found a striking primary defect in replication of the ribosomal DNA (rDNA) array. Each rDNA repeat contains a programmed replication fork barrier (RFB) established by the Fob1 protein. Fob1 removal improves rDNA replication in Smc5/6 depleted cells, implicating Smc5/6 in the management of programmed fork pausing. A similar improvement is achieved by removing the DNA helicase Mph1 whose recombinogenic activity can be inhibited by Smc5/6 under DNA damage conditions. DNA 2D gel analyses further show that Smc5/6 loss increases recombination structures at RFB regions; moreover, mph1∆ and fob1∆ similarly reduce this accumulation. These findings point to an important mitotic role for Smc5/6 in restraining recombination events when protein barriers in rDNA stall replication forks. As rDNA maintenance influences multiple essential cellular processes, Smc5/6 likely links rDNA stability to overall mitotic growth.
Highlights
The conserved Smc5/6 complex is required during normal growth and for coping with genotoxins [1,2,3,4]
To address the roles of Smc5/6 during growth, we rapidly depleted its subunits in yeast and found the main acute effect to be defective ribosomal DNA duplication
The ribosomal DNA (rDNA) contains hundreds of sites that can pause replication forks; these must be carefully managed for cells to finish replication
Summary
The conserved Smc5/6 complex (or Smc5/6) is required during normal growth and for coping with genotoxins [1,2,3,4]. One smc allele (smc6-56) impairs replication of longer chromosomes while another (smc6-9) only diminishes the duplication of chromosome XII (Chr XII), which harbors the entire ribosomal DNA (rDNA) array [5,6,7]. The former defect was interpreted as reflecting Smc5/6 roles in replication fork rotation [6], while the mechanism for the latter defect was unclear [5,7]. Another study proposed that Smc5/6 is essential in G2, but not S phase, as fusion of Smc5/6 with a G2-cyclin, but not S-cyclin, cassette sustained cell growth [8]. It is a challenge to deconvolute chronic mutant phenotypes and derive primary roles for Smc5/6
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