Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes

The function of the essential cohesin-related Smc5-Smc6 complex has remained elusive, though hypomorphic mutants have defects late in recombination, in checkpoint maintenance, and in chromosome segregation. Recombination and checkpoints are not essential for viability, and Smc5-Smc6-null mutants die in lethal mitoses. This suggests that the chromosome segregation defects may be the source of lethality in irradiated Smc5-Smc6 hypomorphs. We show that in smc6 mutants, following DNA damage in interphase, chromosome arm segregation fails due to an aberrant persistence of cohesin, which is normally removed by the Separase-independent pathway. This postanaphase persistence of cohesin is not dependent on DNA damage, since the synthetic lethality of smc6 hypomorphs with a topoisomerase II mutant, defective in mitotic chromosome structure, is also due to the retention of cohesin on undamaged chromosome arms. In both cases, Separase overexpression bypasses the defect and restores cell viability, showing that defective cohesin removal is a major determinant of the mitotic lethality of Smc5-Smc6 mutants.Three essential SMC (structural maintenance of chromosomes) complexes control chromosome dynamics: condensin, cohesin, and the Smc5-Smc6 complex (37). They are composed of SMC heterodimers: Smc2 and -4 in condensin, Smc1 and -3 in cohesin, and Smc5 and -6 in Smc5-Smc6. These are large ATPases with globular N and C termini, which are separated by long coiled-coil domains. The termini interact through an ABC-like coordination of ATP through Walker A and B motifs, with the coiled-coils bending at a flexible “hinge” that acts as the SMC dimerization domain. Each complex contains a number of unique non-Smc subunits, which are likely to contribute to its unique function. Among these is a kleisin subunit, which interacts with both the SMC subunits, closing a potential ring-shaped structure (55, 61).Condensin is localized to chromosomes primarily during mitosis and is essential for mitotic chromosome condensation. Conversely, cohesin is localized primarily to interphase chromosomes and has been postulated to form a ring-shaped structure around sister chromatids to ensure their cohesion, which is important for DNA repair by homologous recombination (HR). As its name suggests, the function of the Smc5-Smc6 complex is relatively poorly understood.Scc2/4 loads cohesin onto chromosomes in G₁, and sister chromatid cohesion is established during replication via the action of the acetyltransferase Eco1. Cohesin must be removed before chromosome segregation, where cleavage of the kleisin subunit Scc1 by the protease Separase is critical (51). In Saccharomyces cerevisiae, Separase-mediated Scc1 cleavage is essential for the removal of cohesin from all loci. In mammals, most cohesin is removed from chromosome arms early in mitosis in a Separase-independent process regulated by cohesin phosphorylation (28, 76). At anaphase, Separase-dependent removal of cohesin at the kinetochores ensures sister chromatid separation. In Schizosaccharomyces pombe, cohesin is thought to be regulated in a manner similar to that in mammals; only a small fraction of the Scc1 homolog Rad21 is cleaved by Separase (70), suggesting that most cohesin is removed by a Separase-independent mechanism.Cohesin-mediated sister chromatid cohesion is required for HR (64). Cohesin is recruited to double-stranded DNA breaks (DSBs) (66) and enforces cohesion genome wide after DNA damage in S. cerevisiae (65, 74). The acetyltransferase activity of Eco1 is essential for genomewide damage-induced cohesion, acting via the acetylation of Smc3 (6, 73, 81). In human cells, small interfering RNA (siRNA) studies have suggested a requirement for Smc5-Smc6 to recruit cohesin to DSBs (57), but this is not the case in S. cerevisiae (65), so the functional relationship between these related complexes also remains to be determined.In S. cerevisiae, Smc5-Smc6 is loaded onto chromatin by the cohesin loader Scc2/4 at loci that overlap with cohesin, including at DSBs (36). Smc5-Smc6-null mutants of S. pombe die in aberrant mitoses (27, 75), though the cause of this is unknown. Genetic analyses of Smc5-Smc6 in these yeasts have focused on its role in DNA repair by utilizing viable hypomorphic mutants that are highly sensitive to DNA damage. Studies with two hypomorphic smc6 mutants, bearing the smc6-X and smc6-74 mutations, have shown that Smc5-Smc6 is required for a late stage of HR subsequent to the recruitment of the Rad51/Rad52 recombination proteins and the formation of recombination intermediates (2). smc6-74 is a mutation (A151T) in the arginine finger motif of the N-terminal globular domain, while smc6-X is a mutation (R706C) in the hinge domain. Overexpression of Brc1, a multi-BRCT domain protein, suppresses the DNA damage sensitivities of several Smc5-Smc6 mutants but does not suppress smc6-X (45, 75). smc6-74 mutants, but not smc6-X mutants, are also defective in an early response to replication fork stalling, involving the recruitment of Rad52 but not Rad51 (30).As with cohesin, the HR defects in Smc5-Smc6 hypomorphic mutants are likely to result from a more general role in chromosome organization than acting as a recombinase. Smc5-Smc6 is required for HR following irradiation or recovery from hydroxyurea (HU)-induced replication arrest (2, 18, 27, 34, 35, 71, 75). However, in contrast to the sustained checkpoint arrest of irradiated HR mutants, S. pombe Smc5-Smc6 hypomorphs, such as that with the smc6-74 mutation, enter highly aberrant mitoses following DNA damage. For DSBs induced by ionizing radiation, smc6 mutants progress into mitosis with wild-type kinetics, but, as shown by pulsed-field gel electrophoresis (PFGE), the chromosomes are highly fragmented (75). In each case, the mitotic defects are blocked by an earlier (upstream) HR defect (2, 27, 43). The chromosome segregation and recombination defects are apparent on each of the three S. pombe chromosomes and are not limited to the ribosomal DNA present on both ends of chromosome III.These aberrant mitoses of Smc5-Smc6 mutants following DNA damage either block segregation completely (the “cut” phenotype, where the division septum bisects the nucleus) or result in partially segregated chromosomes that are incompletely resolved along the division plane, with an elongated mitotic spindle. Since Smc5-Smc6 is required to maintain a damage induced checkpoint arrest, the aberrant mitoses of Smc5-Smc6 mutants could result from attempting to segregate incompletely repaired chromosomes. Alternatively, defects may reflect a role for Smc5-Smc6 in promoting chromosome segregation that is revealed in hypomorphic mutants following exogenous DNA damage but is evident in null mutants without DNA damage or with low-level endogenous lesions. Notably, while viable, the hypomorphic mutants show a high level of spontaneous aneuploidy, which is also consistent with defects in chromosome segregation (35, 75).Another characteristic of smc6 mutants in S. pombe is a strong synthetic lethality with a temperature-sensitive (ts) allele of topoisomerase II (Top2), top2-191, at a permissive temperature for top2-191 of 30°C. This lethality is due to a failure of chromosome segregation that resembles mitoses in irradiated smc6-74 cells (75). top2-191 is a A802V mutation (63), and cells with this mutation show no defects in cell cycle progression at 30°C. At 36°C, top2-191 cells enter mitosis with normal kinetics but fail to segregate chromosomes. The defects of top2-191 cells at the restrictive temperature of 36°C manifest exclusively in mitosis without an interphase delay and include defective chromosome condensation. Therefore, the top2-191 allele may not affect the postreplicative decatenation activity of Top2 in S. pombe. Rather, the smc6-top2-191 interaction may be related to the structural role played by Top2 in mitotic chromosome architecture (12, 14, 79).In vertebrate cells, defective decatenation caused by Top2 inhibition with drugs such as etoposide or doxorubicin block the rejoining of molecules cleaved by Top2. This leaves DSBs that elicit a G₂ DNA damage checkpoint response in many cell types (13, 16, 17, 38). Conversely, human cells in which Top2 has been deleted enter mitosis but show disordered chromosomes that fail to segregate (12). Thus, in S. pombe, top2-191 has a terminal phenotype more closely related to that of human cells with Top2 deleted than to that of cells with chemically inhibited Top2 that are blocked midway in the decatenation reaction.Here we have investigated the mitotic role of Smc5-Smc6 in S. pombe. We find that Smc5-Smc6 is required for the removal of cohesin from damaged chromosome arms prior to anaphase and from undamaged chromosomes when the mitotic function of Top2 is compromised. We show that a defect in cohesin removal is a major determinant of lethality in smc6 mutants and highlight the importance of coordinating Smc5-Smc6 and cohesin function in the maintenance of genome integrity.