Structural maintenance of chromosomes (SMC) complexes, which in eukaryotic cells include cohesin, condensin and the Smc5/6 complex, are central regulators of chromosome dynamics and control sister chromatid cohesion, chromosome condensation, DNA replication, DNA repair and transcription. Even though the molecular mechanisms that lead to this large range of functions are still unclear, it has been established that the complexes execute their functions through their association with chromosomal DNA. A large set of data also indicates that SMC complexes work as intermolecular and intramolecular linkers of DNA. When combining these insights with results from ongoing analyses of their chromosomal binding, and how this interaction influences the structure and dynamics of chromosomes, a picture of how SMC complexes carry out their many functions starts to emerge.

During chromosome duplication the parental DNA molecule becomes overwound, or positively supercoiled, in the region ahead of the advancing replication fork. To allow fork progression, this superhelical tension has to be removed by topoisomerases, which operate by introducing transient DNA breaks. Positive supercoiling can also be diminished if the advancing fork rotates along the DNA helix, but then sister chromatid intertwinings form in its wake. Despite these insights it remains largely unknown how replication-induced superhelical stress is dealt with on linear, eukaryotic chromosomes. Here we show that this stress increases with the length of Saccharomyces cerevisiae chromosomes. This highlights the possibility that superhelical tension is handled on a chromosome scale and not only within topologically closed chromosomal domains as the current view predicts. We found that inhibition of type I topoisomerases leads to a late replication delay of longer, but not shorter, chromosomes. This phenotype is also displayed by cells expressing mutated versions of the cohesin- and condensin-related Smc5/6 complex. The frequency of chromosomal association sites of the Smc5/6 complex increases in response to chromosome lengthening, chromosome circularization, or inactivation of topoisomerase 2, all having the potential to increase the number of sister chromatid intertwinings. Furthermore, non-functional Smc6 reduces the accumulation of intertwined sister plasmids after one round of replication in the absence of topoisomerase 2 function. Our results demonstrate that the length of a chromosome influences the need of superhelical tension release in Saccharomyces cerevisiae, and allow us to propose a model where the Smc5/6 complex facilitates fork rotation by sequestering nascent chromatid intertwinings that form behind the replication machinery.

Abstract3‐Nitrobenzanthrone (3‐NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3‐NBA‐derived DNA adducts in rodent tissues by 32P‐postlabeling, all of which are derived from reductive metabolites of 3‐NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3‐NBA‐derived DNA adduct standards for 32P‐postlabeling by reacting N‐acetoxy‐3‐aminobenzanthrone (N‐Aco‐ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised as N‐(2′‐deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dG3′p‐C8‐N‐ABA), 2‐(2′‐deoxyguanosin‐N2‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dG3′p‐N2‐ABA) and 2‐(2′‐deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dG3′p‐C8‐C2‐ABA), and a deoxyadenosine (dA) adduct was characterised as 2‐(2′‐deoxyadenosin‐N6‐yl)‐3‐aminobenzanthrone‐3′‐phosphate (dA3′p‐N6‐ABA). 3‐NBA‐derived DNA adducts formed experimentally in vivo and in vitro were compared with the chemically synthesised adducts. The major 3‐NBA‐derived DNA adduct formed in rat lung cochromatographed with dG3′p‐N2‐ABA in two independent systems (thin layer and high‐performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3‐aminobenzanthrone (3‐ABA), the major human metabolite of 3‐NBA. Similarly, dG3′p‐C8‐N‐ABA and dA3′p‐N6‐ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3‐NBA or 3‐ABA, whereas dG3′p‐C8‐C2‐ABA did not cochromatograph with any of the adducts found in vivo. Utilizing different enzymatic systems in vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2, N,O‐acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2‐(2′‐deoxyguanosin‐N2‐yl)‐3‐aminobenzanthrone, N‐(2′‐deoxyguanosin‐8‐yl)‐3‐aminobenzanthrone and 2‐(2′‐deoxyadenosin‐N6‐yl)‐3‐aminobenzanthrone in DNA, after incubation with 3‐NBA and/or 3‐ABA. © 2005 Wiley‐Liss, Inc.

The cohesin complex, which is essential for sister chromatid cohesion and chromosome segregation, also inhibits resolution of sister chromatid intertwinings (SCIs) by the topoisomerase Top2. The cohesin-related Smc5/6 complex (Smc5/6) instead accumulates on chromosomes after Top2 inactivation, known to lead to a buildup of unresolved SCIs. This suggests that cohesin can influence the chromosomal association of Smc5/6 via its role in SCI protection. Using high-resolution ChIP-sequencing, we show that the localization of budding yeast Smc5/6 to duplicated chromosomes indeed depends on sister chromatid cohesion in wild-type and top2-4 cells. Smc5/6 is found to be enriched at cohesin binding sites in the centromere-proximal regions in both cell types, but also along chromosome arms when replication has occurred under Top2-inhibiting conditions. Reactivation of Top2 after replication causes Smc5/6 to dissociate from chromosome arms, supporting the assumption that Smc5/6 associates with a Top2 substrate. It is also demonstrated that the amount of Smc5/6 on chromosomes positively correlates with the level of missegregation in top2-4, and that Smc5/6 promotes segregation of short chromosomes in the mutant. Altogether, this shows that the chromosomal localization of Smc5/6 predicts the presence of the chromatid segregation-inhibiting entities which accumulate in top2-4 mutated cells. These are most likely SCIs, and our results thus indicate that, at least when Top2 is inhibited, Smc5/6 facilitates their resolution.

The structural maintenance of chromosome (SMC) protein complexes cohesin and condensin and the Smc5/6 complex (Smc5/6) are crucial for chromosome dynamics and stability. All contain essential ATPase domains, and cohesin and condensin interact with chromosomes through topological entrapment of DNA. However, how Smc5/6 binds DNA and chromosomes has remained largely unknown. Here, we show that purified Smc5/6 binds DNA through a mechanism that requires ATP hydrolysis by the complex and circular DNA to be established. This also promotes topoisomerase 2-dependent catenation of plasmids, suggesting that Smc5/6 interconnects two DNA molecules using ATP-regulated topological entrapment of DNA, similar to cohesin. We also show that a complex containing an Smc6 mutant that is defective in ATP binding fails to interact with DNA and chromosomes and leads to cell death with concomitant accumulation of DNA damage when overexpressed. Taken together, these results indicate that Smc5/6 executes its cellular functions through ATP-regulated intermolecular DNA linking.