Topoisomerase II is a structural component of mitotic chromosome scaffolds.

Abstract

We have obtained a polyclonal antibody that recognizes a major polypeptide component of chicken mitotic chromosome scaffolds. This polypeptide migrates in SDS PAGE with Mr 170,000. Indirect immunofluorescence and subcellular fractionation experiments confirm that it is present in both mitotic chromosomes and interphase nuclei. Two lines of evidence suggest that this protein is DNA topoisomerase II, an abundant nuclear enzyme that controls DNA topological states: anti-scaffold antibody inhibits the strand-passing activity of DNA topoisomerase II; and both anti-scaffold antibody and an independent antibody raised against purified bovine topoisomerase II recognize identical partial proteolysis fragments of the 170,000-mol-wt scaffold protein in immunoblots. Our results suggest that topoisomerase II may be an enzyme that is also a structural protein of interphase nuclei and mitotic chromosomes.It has been known for many years that the chromatin fiber of both meiotic (1-4) and mitotic (5) chromosomes is folded into loops. 1978, Laemmli and co-workers (6-8) proposed that in mitotic chromosomes these loops are formed as a result of interactions between the nucleohistone fiber and a subgroup of chromosomal nonhistone proteins. A residual structure thought to be enriched in these nonhistone proteins was isolated by extraction of nuclease-digested chromosomes under conditions that solubilized >90% of the chromosomal proteins, including all of the histones (9). Because it retained the size and approximate shape of the mitotic chromosome, this residual structure, was termed the "chromosome scaffold." More recent studies have shown that the scaffold fraction is greatly enriched in two high molecular weight polypeptides, Sc-I (170,000 mol wt) and Sc-2 (135,000 mol wt; see reference 10).The existence of a discrete scaffold substructure in intact mitotic chromosomes remains a subject of some debate. The isolated residual scaffold comprises only 3-4% of the chromosome mass (10). Therefore, it is not surprising that direct inspection of whole mounts (11, 12) or thin sections (13) of intact chromosomes does not permit ready visualization of a scaffold substructure, although under certain conditions axial structures can be seen (14). Localization of scaffold components in intact chromosomes has been forced to wait for the availability of anti-scaffold antibodies (15). 1706Results from a number of laboratories suggestthat the chromatin fiber of interphase chromosomes is also topologically constrained in loops (16)(17)(18)(19)(20)(21)(22). ILcorre~t, this model implies that DNA topoisomerases might be required for the termination of replication. For example, the final stages of SV40 replication involve production of catenated dimers (23,24). In the absence of nicks, such strucures can only be separated by a type II DNA topoisomerase. This class of enzyme changes the linking number of circular DNA molecules in steps of two, in an ATP-dependent reaction that proceeds via production of protein-linked double strand DNA breaks ...

Sex chromosomes, synapsis, and cohesins: a complex affair

“…One of these structures is the chromosome scaffold, a structural framework that organizes along mitotic chromosomes (Paulson and Laemmli 1977) and whose main components are DNA topoisomerase IIα and the condensin subunit SMC2 (Earnshaw et al 1985;Saitoh et al 1994). Because DNA topoisomerase IIα is associated to the LEs during meiosis, it has been proposed that the scaffold and the SC may have some degree of interaction (Moens and Earnshaw 1989).…”

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

“…255 meres containing highly condensed chromatin, while the lateral loops consist of decondensed, transcriptionally active chromatin covered with numerous nascent transcripts. Because lampbrush chromosome architecture resembles so closely the proposed organization of somatic interphase chromatin and mitotic chromosomes, it is tempting to speculate that the same molecular components might be involved in the maintenance of chromatin loops.One of the best characterized components of the chromosomal scaffold is DNA topoisomerase 11 (topo 11) [4][5][6][7]. Besides its enzymatic activity that creates transient double-strand breaks in duplex DNA and passes DNA strands through one another, the protein has been shown to interact with the MAR and SAR elements [8].…”

The karyosphere capsule inRana temporariaoocytes contains structural and DNA-binding proteins

“…It can relax supercoiled DNA in an ATP- and Mg 2+ -dependent manner and represents one of the first proteins identified in the scaffold of mitotic chromosomes [51,52], demonstrating highly dynamic behavior during mitosis [53]. In meiotic cells, in particular, in diplotene oocytes of mice, topo II is abundant in the nucleoplasm, but absent in the central body of the mouse karyosphere [30].…”