Topoisomerase II Is a Mitotic Chromosome Structural Scaffolds Component of

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. In 1977 and 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). 1706 Results from a number of laboratories suggestthat the chromatin fiber of interphase chromosomes is also topologically constrained in loops (16-22). 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 (recently reviewed in reference 25). If cellular chromosomes are constrained in closed loops, then topoisomerase 1I might play an essential role in termination of replication and for chromosome segregation. Analysis of conditional lethal topoisomerase II mutants in the unrelated yeasts Saccharomyces cerevisiae (26) and Schizosaccharomyces pornbe (27) confirms that the enzyme is required for chromosome segregation, at least in lower eucaryotes. We describe experiments performed with an antiserum against a 170,000-mol-wt chromosome scaffold polypeptide from chicken cells. Two independent lines of evidence suggest that this scaffold protein is DNA topoisomerase II. These are inhibition of topoisomerase function by anti-scaffold antibody, and comparison of binding of anti-scaffold and antitopoisomerase II antibodies to partial peptides derived from. THE JOURNAL OF CELL BIOLOGY . VOLUME 100 MAY 1985 1706M71'5 © The Rockefeller University Press 0021-952S/&5/05/'I 706/10 $I .00 on A ril 8, 2017 D ow nladed fom Published May 1, 1985