DNA TOPOISOMERASE-II-ALPHA IS THE MAJOR CHROMOSOME PROTEIN RECOGNIZEDBY THE MITOTIC PHOSPHOPROTEIN ANTIBODY MPM-2

We have determined that the major mitotic phosphoprotein in chromosome s recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa i soform of topoisomerase II (topo II), the isoform predominant in proli ferating cells. As a prerequisite to making this discovery, it was nec essary to develop protocols to protect chromosomal proteins from depho sphorylation during cell extraction and chromosome isolation procedure s. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large qu antities of chromosomes from the P388D1 mouse lymphocyte cell line wer e isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold frac tion identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo IIalpha. The 180-kDa is oform, topo IIbeta, present in the isolated chromosomes in much smalle r quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo Il proteins may be critical for proper chromosome condens ation and segregation.