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PMID: 7690961 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 90 ·No. 18 ·1993-09-15 ·Pages 8407-11

Taagepera S, Rao PN, Drake FH, Gorbsky GJ

Abstract

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. 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 quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction 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 II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation.

MeSH Terms
Animals Antibodies Blotting, Western Cell Line Centromere/enzymology,ultrastructure Chromosomes/enzymology,ultrastructure DNA Topoisomerases, Type II/analysis,immunology Deer Epitopes/analysis Fluorescent Antibody Technique Isoenzymes/analysis,immunology Leukemia P388 Lymphocytes Mice Mitosis Phosphoproteins/analysis,immunology Tumor Cells, Cultured
Chemicals
Antibodies Epitopes Isoenzymes Phosphoproteins DNA Topoisomerases, Type II
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Taagepera S
Department of Anatomy and Cell Biology, University of Virginia, Charlottesville 22908.
Rao P N
Drake F H
Gorbsky G J
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.90.18.8407
Published
1993-09-15
Pages
8407-11
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC47365
Subset
IM
Grants
NCRR NIH HHS · 5-SO7-RRO5431-29 · United States
External Links
PubMed source DOI: 10.1073/pnas.90.18.8407 PMC Full Text View journal details
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