Co-localization of chicken DNA topoisomerase IIalpha, but not beta, with sites of DNA replication and possible involvement of a C-terminal region of alpha through its binding to PCNA

Chromosoma. 2001 May;110(2):102-14. doi: 10.1007/s004120100140.


Clones for DNA topoisomerase IIalpha and beta (topo-IIalpha and beta) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIalpha and beta were about 80% identical for the N-terminal ATPase domain and the central core domain but only 37% for the C-terminal domain. Polyclonal antibodies were raised against C-terminal polypeptides specific to topo-IIalpha and beta. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIalpha was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2'-deoxyuridine, whereas topo-IIbeta was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIalpha and beta suggested that a sequence region (residues 1280-1294) in the C-terminal domain of topo-IIalpha was effective in co-localization with sites of DNA replication. This region consists of a QTxhxF motif (x, any residue; h, hydrophobic residue) followed by a KR-rich sequence, which resembles those found in several proteins known to associate with proliferating cell nuclear antigen (PCNA) or targeted to the replication factory. An in vitro pull-down assay with glutathione-S-transferase-PCNA and (His)6-tagged truncated forms of topo-IIalpha demonstrated that polypeptides containing the above region (residues 1158-1553 or 1158-1294) bound to PCNA in vitro.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antigens, Neoplasm
  • Bromodeoxyuridine / analysis
  • Chick Embryo / physiology*
  • Cloning, Molecular
  • DNA Primers / chemistry
  • DNA Replication*
  • DNA Topoisomerases, Type II / metabolism*
  • DNA-Binding Proteins
  • Fibroblasts / cytology
  • Fibroblasts / metabolism*
  • Fluorescent Antibody Technique, Indirect
  • Genetic Vectors
  • Heterochromatin / genetics
  • Heterochromatin / metabolism
  • Humans
  • In Situ Hybridization, Fluorescence
  • Molecular Sequence Data
  • Polymerase Chain Reaction
  • Proliferating Cell Nuclear Antigen / metabolism*
  • Sequence Homology, Amino Acid


  • Antigens, Neoplasm
  • DNA Primers
  • DNA-Binding Proteins
  • Heterochromatin
  • Proliferating Cell Nuclear Antigen
  • DNA Topoisomerases, Type II
  • Bromodeoxyuridine

Associated data

  • GENBANK/AB007445
  • GENBANK/AB007446
  • GENBANK/AB053163