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

Abstract.

Clones for DNA topoisomerase IIα and β (topo-IIα and β) were isolated from a cDNA expression library of chicken MSB-1 cells by immunoscreening. The deduced sequences of chicken topo-IIα and β 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-IIα and β. Indirect immunofluorescence with these antibodies to chicken embryonic fibroblasts demonstrated that topo-IIα was distributed in discrete intranuclear spots, which coincided with sites of DNA replication as indicated by incorporation of 5-bromo-2′-deoxyuridine, whereas topo-IIβ was distributed rather uniformly within a nucleus. Examination of intranuclear distribution patterns of chimeric constructs between topo-IIα and β suggested that a sequence region (residues 1280–1294) in the C-terminal domain of topo-IIα 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-IIα demonstrated that polypeptides containing the above region (residues 1158–1553 or 1158–1294) bound to PCNA in vitro.