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PMID:11453553

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Citation

Niimi, A, Suka, N, Harata, M, Kikuchi, A and Mizuno, S (2001) 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 110:102-14

Abstract

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.

Links

PubMed

Keywords

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

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

CHICK:TOP2A

located_in

GO:0000792: heterochromatin

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TOP2A

located_in

GO:0005694: chromosome

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TOP2B

located_in

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TOP2B

NOT|located_in

GO:0000792: heterochromatin

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:PCNA

enables

GO:0019899: enzyme binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:O42130

F

Seeded From UniProt

complete

CHICK:TOP2A

part_of

GO:0000792: heterochromatin

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TOP2A

part_of

GO:0005694: chromosome

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TOP2A

enables

GO:0030332: cyclin binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:Q9DEA3

F

Seeded From UniProt

complete

CHICK:TOP2A

involved_in

GO:0006260: DNA replication

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

CHICK:TOP2B

NOT|part_of

GO:0000792: heterochromatin

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

CHICK:TOP2B

part_of

GO:0005634: nucleus

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

Notes

See also

References

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