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.
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.
Niimi
A
A
Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, Sendai, Japan.
Suka
N
N
Harata
M
M
Kikuchi
A
A
Mizuno
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000951:Antigens, Neoplasm; D001973:Bromodeoxyuridine / Q000032:analysis; D002642:Chick Embryo / Q000502:physiology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors; D006570:Heterochromatin / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000951:Antigens, Neoplasm; D017931:DNA Primers; D004268:DNA-Binding Proteins; D006570:Heterochromatin; D018809:Proliferating Cell Nuclear Antigen; D004250:DNA Topoisomerases, Type II; D001973:Bromodeoxyuridine