Human DNA replication-related element binding factor (hDREF) self-association via hATC domain is necessary for its nuclear accumulation and DNA binding.
We previously demonstrated that hDREF, a human homologue of Drosophila DNA replication-related element binding factor (dDREF), is a DNA-binding protein predominantly distributed with granular structures in the nucleus. Here, glutathione S-transferase pulldown and chemical cross-linking assays showed that the carboxyl-terminal hATC domain of hDREF, highly conserved among hAT transposase family members, possesses self-association activity. Immunoprecipitation analyses demonstrated that hDREF self-associates in vivo, dependent on hATC domain. Moreover, analyses using a series of hDREF mutants carrying amino acid substitutions in the hATC domain revealed that conserved hydrophobic amino acids are essential for self-association. Immunofluorescence studies further showed that all hDREF mutants lacking self-association activity failed to accumulate in the nucleus. Self-association-defective hDREF mutants also lost association with endogenous importin beta1. Moreover, electrophoretic gel-mobility shift assays revealed that the mutations completely abolished the DNA binding activity of hDREF. These results suggest that self-association of hDREF via the hATC domain is necessary for its nuclear accumulation and DNA binding. We also found that ZBED4/KIAA0637, another member of the human hAT family, also self-associates, again dependent on the hATC domain, with deletion resulting in loss of efficient nuclear accumulation. Thus, hATC domains of human hAT family members appear to have conserved functions in self-association that are required for nuclear accumulation.
Yamashita
Daisuke
D
Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan.
Komori
Hirofumi
H
Higuchi
Yoshiki
Y
Yamaguchi
Tomohiro
T
Osumi
Takashi
T
Hirose
Fumiko
F
D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D017434:Protein Structure, Tertiary; D019895:Transposases / Q000737:chemistry
D004268:DNA-Binding Proteins; D004247:DNA; D019895:Transposases