Abstract
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.
Highlights
Nuclear antigen and DNA polymerase ␣ and dE2F [1,2,3,4]
Because hATC domains of Activator and Hermes have been shown to be involved in dimerization or multimerization [18, 19], we first examined whether the hATC domain of homologue of dDREF (hDREF) could bind to itself in vitro and in vivo
We examined whether full-length hDREF self-associates in vivo
Summary
Nuclear antigen and DNA polymerase ␣ and dE2F [1,2,3,4]. In vivo and in vitro studies from our and other groups have shown that dDREF positively regulates processes involved in DNA replication and cell growth and differentiation [5, 6]. A whole cell extract from HeLa cells simultaneously overexpressing wild-type YFP-hDREF and HAhDREF (⌬hATC), the latter being a mutant lacking the hATC domain (Fig. 1B), was subjected to immunoprecipitation with anti-HA antibodies.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
