DNA Replication-related Element Binding Research Articles

Redox regulation of DNA binding activity of DREF (DNA replication-related element binding factor) in Drosophila

DREF [DRE (DNA replication-related element) binding factor] is an 80 kDa polypeptide homodimer which plays an important role in regulating cell proliferation-related genes. Both DNA binding and dimer formation activities are associated with residues 16-115 of the N-terminal region. However, the mechanisms by which DREF dimerization and DNA binding are regulated remain unknown. Here, we report that the DNA binding activity of DREF is regulated by a redox mechanism, and that the cysteine residues are involved in this regulation. Electrophoretic mobility shift analysis using Drosophila Kc cell extracts or recombinant DREF proteins indicated that the DNA binding domain is sufficient for redox regulation. Site-directed mutagenesis and transient transfection assays showed that Cys59 and/or Cys62 are critical both for DNA binding and for redox regulation, whereas Cys91 is dispensable. In addition, experiments using Kc cells indicated that the DNA binding activity and function of DREF are affected by the intracellular redox state. These findings give insight into the exact nature of DREF function in the regulation of target genes by the intracellular redox state.

Targeted expression of the DNA binding domain of DRE-binding factor, a Drosophila transcription factor, attenuates DNA replication of the salivary gland and eye imaginal disc.

The promoters of Drosophila genes encoding DNA replication-related proteins contain transcription regulatory elements consisting of an 8-bp palindromic DNA replication-related element (DRE) sequence (5'-TATCGATA). The specific DRE-binding factor (DREF), a homodimer of the polypeptide with 709 amino acid residues, is a positive trans-acting factor for transcription of DRE-containing genes. Both DRE binding and dimer formation are associated with residues 16 to 115 of the N-terminal region. We have established transgenic flies expressing the full-length DREF polypeptide or its N-terminal fragment (amino acid residues 1 to 125) under the control of the heat shock promoter, the salivary gland-specific promoter, or the eye imaginal disc-specific promoter. Heat shock induction of the N-terminal fragment during embryonic, larval, or pupal stages caused greater than 50% lethality. This lethality was overcome by coexpression of the full-length DREF. In salivary glands of the transgenic larvae expressing the N-terminal fragment, this fragment formed a homodimer and a heterodimer with the endogenous DREF. Ectopic expression of the N-terminal fragment in salivary gland cells reduced the contents of mRNAs for the 180-kDa subunit of DNA polymerase alpha and for dE2F and the extent of DNA endoreplication. Ectopic expression of the N-terminal fragment in the eye imaginal discs significantly reduced DNA replication in cells at the second mitotic wave. The lines of evidence suggest that the N-terminal fragment can impede the endogenous DREF function in a dominant negative manner and that DREF is required for normal DNA replication in both mitotic cell cycle and endo cycle.

Transcriptional regulation of the Drosophila CycA gene by the DNA replication-related element (DRE) and DRE binding factor (DREF).

The Drosophila gene for cyclin A is expressed in dividing cells throughout development. This expression pattern is similar to those of genes related to DNA replication, suggesting involvement of some common control mechanism(s). In the upstream region (-71 to -64 with respect to the transcription initiation site) of the CycA gene, we found a sequence identical to the DNA replication-related element (DRE; 5'-TATCGATA), which is important for high level expression of replication-related genes such as those encoding DNA polymerase alpha and proliferating cell nuclear antigen. Transient expression assays with chloramphenicol acetyltransferase (CAT) were carried out to examine the function of the DRE sequence of the CycA gene. Deletion or base substitution mutations resulted in an extensive reduction in CAT expression. Furthermore, monoclonal antibodies against DRE binding factor (DREF) diminished or supershifted the complex of the DREF and DRE-containing fragment. The results indicate that the Drosophila CycA gene is under the control of a DRE/DREF system, as are DNA replication-related genes.