Abstract
In this study, we used a photocross-linking method to identify specific contact of CCAAT-binding factor (CBF) subunits in a CBF-DNA complex. The analysis showed that all three subunits in the CBF-DNA complex were cross-linked to DNA and that CBF-B and CBF-C were cross-linked more strongly than CBF-A. None of the CBF-A and CBF-C subunits, which together formed a CBF-A/CBF-C heterodimer, were cross-linked without CBF-B; in contrast, CBF-B was cross-linked in the absence of CBF-A/CBF-C. No subunit of heterotrimeric CBF containing DNA-binding domain mutant of either CBF-B or CBF-C was cross-linked to DNA, and interestingly, cross-linking of CBF-B that occurred without CBF-A/CBF-C was inhibited in presence of mutant CBF-C/CBF-A heterodimer. Altogether, these results indicated that the specific DNA contact surface of each CBF subunit is generated as a result of interaction between CBF-B and CBF-A/CBF-C heterodimer and that the three CBF subunits interact interdependently with DNA to form a CBF-DNA complex. Equilibrium interactions among the three CBF subunits and between CBF subunits and DNA were studied by electrophoretic mobility shift assay. This showed that at equilibrium DNA-binding conditions, the CBF-A/CBF-C heterodimer is very stable, but association between CBF-B and CBF-A/CBF-C is very weak. The nature of the association of CBF-B with CBF-A/CBF-C was also revealed by studying the inhibition of CBF-DNA complex formation by the mutant CBF-B. This study indicated that the association between CBF-B and CBF-A/CBF-C is stabilized upon interaction with DNA, a process likely to favor formation of a high-affinity CBF-DNA complex.
Highlights
In this study, we used a photocross-linking method to identify specific contact of CCAAT-binding factor (CBF) subunits in a CBF-DNA complex
Recombinant CBF subunits used in the DNA binding reactions were purified extensively from bacterial extracts; CBF-B was purified separately, whereas CBF-A and CBF-C were purified as CBF-A/CBF-C heterodimer (Fig. 2A)
BrdUrd-substituted radiolabeled DNA was first incubated with the recombinant CBF subunits; the reaction was irradiated with UV light and the photoreaction was analyzed by SDS-PAGE to detect a covalently cross-linked DNA-protein complex
Summary
We used a photocross-linking method to identify specific contact of CCAAT-binding factor (CBF) subunits in a CBF-DNA complex. No subunit of heterotrimeric CBF containing DNA-binding domain mutant of either CBF-B or CBF-C was cross-linked to DNA, and interestingly, cross-linking of CBF-B that occurred without CBF-A/CBF-C was inhibited in presence of mutant CBFC/CBF-A heterodimer These results indicated that the specific DNA contact surface of each CBF subunit is generated as a result of interaction between CBF-B and CBF-A/CBF-C heterodimer and that the three CBF subunits interact interdependently with DNA to form a CBF-DNA complex. Previous mutational analyses have shown that the conserved segment of both CBF-A and CBF-C consist of overlapping domains of subunit interactions and DNA binding [9, 10] and that the conserved segment of CBF-B, in contrast, contains two separate domains of subunit interaction and DNA binding [11] According to this analysis, large portions of the histonefold motifs of CBF-A and CBF-C interact with each other to form a CBF-A/CBF-C heterodimer, and within the heterodimer, the two subunits together generate a surface that interacts with CBF-B. To evaluate the pathway for DNA binding of the CBF subunits, we
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