When comparing AD1 and AD2 function, we have found that AD1 is Y-27632 dihydrochloride msds responsible for dictating the zinc dose response and induction kinetics of most Zap1 target genes. In contrast, AD2 is required for full induction of only a few Zap1 targets. AD2 appears to play a more important role when zinc deficiency is combined with other stresses such as heat stress or carbon source limitation. Our studies of DNA binding regulation reported here suggest that this level of control also plays a major role in determining the zinc dose response of Zap1; when DNA binding was rendered constitutive by overexpression, Zap1 activity was much less responsive to zinc and zinc homeostasis was disrupted. Another potential role for Zap1 DNA binding regulation is that it may also contribute during transitions in zinc status. However, we found that constitutive Zap1 binding did not affect the rate at which ZRT1 mRNA levels decreased following zinc treatment of Afatinib zinc-limited cells, suggesting this is not the case. How might zinc control Zap1 DNA binding? We mapped the regulation of DNA binding activity to the Zap1 DNA binding domain alone. The five zinc fingers of the DNA binding domain are all high affinity structural zinc sites and these domains have metal bound even in zinc-limited cells. Our previous studies demonstrated that binding of zinc by each of the five fingers is required for ZRE binding. One or more low affinity regulatory zinc-binding sites may also be present in the DNA binding domain. When intracellular zinc rises to sufficiently high levels, this regulatory site could then bind zinc and interfere with DNA binding, perhaps by promoting inhibitory finger-finger interactions. We have tested this model in vitro but these experiments did not support the hypothesis. Using both electrophoretic mobility shift assays and surface plasmon resonance analysis, we have determined that Zn2+ at concentrations as high as 10 mM does not inhibit ZRE binding by purified Zap1. This value is much higher than the nanomolar levels of free zinc found within cells. Higher concentrations of zinc did inhibit Zap1 ZRE binding in vitro but this effect was nonspecific and those levels of zinc also interfered with in vitro DNA binding of control proteins that function well in zinc-replete cells. Thus, inhibition of DNA binding by zinc ions binding directly to the Zap1 DNA binding domain appears unlikely to be the mechanism that operates in vivo. An alternative model is that DNA binding activity is controlled by post-translational modification, such as phosphorylation. This form of DNA binding regulation has been demonstrated for other C2H2 zinc finger transcription factors, such as Adr1. Other work has shown that phosphorylation of the canonical TGEKP zinc finger linker region causes a strong reduction in DNA-binding affinity of C2H2 zinc finger proteins.