The N-terminal fingers of chicken GATA-2 and GATA-3 are independent sequence-specific DNA binding domains.

The GATA family of vertebrate DNA binding regulatory proteins are expressed in diverse tissues and at different times of development. However, the DNA binding regions of these proteins possess considerable homology and recognize a rather similar range of DNA sequence motifs. DNA binding is mediated through two domains, each containing a zinc finger. Previous results have led to the conclusion that although in some cases the N-terminal finger can contribute to specificity and strength of binding, it does not bind independently, whereas the C-terminal finger is both necessary and sufficient for binding. Here we show that although this is true for the N-terminal finger of GATA-1, those of GATA-2 and GATA-3 are capable of strong independent binding with a preference for the motif GATC. Binding requires the presence of two basic regions located on either side of the N-terminal finger. The absence of one of these near the GATA-1 N-terminal finger probably accounts for its inability to bind. The combination of a single finger and two basic regions is a new variant of a motif that has been previously found in the binding domains of other finger proteins. Our results suggest that the DNA binding properties of the N-terminal finger may help distinguish GATA-2 and GATA-3 from GATA-1 and the other GATA family members in their selective regulatory roles in vivo.     

GATA-1 bends DNA in a site-independent fashion

The DNA binding domain of GATA-1 consists of two adjacent homologous zinc fingers, of which only the C-terminal finger binds DNA independently. Solution structure studies have shown that the DNA is bent by about 15 degrees in the complex formed with the single C-terminal finger of GATA-1. The N-terminal finger stabilizes DNA binding at some sites. To determine whether it contributes to DNA bending, we have performed circular permutation DNA bending experiments with a variety of DNA-binding sites recognized by GATA-1. By using a series of full-length GATA-1, double zinc finger, and single C-terminal finger constructs, we show that GATA-1 bends DNA by about 24 degrees, irrespective of the DNA-binding site. We propose that the N- and C-terminal fingers of GATA-1 adopt different orientations when bound to different cognate DNA sites. Furthermore, we characterize circular permutation bending artifacts arising from the reduced gel mobility of the protein-DNA complexes.     

Finger-positional change in three zinc finger protein Sp1: influence of terminal finger in DNA recognition

The connection of functional modules is effective for the design of DNA binding molecules with the desired sequence specificity. C(2)H(2)-type zinc finger proteins have a tandemly repeated array structure consisting of independent finger modules and are expected to recognize any DNA sequences by permutation, multi-connection, and the substitution of various sets of zinc fingers. To investigate the effects of the replacement of the terminal finger on the DNA recognition by other fingers, we have constructed the three zinc finger peptides with finger substitution at the N- or C-terminus, Sp1(zf223), Sp1(zf323), and Sp1(zf321). From the results of gel mobility shift assays, each mutant peptide binds preferentially to the target sequence that is predicted if the fingers act in a modular fashion. The methylation interference analyses demonstrate that in the cases of the N-terminal finger substitution mutants, Sp1(zf223) and Sp1(zf323), the N-terminal finger recognizes bases to different extents from that of the wild-type peptide, Sp1(zf123). Of special interest is the fact that the N-terminal finger of the C-terminal finger substitution mutant, Sp1(zf321), shows a distinct base recognition from those of Sp1(zf123) and Sp1(zf323). DNase I footprinting analyses indicate that the C-terminal finger (active finger) induces a conformational change in the DNA in the region for the binding of the N-terminal finger (passive finger). The present results strongly suggest that the extent of base recognition of the N-terminal finger is dominated by the binding of the C-terminal finger. This information provides an important clue for the creation of a zinc finger peptide with the desired specificity, which is applicable to the design of novel drugs and biological tools.     

Solution structures of two CCHC zinc fingers from the FOG family protein U-shaped that mediate protein-protein interactions

BACKGROUND: Zinc finger domains have traditionally been regarded as sequence-specific DNA binding motifs. However, recent evidence indicates that many zinc fingers mediate specific protein-protein interactions. For instance, several zinc fingers from FOG family proteins have been shown to interact with the N-terminal zinc finger of GATA-1. RESULTS: We have used NMR spectroscopy to determine the first structures of two FOG family zinc fingers that are involved in protein-protein interactions: fingers 1 and 9 from U-shaped. These fingers resemble classical TFIIIA-like zinc fingers, with the exception of an unusual extended portion of the polypeptide backbone prior to the fourth zinc ligand. [15N,(1)H]-HSQC titrations have been used to define the GATA binding surface of USH-F1, and comparison with other FOG family proteins indicates that the recognition mechanism is conserved across species. The surface of FOG-type fingers that interacts with GATA-1 overlaps substantially with the surface through which classical fingers typically recognize DNA. This suggests that these fingers could not contact both GATA and DNA simultaneously. In addition, results from NMR, gel filtration, and sedimentation equilibrium experiments suggest that the interactions are of moderate affinity. CONCLUSIONS: Our results demonstrate unequivocally that zinc fingers comprising the classical betabetaalpha fold are capable of mediating specific contacts between proteins. The existence of this alternative function has implications for the prediction of protein function from sequence data and for the evolution of protein function.     

Functional analysis of the two zinc fingers of SRE, a GATA-type factor that negatively regulates siderophore synthesis in Neurospora crassa

Multiple GATA factors, zinc finger DNA binding proteins that recognize consensus GATA elements, exist in Neurospora crassa. One of them, SRE, is involved in controlling the iron metabolic pathway of N. crassa. In N. crassa, iron transport is mediated by a number of small cyclic peptides, known as siderophores. The siderophore synthesis pathway is negatively regulated by SRE; a loss-of-function sre mutant strain showed partial constitutive synthesis of siderophore. In the research presented here, the negative function of SRE was further confirmed by a heterokaryon test and by gene complementation. SRE was expressed as a GST fusion protein. In vitro EMSA revealed that SRE binds specifically to DNA molecules containing GATA sequence elements. Autoregulation of sre gene expression appears possible because the sre gene promoter itself contains GATA sequences. Mutations were introduced into sre that lead to amino acid substitutions in each of the zinc fingers that will disrupt their function. In vitro EMSA revealed that both N-terminal and C-terminal zinc fingers of SRE are involved in DNA binding. This feature is different from that found with the vertebrate two zinc finger GATA factors. Invivo tests, accomplished by transforming the mutant sre genes into sre rip mutant, showed that SRE with mutations in either or both zinc fingers still maintained its function under low-iron conditions. In contrast, these mutant SRE proteins fail to function under high-iron conditions. Our results predict the presence of other positive or negative regulators of the siderophore synthetic pathway.     

DNA ligase III is recruited to DNA strand breaks by a zinc finger motif homologous to that of poly(ADP-ribose) polymerase. Identification of two functionally distinct DNA binding regions within DNA ligase III.

Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.