Nucleotides within both proximal and distal parts of the consensus sequence are important for specific DNA recognition by the herpes simplex virus regulatory protein ICP4.

The herpes simplex virus type 1 regulatory protein ICP4 is a sequence specific DNA binding protein which associates with a number of different sites, some of which include the consensus ATCGTCnnnnYCGRC. In order to investigate the involvement in DNA binding of conserved bases within the consensus, we have synthesised a family of mutant oligonucleotides and tested their ability to form a complex with ICP4. We have also compared the binding specificities of bacterially expressed fragments of ICP4 which include the DNA binding domain. Mutation of most (but not all) bases in the proximal part of the consensus greatly reduced binding by ICP4, as did a mutation affecting the distal part. Most (but not all) G residues identified in methylation interference assays were required for efficient binding. While a bacterially expressed ICP4 peptide encompassing amino acid residues 252-523 bound to DNA with a specificity similar to that of the whole protein, a shorter protein (residues 275-523) had a slightly relaxed DNA binding specificity.     

Genomic targets of the serendipity beta and delta zinc finger proteins and their respective DNA recognition sites.

The closely related Drosophila serendipity (sry) beta (beta) and delta (delta) Cys2-His2 zinc finger proteins show partly overlapping in vitro DNA binding specificities and distinct patterns of binding sites on polytene chromosomes. Using a newly developed procedure, we identified genomic DNA targets for these two proteins. Both the sry beta and delta proteins protect an 18-22 base region from DNase I digestion within each analysed genomic binding site, that includes a 13 bp consensus sequence. The consensus recognition sites sry beta 5'-YCAGAGATGCGCA-3' and sry delta 5'-YTAGAGATGGRAA-3' thus differ by nucleotides at four out of 13 positions. They are determinant for specific binding of the sry beta and delta proteins, respectively, produced in Escherichia coli or present in Drosophila embryos. We further show that sry beta is the major (if not exclusive) Drosophila nuclear protein that specifically binds the 5'-CAGAGTGCGC-3' sequence. The identified sry beta genomic targets are all contained within single-copy DNA in euchromatic regions of the genome. Two out of the five characterized in detail map at cytological positions coincident with binding sites of the native sry beta protein on polytene chromosomes.     

Separation of primary structural components conferring autoregulation, transactivation, and DNA-binding properties to the herpes simplex virus transcriptional regulatory protein ICP4

A truncated ICP4 peptide which contains the amino-terminal 774 amino acids of the 1,298-amino-acid polypeptide is proficient for DNA binding, autoregulation, and transactivation of some viral genes (N. A. DeLuca and P. A. Schaffer, J. Virol. 62:732-743, 1988) and hence exhibits many of the properties characteristic of intact ICP4. To define the primary sequence important for the activities inherent in the amino-terminal half of the ICP4 molecule, insertional and deletion mutagenesis of the sequences encoding these residues were conducted. The DNA-binding activity of the molecule as assayed by the association with a consensus binding site was sensitive to insertional mutagenesis in two closely linked regions of the molecule. One region between amino acids 445 and 487 is critical for DNA binding and may contain a helix-turn-helix motif. The second region between amino acids 263 and 338 reduces the binding activity to a consensus binding site. When analyzed in the viral background, the DNA-binding activity of a peptide containing an insertion at amino acid 338 to a consensus binding site was reduced while the association with an alternative sequence was eliminated, suggesting a possible mechanism by which ICP4 may recognize a broader range of sequence elements. Mutations which eliminated DNA binding also eliminated or reduced both transactivation and autoregulation, supporting the requirement for DNA binding for these activities. Peptides that retained the deduced DNA-binding domain but lacked amino acids 143 through 210 retained the ability to associate with the consensus site and autoregulatory activity but were deficient for transactivation, demonstrating that the structural requirements for transactivation are greater than those required for autoregulation.     

Characterization of the binding sites of c1 repressor of bacteriophage P1. Evidence for multiple asymmetric sites

The repressor of bacteriophage P1, encoded by the c1 gene, is responsible for maintaining a P1 prophage in the lysogenic state. In this paper we present: (1) the sequence of the rightmost 943 base-pairs of the P1 genetic map that includes the 5'-terminal 224 base-pairs of the c1 gene plus its upstream region; (2) the construction of a plasmid that directs the production of approximately 5% of the cell's protein as P1 repressor; (3) a deletion analysis that establishes the startpoint of P1 repressor translation; (4) filter binding experiments that demonstrate that P1 repressor binds to several regions upstream from the c1 gene; (5) DNase I footprint experiments that directly identify two of the P1 repressor binding sites. Sequences very similar to the identified binding sites occur in at least 11 sites in P1, in most cases near functions known, or likely, to be controlled by repressor. From these sites we have derived the consensus binding site sequence ATTGCTCTAATAAATTT. We suggest that, unlike other phage operators, the P1 repressor binding sites lack rotational symmetry.