Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.     

The role of surface-exposed lysines in wrapping DNA about the bacterial histone-like protein HU

Several basic proteins, including the ubiquitous HU proteins, serve histone-like functions in prokaryotes. Significant sequence conservation exists between HU homologues; yet binding sites varying from 9 to 37 bp have been reported. TF1, an HU homologue with a 37 bp binding site that is encoded by the Bacillus subtilis bacteriophage SPO1, binds with nM affinity to DNA that contains 5-hydroxymethyluracil (hmU) in place of thymine and to T-containing DNA with loops. We evaluated the contribution of three conserved lysines to specifying the length of the binding site and show that Lys3 is critical for maintaining a long binding site in T-containing DNA: A mutant protein in which Lys3 is replaced with Gln(TF1-K3Q) is completely deficient in forming a stable complex. The affinity for 37 bp hmU-containing DNA is also reduced, from approximately 3 nM for wild-type TF1 to approximately 90 nM for TF1-K3Q. The decrease in affinity of TF1-K3Q for hmU-containing DNA > or = 25 bp suggests that Lys3 contacts DNA 8-9 bp distal to the sites of kinking. We propose that Lys3 forms an internal saltbridge to Asp26 in HU homologues characterized by shorter binding sites and that its surface exposure, and hence a longer binding site, may correlate with absence of this aspartate.     

Target Detection Assay (TDA): a versatile procedure to determine DNA binding sites as demonstrated on SP1 protein.

We developed a rapid method designated Target Detection Assay (TDA) to determine DNA binding sites for putative DNA binding proteins. A purified, functionally active DNA binding protein and a pool of random double-stranded oligonucleotides harbouring PCR primer sites at each end are included the TDA cycle which consists of four separate steps: a DNA protein incubation step, a protein DNA complex separation step, a DNA elution step and a polymerase chain reaction (PCR) DNA amplification step. The stringency of selection can be increased in consecutive TDA cycles. Since tiny amounts of retained DNA can be rescued by PCR, buffer systems, salt concentrations and competitor DNA contents can be varied in order to determine high affinity binding sites for the protein of choice. To test the efficiency of the TDA procedure potential DNA binding sites were selected by the DNA binding protein SP1 from a pool of oligonucleotides with random nucleotides at 12 positions. Target sites selected by recombinant SP1 closely matched the SP1 consensus site. If DNA recognition sites have to be determined for known, mutated or putative DNA binding proteins, the Target Detection Assay (TDA) is a versatile and rapid technique for consideration.     

Surface salt bridges modulate DNA wrapping by the type II DNA-binding protein TF1

The histone-like protein HU is involved in compaction of the bacterial genome. Up to 37 bp of DNA may be wrapped about some HU homologues in a process that has been proposed to depend on a linked disruption of surface salt bridges that liberates cationic side chains for interaction with the DNA. Despite significant sequence conservation between HU homologues, binding sites from 9 to 37 bp have been reported. TF1, an HU homologue that is encoded by Bacillus subtilis bacteriophage SPO1, has nM affinity for 37 bp preferred sites in DNA with 5-hydroxymethyluracil (hmU) in place of thymine. On the basis of electrophoretic mobility shift assays, we show that TF1-DNA complex formation is associated with a net release of only approximately 0.5 cations. The structure of TF1 suggests that Asp13 can form a dehydrated surface salt bridge with Lys23; substitution of Asp13 with Ala increases the net release of cations to approximately 1. These data are consistent with complex formation linked to disruption of surface salt bridges. Substitution of Glu90 with Ala, which would expose Lys87 predicted to contact DNA immediately distal to a proline-mediated DNA kink, causes an increase in affinity and an abrogation of the preference for hmU-containing DNA. We propose that hmU preference is due to finely tuned interactions at the sites of kinking that expose a differential flexibility of hmU- and T-containing DNA. Our data further suggest that the difference in binding site size for HU homologues is based on a differential ability to stabilize the DNA kinks.     

Digestion of highly modified bacteriophage DNA by restriction endonucleases.

The ability of thirty Type II restriction endonucleases to cleave five different types of highly modified DNA has been examined. The DNA substrates were derived from relatively large bacteriophage genomes which contain all or most of the cytosine or thymine residues substituted at the 5-position. These substituents were a proton (PBS1 DNA), a hydroxymethyl group (SP01 DNA), a methyl group (XP12 DNA), a glucosylated hydroxymethyl group (T4 DNA), or a phosphoglucuronated, glucosylated 4,5-dihydroxypentyl group (SP15 DNA). Although PBS1 DNA and SP01 DNA were digested by most of the enzymes, they were cleaved much more slowly than was normal DNA by many of them. 5-Methylcytosine-rich XP12 DNA and the multiply modified T4 and SP15 DNAs were resistant to most of these endonucleases. The only enzyme that cleaved all five of these DNAs was TaqI, which fragmented them extensively.