T7 RNA polymerase interacts with its promoter from one side of the DNA helix

The interactions of T7 RNA polymerase with its promoter DNA have been previously probed in footprinting experiments with either DNase I or (methidiumpropyl-EDTA)-Fe(II) to cleave unprotected DNA [Basu, S., & Maitra, U. (1986) J. Mol. Biol. 190, 425-437. Ikeda, R. A., & Richardson, C. C. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3614-3618]. Both of these reagents have drawbacks; DNase I is a bulky reagent and so provides low resolution, and (methidiumpropyl-EDTA)-Fe(II) intercalates into DNA and is therefore biased toward cleavage of double-stranded DNA. In this study, the interaction between the polymerase and the promoter has been probed with Fe(II)-EDTA. This reagent generates reactive hydroxyl radicals free in solution, which produces a more detailed picture of the polymerase-promoter complex. Two protected regions are observed on each of the two promoter DNA strands: from position -17 to position -13 and from position -7 to position -1 on the coding strand and from position -14 to position -9 and from position -3 to position +2 on the noncoding strand. From this pattern it is clear that if recognition occurs via double-stranded B-form DNA, then the protected regions lie on one face of the DNA helix, and therefore the enzyme must interact predominantly from one side of the DNA helix. Digestion of the DNA in a polymerase-promoter complex with a single-strand-specific endonuclease shows that a small region of the noncoding strand near position -5 is susceptible to cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Strand specificity of the topoisomerase II mediated double-stranded DNA cleavage reaction

The strand specificity of topoisomerase II mediated DNA cleavage was analyzed at the nucleotide level by characterizing the enzyme's interaction with a strong DNA recognition site. This site was isolated from the promoter region of the extrachromosomal rRNA genes of Tetrahymena thermophila and was recognized by type II topoisomerases from a variety of phylogenetically diverse eukaryotic organisms, including Drosophila, Tetrahymena, and calf thymus. When incubated with this site, topoisomerase II was found to introduce single-stranded breaks (i.e., nicks) in addition to double-stranded breaks in the nucleic acid backbone. Although the nucleotide position of cleavage on both the noncoding and coding strands of the rDNA remained unchanged, the relative ratios of single- and double-stranded DNA breaks could be varied by altering reaction conditions. Under all conditions which promoted topoisomerase II mediated DNA nicking, the enzyme displayed a 3-10-fold specificity for cleavage at the noncoding strand of its recognition site. To determine whether this specificity of topoisomerase II was due to a faster forward rate of cleavage of the noncoding strand or a slower rate of its religation, a DNA religation assay was performed. Results indicated that both the noncoding and coding strands were religated by the enzyme at approximately the same rate. Therefore, the DNA strand preference of topoisomerase II appears to be embodied in the enzyme's forward cleavage reaction.     

Contacts between 5 S DNA and Xenopus TFIIIA identified using 5-azido-2'-deoxyuridine-substituted DNA

A highly photosensitive analogue of thymidine, 5-azidodeoxyuridine 5'-triphosphate, has been incorporated into 61-base pair (bp) DNA fragments corresponding to the central region of Xenopus somatic-type 5 S RNA genes such that 5-azidodeoxyuridine replaces some or all T residues in either the coding or noncoding strand of the TFIIIA binding site. Photolysis of TFIIIA.DNA complexes formed with these probes results in efficient, sequence-specific cross-linking to the Zn-finger protein providing direct evidence that this class of proteins have contacts in the major groove of their target sequence. Of the 20 T residues present in the 61-bp probes, greater than 90% of the cross-linking occurs from two sites in the 5 S RNA gene corresponding to T residues at positions 84 and 88 in the noncoding and coding strands, respectively. Digestion by V8 protease of the complex formed with the noncoding strand probe releases peptides not bound to the DNA. Amino acid sequence analysis of the remaining, cross-linked peptides indicates the region including zinc-finger 2 plus the finger 2-3 linker is in contact with position 84. The linker region between fingers 5 and 6 is also in close proximity to the major groove somewhere upstream from position 84.     

Nuclease activity of 1,10-phenanthroline-copper ion. Conformational analysis and footprinting of the lac operon

The nuclease activity of 1,10-phenanthroline-copper [(OP)2Cu+] preferentially nicks the wild-type, Ps, and L8-UV-5 lac promoters in the conserved promoter specific sequence (Pribnow box). The preferred sites of attack of the wild-type fragment within this region are at positions -13 and -12 on the template strand. When the comparable fragment from the Ps promoter, which differs from the wild type at position -9 (T instead of C), is cleaved with (OP)2Cu+, a new strong band at position -10 in the gel patterns is clear. An apparent increase in cutting at position -11 can also be observed. The conversion of the Ps promoter to the L8-UV-5 promoter (a change from an A to a T at position -8 and a change from a C to a T at position -66) results in alteration of the relative intensities of the four prominent bands at positions -13 to -10. Most notably, the intensity at position -10 is attenuated in L8-UV-5. The hypersensitivity of the Pribnow box region to the coordination complex is also apparent if the cutting of the missense strand is analyzed. The region of strong nicking in this case ranges from positions -11 to -3, and the relative intensities of the bands depend on the primary sequence of the promoters. These data suggest that a single base change induces local variation in the DNA structure. This new structure may be responsible for the notable difference in the efficiency of the promoters. Pancreatic deoxyribonuclease I (DNase I) does not preferentially cleave the Pribnow box relative to other regions of the sequence.(ABSTRACT TRUNCATED AT 250 WORDS)