The broad, long-term objectives of this proposal are (1) to understand the variety of structures formed by homopurine sequences in DNA, and their roles in biological processes such as transcription and recombination; (2) to develop simple methods for detecting bends in DNA at the nucleotide level, to then determine the sequence requirements for bending and the exact positions of bends, for both naked DNA and DNA which is bent by wrapping around proteins, particularly nucleosome cores; (3) to determine the structure of intermediates in homologous recombination; and (4) to develop a method for designing artificial """"""""restriction enzymes"""""""" having any desired sequence specificity. Each of the first three objectives would contribute significantly to an understanding of how DNA functions within cells, which is vital for a systemic attack on such major health problems as AIDS and cancer. The fourth goal would provide an important tool for mapping the human genome. Homopurine-homopyrimidine sequences are often associated with transcriptional control elements, and form altered structures sensitive to SI nuclease. I have recently obtained strong evidence, based on reactivity to chemical probes which are sensitive to DNA conformation, in support of a triple-helical model for this altered structure. This model, which requires mirror symmetry in each DNA strand, will be tested by synthesizing sequences which deviate from perfect mirror symmetry in various ways, and examining their ability to form a triple-helix, using 2-D gel electrophoresis and chemical probing. Attempts will be made to form triplexes having two parallel pyrimidine strands, which need not have symmetric sequences, and which might be formed during transcription termination or homologous recombination. Attempts will be made to detect triple-helical structures in cells by injecting antibodies to triplexes, and to stabilize the structures by UV light or chemical crosslinking. Preliminary results indicate that a particular homopurine sequence which is associated with DNA bending, transcriptional control elements and replication origins. (dA)n (dT)n, exhibits hyperreactivity to osmium tetroxide at the 3'-most thymine. Further experiments will aim at developing this observation into a simple assay for bent DNA, as well as providing information on the exact position of the bend, which is controversial. This will also permit an examination of sites of bending in the DNA wrapped around nucleosomes. Crystal structures indicate that sharp bends occur preferentially at certain positions; if these correlate with positions of natural bending or unusual flexibility, we may be able to derive a set of rules guiding the positioning of nucleosomes on DNA. A major goal is the elucidation of the structure of the paranemic joint, an intermediate in general recombination in which two homologous DNA molecules have formed a heteroduplex without the need for ends or strand breakage. There is no net linking of the heteroduplex strands; therefore any right- handed turns must be compensated for by left-handed turns. Paranemic joints are formed in vitro by both the E. coli recA protein and the eukaryotic recombinase recl. Chemical probing experiments will be used to probe the structure of joints formed by each of these recombinases, to determine whether Z-DNA is used to absorb left-handed turns, and where the rec proteins are bound. To design an artificial """"""""restriction enzyme,"""""""" oligonucleotides consisting of any desired recognition sequence will be used together with recA to form D-loops with the target DNA. These will then be cut, using either endonuclease VII from phage T4, a resolvase which cuts at D-loops, or a chemical cutting function attached to the oligonucleotide.
| Jayasena, S D; Johnston, B H (1993) Sequence limitations of triple helix formation by alternate-strand recognition. Biochemistry 32:2800-7 |
| Jayasena, V K; Johnston, B H (1993) Complement-stabilized D-loop. RecA-catalyzed stable pairing of linear DNA molecules at internal sites. J Mol Biol 230:1015-24 |
| Johnston, B H (1992) Hydroxylamine and methoxylamine as probes of DNA structure. Methods Enzymol 212:180-94 |
| Jayasena, S D; Johnston, B H (1992) Oligonucleotide-directed triple helix formation at adjacent oligopurine and oligopyrimidine DNA tracts by alternate strand recognition. Nucleic Acids Res 20:5279-88 |
| Johnston, B H (1992) Generation and detection of Z-DNA. Methods Enzymol 211:127-58 |
| Johnston, B H; Quigley, G J; Ellison, M J et al. (1991) The Z-Z junction: the boundary between two out-of-phase Z-DNA regions. Biochemistry 30:5257-63 |
