Site specific cleavage of genomic DNA mediated by triple helix formation

Physical isolation of large segments of chromosomal DNA is a major goal of human genetics. This would be greatly assisted by a generalizable technique for the cleavage of chromosomal DNA at a single site. Pyrimidine oligonucleotide directed triple helix formation is a generalizable motif for the site specific recognition of duplex DNA. The pyrimidine oligodeoxyribonucleotide is bound in the major groove parallel to the purine strand through formation of specific Hoogsteen hydrogen bonds. Specificity is derived from thymine (T) recognition of adenine-thymine (AT) base pairs (T•AT triplet) and N3 protonated cytosine (C+) recognition of guaninecytosine (GC triplet). Theoretically, the binding site size is sufficient to specify a unique site in gigabase DNA, yet the binding motif is sufficiently generalizable to recognize an endogenous site every 1000 base pairs. This thesis describes the application of oligonucleotide directed triple helix formation to bind unique target sites in bacteriophage λ, yeast, and human genomic DNA. Cleavage at the binding sites are achieved by affinity cleaving with EDTA•Fe(II) derivatized oligonucleotides, alkylation with bromoacetyl derivatized oligonucleotides, and by site specific triple helix mediated methylase inhibition followed by digestion with the cognate endonuclease. Cleavage of genomic substrates with progressively greater complexity is described. Bacteriophage λ genomic DNA (48.5 kilobase pairs) was targeted at a single endogenous homopurine site within the origin of replication. This substrate was also used to demonstrate cooperative binding of heterologous oligonucleotides to duplex DNA at contiguous binding sites. An engineered target site on yeast chromosome III (340 kilobase pairs) was cut quantitatively at a single site within total yeast genomic DNA (14 megabase pairs) by both chemical and enzymatic techniques. Techniques for the identification of endogenous triple helix target sites within unsequenced genetic markers were developed and successfully used to characterize a target site on human chromosome 4, proximal to the Huntington disease gene. As a test for the site specific cleavage of gigabase DNA, this site near the end of human chromosome 4 was cleaved by triple helix mediated enzymatic cleavage. This generated a specific 3.6 Mb fragment in greater than 80% yield that contained the entire candidate region for the Huntington mutation.