Native DNA targeting methods can be applied to duplex DNA in solution and nuclear chromatin in situ. The PI is developing native DNA targeting reactions mediated by E. coli RecA protein which catalyze the hybridization of pairs of complementary single-stranded (css) DNA probes to homologous dsDNA targets in the presence of very large excesses of heterologous DNA. cssDNA probes specifically and efficiently target any pre-selected homologous dsDNA sequence and form a novel, stable four- strand-containing hybrid DNA structure, the double-D-loop. The native dsDNA targeting reaction and its stable hybrid intermediates will be further characterized and developed in situ. The proposed research program will examine this targeting reaction in duplex DNA in vitro by studies of the structures and thermal stabilities of double-D-loop DNA. It is also proposed to examine RecA-mediated native in situ targeting to single copy mutant genes, oncogenes, tumor suppressor and human hepatitis B viral DNA target sequences in chromatin of metabolically active nuclei and human tumor cells. A spectrum of DNA insertions, deletions, and multiple DNA base mismatches in native dsDNA targets will be characterized and homologous or mismatched double-D-loop DNA hybrids will be analyzed by chemical and enzymatic probes of DNA structure. The principle of in vitro targeting will be extended to targeting in native chromatin of metabolically active permeablized interphase nuclei and transfected human cells. RecA-protein-mediated targeting of css DNA probes to native dsDNA targets in interphase nuclei will emphasize studies of p53 tumor suppressor, ERRB2 and c-myc oncogene, and mutant HPRT chromosomal gene targets. The optimal DNA probe sizes, ends, number of strands, and reaction co-factor and accessory protein requirements will be measured. Target DNA composition, location and topological requirements will also be determined in nuclear chromatin. Chromosomal damage mediated by exogenously added nucleases and the effects of cellular DNA replication and transcription on native targeting dsDNA will be examined with emphasis on p53 and c-myc hybrid DNA targets. The specificity and efficiency of hybridization of labeled cssDNA probes with native dsDNA will be monitored directly by fluorescence in situ hybridization in conjunction with confocal laser scanning microscopy and digital image analysis. These studies will provide important new understanding of DNA targeting to native genes in metabolically active nuclei and human cells and would enable homologous DNA targeting of oncogenes, tumor suppressor genes or mutant genes in native human chromatin and cells.
