TARGETING NATIVE DOUBLE HELIX STRUCTURES AND FUNCTIONS

描述：天然DNA靶向方法可应用于双链体DNA 在溶液中和核染色质原位。 PI正在开发本地 E.大肠杆菌RecA蛋白 互补单链（css）DNA的杂交 在存在非常大过量的情况下， 异源DNA cssDNA探针特异性且有效地靶向 任何预先选择的同源dsDNA序列，并形成一个新的，稳定的四- 含链的杂交DNA结构，即双D环。 天然 dsDNA靶向反应及其稳定的杂合中间体将是 在原地进一步表征和开发。 拟议研究 该计划将在体外双链体DNA中检测这种靶向反应， 双D环DNA的结构和热稳定性研究。 还建议检查RecA介导的天然原位靶向 单拷贝突变基因、癌基因、肿瘤抑制基因和人类 代谢性肝细胞染色质中B型肝炎病毒DNA靶序列 活性细胞核和人类肿瘤细胞。 一系列DNA插入、缺失和多个DNA碱基错配 在天然dsDNA中，靶将被表征和同源，或 错配的双D环DNA杂交体将通过化学和 DNA结构的酶探针。 体外靶向原理 将被扩展到靶向天然染色质的代谢 活性透化的间期核和转染的人细胞。 RecA蛋白介导的css DNA探针靶向天然dsDNA 间期细胞核靶点将成为p53肿瘤研究的重点 抑制基因、ERRB 2和c-myc癌基因以及突变型HPRT染色体基因 目标的 最佳的DNA探针大小、末端、链数和 将测量反应辅因子和辅助蛋白的需要量。 目标DNA组成、位置和拓扑要求也将 在核染色质中确定。 介导的染色体损伤 外源性核酸酶对细胞DNA复制的影响 和转录的天然靶向dsDNA将检查与 强调p53和c-myc杂交DNA靶点。 的特异性和 标记的cssDNA探针与天然dsDNA的杂交效率 将通过荧光原位杂交直接监测， 与共聚焦激光扫描显微镜和数字图像相结合 分析. 这些研究将为DNA提供重要的新认识 靶向代谢活性细胞核和人类细胞中的天然基因 并将使同源DNA靶向癌基因，肿瘤抑制基因， 基因或突变基因。

DESCRIPTION: 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.