TARGETING NATIVE DOUBLE HELIX STRUCTURES AND FUNCTIONS

描述：天然DNA靶向方法可应用于双链DNA 在溶液和核染色质中原位染色。PI正在发展为本地的 大肠杆菌RecA蛋白催化的DNA靶向反应 互补单链DNA对的杂交 在存在非常大过量的情况下对同源dsDNA靶标的探测 异源DNA。CssDNA探针特异高效地靶向 任何预先选择的同源dsDNA序列并形成新的、稳定的四个 含有链的杂交DNA结构，双D环。原住民 DsDNA靶向反应及其稳定的杂化中间体 进一步表征并就地开发。拟议的研究 计划将通过以下方法在体外检查双链DNA中的靶向反应 双D-环DNA的结构和热稳定性研究。 还建议研究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 乙肝病毒DNA靶序列在核染色质中的代谢 活跃的细胞核和人类肿瘤细胞。 DNA插入、缺失和多个DNA碱基不匹配的光谱 在原生dsDNA中，靶标将被表征并同源或 不匹配的双D环DNA杂交将通过化学和 DNA结构的酶探针。体外靶向的原理 将扩展到以天然染色质为靶点的代谢 活性通透性间期核和转染人细胞。 RecA蛋白介导的css DNA探针靶向天然dsDNA的研究 间期核靶点将侧重于对P53肿瘤的研究 抑癌基因ERRB2、c-myc和突变型HPRT染色体基因 目标。最佳的DNA探针大小、末端、链数和 将测量反应辅助因子和辅助蛋白质的需要量。 目标DNA组成、位置和拓扑要求也将 在核染色质中被测定。细胞因子介导的染色体损伤 外源添加的核酸酶与细胞DNA复制的影响 和原生靶向dsDNA的转录将用 强调p53和c-myc杂合DNA靶点。它的特殊性和 标记单链DNA探针与天然双链DNA杂交效率的研究 将通过荧光原位杂交直接监测 与激光共聚焦扫描显微镜和数字图像相结合 分析。这些研究将提供对DNA的重要的新理解 靶向代谢活跃的细胞核和人类细胞中的天然基因 并将实现癌基因、肿瘤抑制因子的同源DNA靶向 天然人类染色质和细胞中的基因或突变基因。

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.