ENZYMOLOGY OF DNA DOUBLE STRAND BREAK REJOINING

电离辐射和一些拟辐射物质诱导DNA倍增 链断裂（DSB）。细胞机制重新连接这些断裂的末端 偶尔导致染色体畸变如易位。 染色体易位可导致基因表达改变， 转化的细胞表型和肿瘤发生。参与的蛋白质 DSB重新加入尚未完全确定;然而， 蛋白质如DNA聚合酶、Ku和p350（DNA-pk）已经被 与反应有关。DNA聚合酶已被证明参与其中， 在末端连接反应的后期，在单链间隙的填充中。的 人Ku异源二聚体和p350，DNA依赖蛋白组分 激酶（DNA-pk）被认为参与了DSB再连接反应 因为缺乏这些因子的突变细胞也对 电离辐射，并显示DNA末端连接水平降低。虽然 这些蛋白质与DSB的重新连接有关， 在DSB重新加入中的作用尚未明确确定。 这项提案的总体目标是确定哺乳动物中的因素， 参与重新连接DNA双链断裂（DSB）两端的细胞。 使用纯化的无核酸外切酶的Klenow片段（KF）的研究表明， 它能够使DSB的两端对齐，并合成跨越差距的DNA 体外（King等人，1994）。此外，结果的排序 这种聚合酶留下的连接显示出惊人的相似性， 限制性内切酶诱导的非同源 中国人卵巢（CHO）细胞中APRT基因座的重组 （菲利普斯和摩根，1994）。 1.基于这一点和其他证据（见初步结果），我们 假设哺乳动物DNA聚合酶的作用是将DSB的两端 一起 2.我们还假设一些其他已知或未知的因素 可以与DNA聚合酶相互作用，以促进DNA末端连接， 哺乳动物细胞 3.此外，我们假设DNA末端的某些特征 (e.g.序列同源性或错配的小片段）将影响 反式作用因子连接DNA末端的效率。

Ionizing Radiation and a number of radiomimetic agents induce DNA double strand breaks (DSB). Cellular mechanisms rejoin these broken ends occasionally resulting in chromosomal aberrations such as translocations. Chromosomal translocations can lead to altered gene expression, transformed cellular phenotype and oncogenesis. The proteins involved in DSB rejoining have not been fully identified; however, a number of proteins such as DNA polymerase, Ku and p350(DNA -pk) have been implicated in the reaction. DNA polymerase has been shown to be involved, late in the end-joining reaction, in filling in single strand gaps. The human Ku heterodimer and p350, components of a DNA dependent protein kinase (DNA-pk) are thought to be involved in DSB rejoining reaction because mutant cells deficient in these factors are also sensitive to ionizing radiation and show decreased levels of DNA end joining. Although these proteins have been implicated in DSB rejoining, their individual roles in DSB rejoining have not been clearly established. The overall goal of this proposal is to identify the factors in mammalian cells involved in rejoining two ends of a DNA double strand break (DSB). Studies using purified exonuclease-free Klenow fragment (KF) demonstrated its capacity to align two ends of a DSB and synthesize DNA across the gap in vitro (King et al. 1994). Furthermore, sequencing of the resultant junctions left by this polymerase showed striking similarities to the mutational spectra seen for restriction enzyme-induced nonhomologous recombination in the APRT locus in Chinese Hamster Ovary (CHO) cells (Phillips and Morgan 1994). 1. Based on this and other evidence (see Preliminary Results), we hypothesize that mammalian DNA polymerase acts to bring two ends of a DSB together. 2. We also hypothesize that a number of other known or unknown factors may interact with DNA polymerase to facilitate DNA end-joining in mammalian cells. 3. Furthermore, we hypothesize that certain features of the DNA ends (e.g. small stretches of sequence homology or mismatches)will affect the efficiency of trans-acting factors to join DNA ends.