Observations of a Genome Carrying an Unreplicatable Region

携带不可复制区域的基因组的观察

基本信息

批准号：
02808049
负责人：
HORIUCHI Takashi
金额：
$ 1.09万
依托单位：
Okazaki National Research Institutes (1991)Kyushu University (1990)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

To block the progression of the DNA replication fork in E. coli cells, at least two factors are required ; one is the specific terminus (ter) sequence (-22bp) and the other is ter-binding protein. (1) To investigate behavior of an E. coli genome carrying the region unreplicated after DNA synthesis period and effects this genome have on host cells, we did the following experiments ; an E. coli strain, the genome of which carried a region flanked by two ter sequences, was constructed from a strain deficient in ter-binding protein. We expected that in the presence of ter-binding protein this strain would be lethal or would show poor growth due to the presence of an unreplicatable region on the genome. As expected, when the tau gene which codes for ter-binding protein was introduced into the E. coli strain, growth rate of the strain was greatly reduced, in comparison with that of the control strain. This suggested that blockage against' the DNA replication fork at the ter site is leaky. A greater inhibition for the replication fork at the ter site is needed to assess the fate of the genome with an unreplicatable region. (2) To determine whether or not E. coli ter system is functional on eucaryotic DNA replication, we investigated the DNA synthesis of SV40 DNA carrying the ter sequence, in both crude and purified enzyme in vitro, in the presence of ter-binding protein. As well as in the E. coli in vitro DNA replication system, blockage of the DNA replication fork at the ter site was evident under both conditions. The ter sequence-ter binding protein complex could impede the helicase action of SV40 large T antigen, in a polar fashion. A similar activity was observed previously when we used 3 types of E. coli helicases.

为了阻止大肠杆菌细胞中DNA复制叉的进展，至少需要两个因素。一个是特定的末端（TER）序列（-22bp），另一个是结合蛋白。（1）研究携带DNA合成期后未复制区域的大肠杆菌基因组的行为，并影响该基因组对宿主细胞的影响，我们进行了以下实验；大肠杆菌菌株是由两个TER序列两侧的区域的基因组，是由缺乏TER结合蛋白的菌株构成的。我们预计，在存在结合蛋白的情况下，由于基因组上存在不可重复的区域，这种菌株将致命或生长不佳。正如预期的那样，与对照菌株相比，当将TER结合蛋白的TAU基因引入大肠杆菌菌株中时，菌株的生长速率大大降低。这表明对TER位点的DNA复制叉的阻塞是泄漏的。需要更大的抑制在TER位点的复制叉来评估基因组的命运，并具有不可替代的区域。（2）为了确定大肠杆菌系统是否对嗜酸性DNA复制起作用，我们研究了在体外和纯化的酶中携带TER序列的SV40 DNA的DNA合成，在Ter结合蛋白的情况下。以及在大肠杆菌体外DNA复制系统中，在两种情况下，TER位点的DNA复制叉的阻塞都显而易见。 TER序列结合蛋白复合物可以以极性方式阻止SV40大T抗原的解旋酶作用。当我们使用3种类型的大肠旋转酶时，先前观察到类似的活性。