Observations of a Genome Carrying an Unreplicatable Region

阻断E.大肠杆菌中，至少需要两个因子：一个是特异性末端（ter）序列（~ 22 bp），另一个是ter结合蛋白。(1)为了研究E.大肠杆菌基因组DNA合成期后未复制区的存在及其对宿主细胞的影响，我们进行了以下实验：大肠杆菌菌株，其基因组携带侧翼有两个ter序列的区域，从一个缺乏ter结合蛋白的菌株构建。我们预期，在ter-binding蛋白存在下，该菌株将是致命的，或者由于基因组上存在不可复制的区域而显示出较差的生长。如预期的那样，当编码ter-binding蛋白的tau基因被导入E.大肠杆菌菌株，与对照菌株相比，该菌株的生长速率大大降低。这表明在ter位点对DNA复制叉的阻断是有漏洞的。为了评估具有不可复制区域的基因组的命运，需要对ter位点的复制叉进行更大的抑制。(2)为了确定E. coli ter系统对真核DNA的复制具有重要作用，我们研究了在体外条件下，在ter-binding蛋白存在下，在粗酶和纯化酶中，携带ter序列的SV 40 DNA的DNA合成。在E. coli体外DNA复制系统中，在两种条件下，DNA复制叉在ter位点的阻断是明显的。ter序列-ter结合蛋白复合物可以以极性方式阻碍SV 40大T抗原的解旋酶作用。当我们以前使用3种类型的E.大肠杆菌解旋酶。

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.