PURIFIED DNAA PROTEIN IN INITIATION OF REPLICATION AT THE ESCHERICHIA-COLI CHROMOSOMAL ORIGIN OF REPLICATION
PURIFIED DNAA PROTEIN IN INITIATION OF REPLICATION AT THE ESCHERICHIA-COLI CHROMOSOMAL ORIGIN OF REPLICATION
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DOI：
10.1073/pnas.80.19.5817
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发表时间：
1983-01-01
期刊：
影响因子：
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通讯作者：
KORNBERG, A
中科院分区：
文献类型：
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作者：
FULLER, RS;KORNBERG, A
大肠杆菌dnaA+细胞而非dnaA温度敏感细胞的可溶性蛋白片段复制含有大肠杆菌染色体复制起源（oriC）的质粒。这些突变体部分的互补提供了在oriC上开始复制时dna蛋白活性的测定。从过量产生超过200倍dna蛋白的菌株（体外构建）中，纯化了52,000道尔顿多肽，接近均匀性。虽然蛋白质倾向于聚集，但通过高效液相色谱纯化的单体大小的蛋白质具有充分的复制活性。通过Millipore过滤器结合试验和oriC序列中独特的HindIII位点的保护，可以判断它与超螺旋质粒中的oriC特异性和紧密结合。在DNA复制反应中，DNA蛋白在DNA合成之前的早期阶段起作用。
Soluble protein fractions from E. coli dnaA+ cells but not dnaA temperature-sensitive cells replicate plasmids containing the E. coli chromosomal origin of replication (oriC). Complementation of these mutant fractions provided an assay for dnaA protein activity in initiation of replication at oriC. From a strain (constructed in vitro) that overproduces than dnaA protein > 200-fold, the 52,000-dalton polypeptide was purified to near homogeneity. Although the protein tends to aggregate, monomer-sized protein purified by high-performance liquid chromatography is fully active for replication. It binds specifically and tightly to oriC in a supercoiled plasmid as judged by a Millipore filter-binding assay and by protection of the unique HindIII site within the oriC sequence. In the oriC replication reaction, dnaA protein acts at an early step preceding DNA synthesis.