Intramolecular DNA triplexes, bent DNA and DNA unwinding elements in the initiation region of an amplified dihydrofolate reductase replicon.
Intramolecular DNA triplexes, bent DNA and DNA unwinding elements in the initiation region of an amplified dihydrofolate reductase replicon.
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扩增的二氢叶酸还原酶复制子起始区的分子内 DNA 三链体、弯曲 DNA 和 DNA 解旋元件。
DOI：
10.1016/0022-2836(90)90008-a
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发表时间：
1990
影响因子：
5.6
通讯作者：
Heintz,NH
中科院分区：
文献类型：
--
作者：
Caddle,MS;Lussier,RH;Heintz,NH
已经确定了6.2 kb （1 kb = 103碱基对）DNA的核苷酸序列，该序列包含了CHOC 400细胞扩增的二氢叶酸还原酶结构域的最早复制部分。起源区DNA包含两个alui家族重复序列，一个新的重复元件（称为ORR-1），一个富含tgggt的区域，以及几个同嘌呤/同嘧啶和嘌呤/嘧啶交替的区域，包括由（G- c）5, (a - c)18, (a -G)21, (G)9, (CAGA)4, gaggg - agaggcagagaggg, (a -G)27组成的不寻常的简单重复序列簇。含有起源区序列的重组质粒被检查DNA结构构象先前涉及起源激活。绿豆核酸酶对DNA解绕元件的敏感性试验表明，在中性pH下，超卷曲质粒中核酸酶的首选裂解顺序为：（A- t）23⪢（A- g）簇⪢(A)38⪢载体= (AATT)n。在酸性pH下，切割偏好的层次变化为：（A- g）簇⪢（A- t）23⪢（AATT）n>向量= (A)38。在酸性或中性pH下，绿豆核酸酶解绕实验中发现了一个稳定弯曲的DNA区域，并表明该区域没有反应。分子间杂交研究表明，在pH 5.2下存在扭转应力时，（A- g）簇形成三链DNA。这些结果表明，扩增的染色体复制子的起源区域包含一个新的重复元件和多个序列元件，促进DNA弯曲，DNA解绕和分子内三链DNA的形成。
The nucleotide sequence of 6.2 kb (1 kb = 103base-pairs) of DNA that encompasses the earliest replicating portion of the amplified dihydrofolate reductase domains of CHOC 400 cells has been determined. Origin region DNA contains twoAluI family repeats, a novel repetitive element (termed ORR-1), a TGGGT-rich region, and several homopurine/ homopyrimidine and alternating purine/pyrimidine tracts, including an unusual cluster of simple repeating sequences composed of (G-C)5, (A-C)18, (A-G)21, (G)9, (CAGA)4, GAGGG-AGAGAGGCAGAGAGGG, (A-G)27. Recombinant plasmids containing origin region sequences were examined for DNA structural conformations previously implicated in origin activation. Mung bean nuclease sensitivity assays for DNA unwinding elements show the preferred order of nuclease cleavage at neutral pH in supercoiled origin plasmids to be: (A-T)23⪢ the (A-G) cluster ⪢(A)38⪢ vector = (AATT)n. At acid pH, the hierarchy of cleavage preferences changes to: the (A-G) cluster ⪢(A-T)23⪢ (AATT)n> vector = (A)38. A region of stably bent DNA was identified and shown not to be reactive in the mung bean nuclease unwinding assay at either acid or neutral pH. Intermolecular hybridization studies show that, in the presence of torsional stress at pH 5.2, the (A-G) cluster forms triple-stranded DNA. These results show that the origin region of an amplified chromosomal replicon contains a novel repetitive element and multiple sequence elements that facilitate DNA bending, DNA unwinding and the formation of intramolecular triple-stranded DNA.