Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5′- and 3′-tails of the forked DNA substrate
Asymmetric interactions of hexameric bacteriophage T7 DNA helicase with the 5′- and 3′-tails of the forked DNA substrate
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DOI：
10.1074/jbc.272.51.32267
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发表时间：
1997-12-19
影响因子：
4.8
通讯作者：
Patel, SS
中科院分区：
文献类型：
--
作者：
Ahnert, P;Patel, SS
噬菌体T7 DNA解旋酶需要两个非互补的单链DNA（ssDNA）尾，紧挨着双链DNA（dsDNA）区域来启动DNA解旋。使用一系列分叉的DNA研究解旋酶与DNA的相互作用。我们的研究结果表明，解旋酶与分叉DNA的两个尾部不对称地相互作用。当解旋酶在解旋开始之前预组装在分叉的DNA上时，具有15个核苷酸（nt）3 '-尾和35个nt 5'-尾的DNA在18 ℃下以接近60个碱基对/秒的最佳速率解旋。当解旋酶未预组装在DNA上时，需要一个>65 nt长的5 '-尾才能达到12个碱基对/秒的最大解旋速率。我们表明，解旋酶与ssDNA区域特异性相互作用，并在DNA解旋过程中与两条ssDNA链保持接触，因为两条ssDNA尾向dsDNA结构的转化极大地抑制了解旋，并且解旋酶无法解旋通过dsDNA区域中的切口。这些研究为DNA解旋机制提供了新的见解。我们提出了一个DNA解旋的排除模型，其中T7解旋酶六聚体在DNA解旋过程中主要与ssDNA链相互作用，包围5 '链，并将3'链从孔中排除。
Bacteriophage T7 DNA helicase requires two noncomplementary single-stranded DNA (ssDNA) tails next to a double-stranded DNA (dsDNA) region to initiate DNA unwinding. The interactions of the helicase with the DNA were investigated using a series of forked DNAs. Our results show that the helicase interacts asymmetrically with the two tails of the forked DNA. When the helicase was preassembled on the forked DNA before the start of unwinding, a DNA with 15-nucleotide (nt) 3'-tail and 35-nt 5'-tail was unwound with optimal rates close to 60 base pairs/s at 18 degrees C. When the helicase was not preassembled on the DNA, a >65-nt long 5'-tail was required for maximal unwinding rates of 12 base pairs/s. We show that the helicase interacts specifically with the ssDNA region and maintains contact with both ssDNA strands during DNA unwinding, since conversion of the two ssDNA tails to dsDNA structures greatly inhibited unwinding, and the helicase was unable to unwind past a nick in the dsDNA region. These studies have provided new insights into the mechanism of DNA unwinding. We propose an exclusion model of DNA unwinding in which T7 helicase hexamer interacts mainly with the ssDNA strands during DNA unwinding, encircling the 5'-strand and excluding the 3'-strand from the hole.