Runyon G T, Lohman T M
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
J Biol Chem. 1989 Oct 15;264(29):17502-12.
We have examined the duplex DNA unwinding (helicase) properties of the Escherichia coli helicase II protein (uvrD gene product) over a wide range of protein concentrations and solution conditions using a variety of duplex DNA substrates including fully duplex blunt ended and nicked circular molecules. We find that helicase II protein is able to initiate on and completely unwind fully duplex DNA molecules without the requirement for a covalently attached 3' single-stranded DNA tail. This DNA unwinding activity is dependent upon Mg2+ and ATP and requires that the amount of protein be in excess of that needed to saturate the resulting single-stranded DNA. Unwinding experiments on fully duplex blunt ended DNA with lengths of 341, 849, 1625, and 2671 base pairs indicate that unwinding occurs at the same high ratios of helicase II protein/nucleotide, independent of DNA length (50% unwinding requires approximately 0.6 helicase II monomers/nucleotide in 2.5 mM MgCl2, 10% glycerol, pH 7.5, 37 degrees C). Helicase II protein is also able to unwind completely a nicked circular DNA molecule containing 2671 base pairs. At lower but still high molar ratios of helicase II protein to DNA, duplex DNA molecules containing a single-stranded (ss) region attached to a 3' end of the duplex are preferentially unwound in agreement with the results obtained by S. W. Matson [1986) J. Biol. Chem. 261, 10169-10175). This preferential unwinding of duplex DNA with an attached 3' ssDNA most likely reflects the availability of a high affinity site (ssDNA) with the proper orientation for initiation; however, this may not reflect the type of DNA molecule upon which helicase II protein initiates DNA unwinding in vivo. The effects of changes in NaCl, NaCH3COO, and MgCl2 concentration on the ability of helicase II protein to unwind fully duplex DNA and duplex DNA with a 3' ssDNA tail have also been examined. Although the unwinding of fully duplex and nicked circular DNA molecules reported here occurs at higher helicase II protein to DNA ratios than have been previously used in most studies of this protein in vitro, this activity is likely to be relevant to the function of this protein in vivo since very high levels of helicase II protein accumulate in E. coli during the SOS response to DNA damage (approximately 2-5 x 10(4) copies/cell).(ABSTRACT TRUNCATED AT 400 WORDS)
我们使用多种双链 DNA 底物，包括完全双链平端和带切口的环状分子，在广泛的蛋白质浓度和溶液条件下，研究了大肠杆菌解旋酶 II 蛋白（uvrD 基因产物）的双链 DNA 解旋（解旋酶）特性。我们发现解旋酶 II 蛋白能够起始并完全解开完全双链 DNA 分子，而无需共价连接的 3' 单链 DNA 尾巴。这种 DNA 解旋活性依赖于 Mg2+ 和 ATP，并且要求蛋白质的量超过饱和所得单链 DNA 所需的量。对长度为 341、849、1625 和 2671 个碱基对的完全双链平端 DNA 进行的解旋实验表明，解旋以相同的高解旋酶 II 蛋白/核苷酸比例发生，与 DNA 长度无关（在 2.5 mM MgCl2、10%甘油、pH 7.5、37℃条件下，50%解旋需要约 0.6 个解旋酶 II 单体/核苷酸）。解旋酶 II 蛋白也能够完全解开一个含有 2671 个碱基对的带切口的环状 DNA 分子。在较低但仍然较高的解旋酶 II 蛋白与 DNA 的摩尔比下，含有连接在双链 3' 末端的单链（ss）区域的双链 DNA 分子优先被解旋，这与 S. W. Matson [1986] J. Biol. Chem. 261, 10169 - 10175 所获得的结果一致。这种带有连接的 3' ssDNA 的双链 DNA 的优先解旋很可能反映了具有适当起始方向的高亲和力位点（ssDNA）的可用性；然而，这可能并不反映解旋酶 II 蛋白在体内起始 DNA 解旋的 DNA 分子类型。还研究了 NaCl、NaCH3COO 和 MgCl2 浓度变化对解旋酶 II 蛋白解开完全双链 DNA 和带有 3' ssDNA 尾巴的双链 DNA 能力的影响。尽管此处报道的完全双链和带切口的环状 DNA 分子的解旋发生在比该蛋白大多数体外研究中先前使用的更高的解旋酶 II 蛋白与 DNA 比例下，但这种活性可能与该蛋白在体内的功能相关，因为在大肠杆菌对 DNA 损伤的 SOS 应答过程中会积累非常高水平的解旋酶 II 蛋白（约 2 - 5×10(4) 个拷贝/细胞）。（摘要截短至 400 字）
