Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated
Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated
复制标题
DOI：
10.1128/jb.00154-10
复制
发表时间：
2010-07-01
影响因子：
3.2
通讯作者：
Schildbach, Joel F.
中科院分区：
文献类型：
--
作者：
Dostal, Lubomir;Schildbach, Joel F.
接合质粒的转移需要松弛酶，即特异性切割一条质粒链序列的蛋白质。 F 质粒松弛酶 TraI（1,756 个氨基酸）也是一种高度持续性的 DNA 解旋酶。 TraI 松弛酶活性位于 N 末端，类似于 300 个氨基酸，而解旋酶基序位于包含位置 990 至 1450 的区域。为了有效的 F 转移，这两个活性必须物理连接。这两个 TraI 活动可能用于不同的迁移阶段；该蛋白质如何调节活性之间的转换尚不清楚。我们检查了 TraI 解旋酶单链 DNA (ssDNA) 识别，以补充之前对松弛酶 ssDNA 结合的探索。在这里，我们表明 TraI 解旋酶相关的 ssDNA 结合独立于所有解旋酶基序，并且位于所有解旋酶基序的 N 端。解旋酶相关位点结合 ssDNA 寡核苷酸，具有 nM 范围的平衡解离常数和一些序列特异性。值得注意的是，我们观察到松弛酶和解旋酶相关位点之间的 ssDNA 结合存在明显的强负协同性。我们检查了三个 TraI 变体，它们在解旋酶相关的 ssDNA 结合位点内或附近插入了 31 个氨基酸。 B. A. Traxler 及其同事 (J. Bacteriol. 188: 6346-6353) 表明，在某些条件下，这些变体从某种形式的负调控中释放出来，使它们比野生型 TraI 更有效地促进转移。我们发现，相对于野生型 TraI，这些变体通过解旋酶相关的结合位点显示出对 ssDNA 的亲和力适度降低，并且结合的表观负协同性显着降低。这些结果表明，与 TraI 的两个 ssDNA 结合位点结合的明显负协同性在 F 转移中发挥着主要的调节功能。
Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal similar to 300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188: 6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.