Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB, DnaC complex
Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB, DnaC complex
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DOI：
10.1021/bi060118d
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发表时间：
2006-09-26
期刊：
影响因子：
2.9
通讯作者：
Biswas-Fiss, Esther E.
中科院分区：
文献类型：
--
作者：
Biswas, Subhasis B.;Biswas-Fiss, Esther E.
DNA解旋酶在染色体DNA复制过程中负责解绕双链DNA，是大肠杆菌DNA复制装置的重要组成部分。我们分析了DnaB、DnaC复合物和DnaB解旋酶与单链DNA （ssDNA）的结合机制。ssDNA与DnaB解旋酶的结合受到核苷酸辅助因子的显著调节，其与DnaC复合物的调节明显不同。DnaB解旋酶仅在ATP γ S （ATP的不可水解类似物）存在的情况下才能以高亲和力[K-d = (5.09 +/- 0.32) × 10(-8) M]结合ssDNA，而不存在其他核苷酸。在30 ~ 37℃范围内，这种结合对离子强度敏感，但对温度变化不敏感。另一方面，ADP存在时ssDNA的结合比ATP γ S存在时弱，并且对离子强度不敏感。DnaC蛋白与DnaB六聚体形成1:1的复合物，并通过形成DnaB(6)中心点DnaC(6)十二聚体复合物将其装载到ssDNA上。我们的研究结果表明，与DnaB六聚体不同，DnaB(6)中心点DnaC(6)复合物在ATP存在下以高亲和力[K-d = (6.26 +/- 0.65) × 10(-8) M]结合ssDNA。在ATP γ S或ADP存在的情况下，DnaB(6)中心点DnaC(6)复合物结合ssDNA是一个低亲和力的过程。综上所述，我们的研究结果表明，在体内存在ATP的情况下，DnaB(6)中心点DnaC(6)复合体在结合DNA以及将DnaB装载到ssDNA上的效率应该比DnaB解旋酶本身更高。
DnaB helicase is responsible for unwinding duplex DNA during chromosomal DNA replication and is an essential component of the DNA replication apparatus in Escherichia coli. We have analyzed the mechanism of binding of single-stranded DNA (ssDNA) by the DnaB, DnaC complex and DnaB helicase. Binding of ssDNA to DnaB helicase was significantly modulated by nucleotide cofactors, and the modulation was distinctly different for its complex with DnaC. DnaB helicase bound ssDNA with a high affinity [K-d = (5.09 +/- 0.32) x 10(-8) M] only in the presence of ATP gamma S, a nonhydrolyzable analogue of ATP, but not other nucleotides. The binding was sensitive to ionic strength but not to changes in temperature in the range of 30-37 degrees C. On the other hand, ssDNA binding in the presence of ADP was weaker than that observed with ATP gamma S, and the binding was insensitive to ionic strength. DnaC protein hexamerizes to form a 1: 1 complex with the DnaB hexamer and loads it onto the ssDNA by forming a DnaB(6)center dot DnaC(6) dodecameric complex. Our results demonstrate that the DnaB(6)center dot DnaC(6) complex bound ssDNA with a high affinity [K-d = (6.26 +/- 0.65) x 10(-8) M] in the presence of ATP, unlike the DnaB hexamer. In the presence of ATP gamma S or ADP, binding of ssDNA by the DnaB(6)center dot DnaC(6) complex was a lower-affinity process. In summary, our results suggest that in the presence of ATP in vivo, the DnaB(6)center dot DnaC(6) complex should be more efficient in binding DNA as well as in loading DnaB onto the ssDNA than DnaB helicase itself.