Saccharomyces cerevisiae replication protein a binds to single-stranded DNA in multiple salt-dependent modes
Saccharomyces cerevisiae replication protein a binds to single-stranded DNA in multiple salt-dependent modes
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DOI：
10.1021/bi060994r
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发表时间：
2006-10-03
期刊：
影响因子：
2.9
通讯作者：
Lohman, Timothy M.
中科院分区：
文献类型：
--
作者：
Kumaran, Sangaralingam;Kozlov, Alexander G.;Lohman, Timothy M.
我们用荧光滴定、恒温滴定和沉淀量热法检测了酿酒酵母复制蛋白A(ScRPA)的单链DNA(SsDNA)结合特性，以确定scRPA是否能以多种结合模式与ssDNA结合。我们测量了scRPA结合聚(DT)的封闭位置大小，以及scRPA-(DT)(L)络合物的化学计量、平衡结合常数和结合热随寡核苷酸长度的变化，L沉降平衡研究表明，scRPA在所研究的[NaCl0.02-1.5M]范围内是一个稳定的杂三聚体。然而，闭塞部位大小n在低[NaC l]时n=18-20个核苷酸和高[NaC l]时n=26-28个核苷酸之间经历了一个依赖于盐的转变，过渡中点接近0.36M NaC l(25.0℃，pH 8.1)。SCRPA-(DT)(L)络合物的化学计量学测量也显示出依赖于氯化钠的化学计量学变化，与观察到的闭塞位置大小的变化一致。在1.5M的氯化钠溶液中测量单链RPA与(DT)(L)结合的Delta H-OBSD得到的接触位点大小为28个核苷酸，与在此[氯化钠]下确定的封闭位点大小相似。总之，这些数据支持一个模型，在该模型中，scRPA可以至少两种结合模式与单链DNA结合，一种是低位点模式(n=18+/-1个核苷酸)，稳定在低[NaC l]，其中只使用三个其寡核苷酸/寡糖结合折叠(OB-折叠)，另一种是较高位点模式(n=27+/-1个核苷酸)，稳定在较高的[NaC l]，它使用四个OB-折叠。在所考察的任何条件下，均未发现scRPA与单链DNA高度协同结合的证据。因此，scRPA表现出与大肠杆菌SSB同源四聚体相似的行为，也表现出结合模式的转变，但也存在一些显著的差异。
We have examined the single-stranded DNA (ssDNA) binding properties of the Saccharomyces cerevisiae replication protein A (scRPA) using fluorescence titrations, isothermal titration calorimetry, and sedimentation equilibrium to determine whether scRPA can bind to ssDNA in multiple binding modes. We measured the occluded site size for scRPA binding poly(dT), as well as the stoichiometry, equilibrium binding constants, and binding enthalpy of scRPA-(dT)(L) complexes as a function of the oligodeoxynucleotide length, L. Sedimentation equilibrium studies show that scRPA is a stable heterotrimer over the range of [NaCl] examined (0.02-1.5 M). However, the occluded site size, n, undergoes a salt-dependent transition between values of n = 18-20 nucleotides at low [NaCl] and values of n = 26-28 nucleotides at high [NaCl], with a transition midpoint near 0.36 M NaCl (25.0 degrees C, pH 8.1). Measurements of the stoichiometry of scRPA-(dT)(L) complexes also show a [NaCl]-dependent change in stoichiometry consistent with the observed change in the occluded site size. Measurements of the Delta H-obsd for scRPA binding to (dT)(L) at 1.5 M NaCl yield a contact site size of 28 nucleotides, similar to the occluded site size determined at this [NaCl]. Altogether, these data support a model in which scRPA can bind to ssDNA in at least two binding modes, a low site size mode (n = 18 +/- 1 nucleotides), stabilized at low [NaCl], in which only three of its oligonucleotide/oligosaccharide binding folds (OB-folds) are used, and a higher site size mode (n = 27 +/- 1 nucleotides), stabilized at higher [NaCl], which uses four of its OB-folds. No evidence for highly cooperative binding of scRPA to ssDNA was found under any conditions examined. Thus, scRPA shows some behavior similar to that of the E. coli SSB homotetramer, which also shows binding mode transitions, but some significant differences also exist.