Sarai A, Uedaira H, Morii H, Yasukawa T, Ogata K, Nishimura Y, Ishii S
Life Science Center, Institute of Chemical & Physical Research (RIKEN), Ibaraki, Japan.
Biochemistry. 1993 Aug 3;32(30):7759-64. doi: 10.1021/bi00081a022.
The DNA-binding domain of the c-myb protooncogene product consists of three homologous tandem repeats of 51-52 amino acids (denoted as R1, R2, and R3 from the N-terminal side). In order to analyze conformational and thermodynamic characteristics of the homologous repeats, we have examined the DNA-binding domain by circular dichroism (CD) and differential scanning calorimetry (DSC). The CD spectra for the three individual repeats are significantly different in the fine profiles, indicating subtle differences in their conformations. The melting analyses for the fragments show that the thermal stability of each fragment is different from one another, with the following order of stability: R1(Tm = 61 degrees C) approximately greater than R3(57 degrees C) >> R2(43 degrees C), where R2 is much less stable than the other repeats. The denaturing process for the whole DNA-binding domain, measured by DSC, is characterized by a very broad transition ranging from 30 to 80 degrees C. The denaturation curve can be fit well by a three-state transition with one intermediate state. The transition temperature for the native-to-intermediate transition coincides with the melting temperature of R2, indicating that the intermediate state corresponds to the unfolding of unstable R2. The CD spectrum of the whole domain is almost identical to the sum of the individual spectra. Thus, these results suggest that the individual repeats in the whole DNA-binding domain behave independently in terms of conformation and stability. The addition of DNA to the DNA-binding fragment drastically changed the melting profile, in which the broad transition curve was replaced by a sharp peak at 58 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
c-myb原癌基因产物的DNA结合结构域由三个51-52个氨基酸的同源串联重复序列组成（从N端起分别记为R1、R2和R3）。为了分析这些同源重复序列的构象和热力学特征，我们通过圆二色性（CD）和差示扫描量热法（DSC）对DNA结合结构域进行了研究。三个单独重复序列的CD光谱在精细轮廓上有显著差异，表明它们的构象存在细微差别。片段的熔解分析表明，每个片段的热稳定性彼此不同，稳定性顺序如下：R1（熔解温度Tm = 61℃）约大于R3（57℃）>> R2（43℃），其中R2比其他重复序列稳定性低得多。通过DSC测量的整个DNA结合结构域的变性过程的特征是在30至80℃范围内有非常宽的转变。变性曲线可以很好地用具有一个中间状态的三态转变来拟合。天然态到中间态转变的转变温度与R2的熔解温度一致，表明中间状态对应于不稳定的R2的展开。整个结构域的CD光谱几乎与各个光谱的总和相同。因此，这些结果表明，整个DNA结合结构域中的各个重复序列在构象和稳定性方面表现独立。向DNA结合片段中加入DNA会显著改变熔解曲线，其中宽的转变曲线被58℃处的尖锐峰所取代。（摘要截短于250字）
