Krishnamurthy G, Brenowitz M D, Ellestad G A
Natural Products Research Section, American Cyanamid Co., Lederle Laboratories, Pearl River, New York 10965.
Biochemistry. 1995 Jan 24;34(3):1001-10. doi: 10.1021/bi00003a035.
Calicheamicin gamma 1I site-specifically binds and cleaves three closely spaced tetranucleotide sequences embedded in an AT-rich region of a 142 base pair DNA restriction fragment. Cleavage is observed predominantly at the TCCT, TTGT, and ATCT sequences, of which TCCT is the primary cleavage site. The Gibbs free energies required to bind calicheamicin to these sequences within the DNA restriction fragment have been determined as a function of NaCl concentration at pH 8.1 and 0 degrees C and at pH 7.5 and 23 and 0 degrees C. Between 150 mM and 1 M NaCl, calicheamicin binding to all three sequences is insensitive to salt. The insensitivity of calicheamicin binding to salt continues to 50 mM NaCl for the TTGT and ATCT sequences; the delta G values for calicheamicin binding to these sequences are on the order of -7.8 to -7.9 kcal mol-1 over the entire range of NaCl concentrations studied. However, between 150 and 125 mM NaCl, the TCCT sequence displays a sharp transition in the delta G of calicheamicin binding from -7.6 to -8.9 kcal mol-1. Below 125 mM NaCl, the delta G values for calicheamicin binding to the TCCT sequence again are invariant. An analysis of the data in terms of polyelectrolyte theory suggests that counterion release from DNA does not contribute significantly to the energetics of the association and that the association of calicheamicin with specific DNA sequences is dominated by nonionic rather than electrostatic forces. Our results further suggest that some calicheamicin binding/cleavage sites are dependent on flanking sequences.
加利车霉素γ1I能位点特异性地结合并切割位于一个142个碱基对的DNA限制性片段富含AT区域内的三个紧密相邻的四核苷酸序列。切割主要发生在TCCT、TTGT和ATCT序列处，其中TCCT是主要切割位点。在pH 8.1、0℃以及pH 7.5、23℃和0℃条件下，已测定了加利车霉素与DNA限制性片段内这些序列结合所需的吉布斯自由能随NaCl浓度的变化情况。在150 mM至1 M NaCl之间，加利车霉素与所有三个序列的结合对盐不敏感。对于TTGT和ATCT序列，加利车霉素结合对盐的不敏感性一直持续到50 mM NaCl；在所研究的整个NaCl浓度范围内，加利车霉素与这些序列结合的ΔG值在-7.8至-7.9 kcal mol-1左右。然而，在150至125 mM NaCl之间，TCCT序列在加利车霉素结合的ΔG方面显示出从-7.6至-8.9 kcal mol-1的急剧转变。在125 mM NaCl以下，加利车霉素与TCCT序列结合的ΔG值再次不变。根据聚电解质理论对数据进行分析表明，DNA中抗衡离子的释放对结合能的贡献不大，并且加利车霉素与特定DNA序列的结合主要由非离子力而非静电力主导。我们的结果进一步表明，一些加利车霉素结合/切割位点依赖于侧翼序列。
