Tenen D G, Taylor T S, Haines L L, Bradley M K, Martin R G, Livingston D M
J Mol Biol. 1983 Aug 25;168(4):791-808. doi: 10.1016/s0022-2836(83)80075-8.
The binding of purified simian virus 40 (SV40) large T antigen (T) from monkey cells infected with wild-type SV40 virus to viral replication origin-containing DNA fragments was studied by DNase footprinting and restriction endonuclease protection methods. A strong affinity binding site (site 1) of 30 base-pairs and a second, adjacent 40 base-pair lower affinity binding site (site 2), which includes the origin of replication, were detected in these assays. These sites appear identical to those previously noted in similar assays performed with the Ad2 + D2 (D2) T protein. Heating T prior to incubation with DNA significantly increased the binding to these two sites, and the order of binding did not change. Moreover, protection of sequences was observed on both strands in these two sites suggesting that both strands can participate in binding of T to these two sites. Studies with DNAs from two internal site 2 deletion mutants as well as with a DNA fragment lacking the distal 13 base-pairs of site 2 revealed that sequences in the "early" portion of site 2 are sufficient for T binding to the intact site. Furthermore, use of a new assay that measures protection of DNA sequences from specific restriction enzyme cleavage revealed that site 2 can be subdivided into two subsites, 2A and 2B, where 2A corresponds to the above-noted early segment of this locus. In titration experiments, the affinity of 2A for T was greater than that of 2B. Hence, binding to a major portion of the replication initiation sequence (i.e. site 2) is the product of at least two interactions. Finally, analyses performed with DNA from a site 1 deletion mutant, cs1085, revealed that prior binding of T to this locus did not facilitate its binding to site 2. The opposite effect was observed when D2T was employed in these assays. Thus, although similar in many respects, these proteins display a detectable difference in their DNA binding mechanisms.
通过DNA酶足迹法和限制性内切核酸酶保护法，研究了从感染野生型猴病毒40（SV40）的猴细胞中纯化得到的SV40大T抗原（T）与含病毒复制起点的DNA片段的结合情况。在这些实验中，检测到一个30个碱基对的强亲和力结合位点（位点1）和第二个相邻的、亲和力较低的40个碱基对结合位点（位点2），后者包含复制起点。这些位点似乎与之前用腺病毒2 + D2（D2）T蛋白进行的类似实验中所发现的位点相同。在与DNA孵育之前加热T，可显著增加其与这两个位点的结合，且结合顺序不变。此外，在这两个位点的两条链上均观察到序列受到保护，这表明两条链均可参与T与这两个位点的结合。对两个内部位点2缺失突变体的DNA以及一个缺少位点2远端13个碱基对的DNA片段进行的研究表明，位点2“早期”部分的序列足以使T结合到完整位点。此外，使用一种新的检测方法来测量DNA序列免受特定限制性酶切割的保护情况，结果显示位点2可细分为两个亚位点，2A和2B，其中2A对应于该位点上述的早期片段。在滴定实验中，2A对T的亲和力大于2B。因此,与复制起始序列的主要部分（即位点2）的结合是至少两种相互作用的结果。最后，对位点1缺失突变体cs1085的DNA进行的分析表明，T预先结合到该位点并不会促进其与位点2的结合。在这些实验中使用D2T时，观察到的是相反的效果。因此，尽管这些蛋白质在许多方面相似，但它们在DNA结合机制上存在可检测到的差异。
