Malkas L H, Baril E F
Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Proc Natl Acad Sci U S A. 1989 Jan;86(1):70-4. doi: 10.1073/pnas.86.1.70.
A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.
已鉴定出一种能识别脱氧腺嘌呤和/或脱氧胸腺嘧啶连续序列（dA/dT序列）的DNA结合蛋白，并通过在寡聚（dT）-纤维素和Q-琼脂糖上的连续色谱步骤，从含有HeLa细胞多蛋白DNA聚合酶α-引发酶复合物的色谱级分中纯化得到。在十二烷基硫酸钠（NaDodSO4）存在下，对纯化的dA/dT序列结合蛋白进行聚丙烯酰胺凝胶电泳，显示出一条62 kDa的单一蛋白条带。使用同聚脱氧核苷酸的硝酸纤维素滤膜结合试验表明，纯化的蛋白优先结合单链或双链DNA中的dA/dT序列。用多种DNA进行的凝胶迁移率变动分析表明，纯化的蛋白特异性结合含有最小（核心）复制起点的猿猴病毒40 DNA片段。这种结合以蛋白依赖的方式发生，且在存在大量缺乏猿猴病毒复制起点的竞争性DNA的情况下也能发生。然而，当在分析中使用来自猿猴病毒40缺失突变体的DNA片段时，其核心DNA复制起点中富含17个碱基对的A + T区域内存在缺失，此时起点结合减少。这些结果表明，dA/dT序列结合蛋白优先结合富含17个碱基对的A + T区域，并提示该蛋白在DNA复制起始中可能发挥作用。
