Frick D N, Richardson C C
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1999 Dec 10;274(50):35889-98. doi: 10.1074/jbc.274.50.35889.
The primase fragment of the bacteriophage T7 63-kDa gene 4 helicase/primase protein contains the 271 N-terminal amino acid residues and lacks helicase activity. The primase fragment catalyzes the synthesis of oligoribonucleotides at rates similar to those catalyzed by the full-length protein in the presence of a 5-nucleotide DNA template containing a primase recognition site (5'-GGGTC-3', 5'-TGGTC-3', 5'-GTGTC-3', or 5'-TTGTC-3'). Although it is not copied into the oligoribonucleotides, the cytosine at the 3'-position is essential for synthesis and template binding. Two nucleotides flanking the 3'-end of the recognition site are required for tight DNA binding and rapid oligoribonucleotide synthesis. Nucleotides added to the 5'-end have no effect on the rate of oligoribonucleotide synthesis or the affinity of the primase for DNA. The binding of either ATP or CTP significantly increases the affinity of the primase for its DNA template. DNA lacking a primase recognition site does not inhibit oligoribonucleotide synthesis, suggesting that the primase binds DNA in a sequence-specific manner. The affinity of the primase for templates is weak, ranging from 10 to 150 microM. The tight DNA binding (<1 microM) observed with the 63-kDa gene 4 protein occurs via interactions between DNA templates and the helicase domain.
噬菌体T7 63-kDa基因4解旋酶/引发酶蛋白的引发酶片段包含271个N端氨基酸残基，且缺乏解旋酶活性。在含有引发酶识别位点（5'-GGGTC-3'、5'-TGGTC-3'、5'-GTGTC-3'或5'-TTGTC-3'）的5核苷酸DNA模板存在的情况下，引发酶片段催化寡核糖核苷酸的合成，其速率与全长蛋白催化的速率相似。尽管3'-位的胞嘧啶不被复制到寡核糖核苷酸中，但它对于合成和模板结合至关重要。识别位点3'-端两侧的两个核苷酸是紧密DNA结合和快速寡核糖核苷酸合成所必需的。添加到5'-端的核苷酸对寡核糖核苷酸合成速率或引发酶与DNA的亲和力没有影响。ATP或CTP的结合显著增加了引发酶对其DNA模板的亲和力。缺乏引发酶识别位点的DNA不会抑制寡核糖核苷酸的合成，这表明引发酶以序列特异性方式结合DNA。引发酶对模板的亲和力较弱，范围为10至150微摩尔。63-kDa基因4蛋白观察到的紧密DNA结合（<1微摩尔）是通过DNA模板与解旋酶结构域之间的相互作用发生的。
