Interaction of bacteriophage T7 gene 4 primase with its template recognition site
Interaction of bacteriophage T7 gene 4 primase with its template recognition site
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DOI：
10.1074/jbc.274.50.35889
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发表时间：
1999-12-10
影响因子：
4.8
通讯作者：
Richardson, CC
中科院分区：
文献类型：
--
作者：
Frick, DN;Richardson, CC
噬菌体T763-kDa基因4解旋酶/启动酶蛋白的启动酶片段含有271个N端氨基酸残基，缺乏解旋酶活性。在含有引物酶识别位点的5-核苷酸DNA模板(5‘-GGGTC-3’、5‘-TGGTC-3’、5‘-GTGTC-3’或5‘-TTGTC-3’)的存在下，该引物酶片段催化合成寡核苷酸的速率与全长蛋白质催化的速率相似。虽然它没有复制到寡核苷酸中，但3‘-位的胞嘧啶对于合成和模板结合是必不可少的。识别位点3‘端两侧的两个核苷酸是紧密结合DNA和快速合成寡核苷酸所必需的。在5‘端添加核苷酸对寡核苷酸合成速率或引物酶与DNA的亲和力没有影响，ATP或CTP的结合显著增加了引物酶对其DNA模板的亲和力。缺乏底物酶识别位点的DNA不会抑制寡核苷酸的合成，这表明底物酶以一种序列特异性的方式与DNA结合。底物酶对模板的亲和力很弱，在10~150 mM之间。DNA紧密结合(
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 mu M. The tight DNA binding (