Sheaff R J, Kuchta R D
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Biochemistry. 1993 Mar 30;32(12):3027-37. doi: 10.1021/bi00063a014.
The mechanism by which calf thymus DNA primase synthesizes RNA primers was examined. Primase first binds a single-stranded DNA template (KD << 100 nM) and can then slide along the DNA in order to find a start for initiating primer synthesis. NTP binding appears ordered, such that the NTP which eventually becomes the second nucleotide of the primer binds the E.DNA complex first. The NTP that becomes the second nucleotide of the primer thereby influences where primase initiates. Primer synthesis is remarkably slow (0.0027 s-1 at 20 microM NTP). The rate-limiting step is after formation of the E.DNA.NTP.NTP complex and before or during dinucleotide synthesis. After synthesis of the dinucleotide, additional NTPs are rapidly polymerized. Primase products are 2-10 nucleotides long. If the enzyme fails to synthesize a primer at least 7 nucleotides long, it reinitiates rather than dissociating from the template. Once a primer at least 7 nucleotides long has been generated, however, subsequent primase activity is inhibited. This inhibition is due to the generation of a stable primer-template complex, which likely remains associated with pol alpha.primase. The role of primase is to synthesize primers that pol alpha can elongate. The ability of primase to distinguish between primers at least 7 nucleotides long and shorter products therefore likely reflects the fact that pol alpha only utilizes primers at least 7 nucleotides long.
对小牛胸腺DNA引发酶合成RNA引物的机制进行了研究。引发酶首先结合单链DNA模板（解离常数KD << 100 nM），然后可以沿着DNA滑动以寻找启动引物合成的起始点。NTP的结合似乎是有序的，使得最终成为引物第二个核苷酸的NTP首先结合E.DNA复合物。成为引物第二个核苷酸的NTP从而影响引发酶的起始位置。引物合成非常缓慢（在20 microM NTP时为0.0027 s-1）。限速步骤在E.DNA.NTP.NTP复合物形成之后且在二核苷酸合成之前或期间。二核苷酸合成后，额外的NTP会迅速聚合。引发酶产物长度为2 - 10个核苷酸。如果该酶未能合成至少7个核苷酸长的引物，它会重新起始而不是从模板上解离。然而，一旦生成了至少7个核苷酸长的引物，随后的引发酶活性就会受到抑制。这种抑制是由于形成了稳定的引物 - 模板复合物，该复合物可能仍然与pol alpha.引发酶相关联。引发酶的作用是合成pol alpha能够延伸的引物。因此，引发酶区分至少7个核苷酸长的引物和较短产物的能力可能反映了pol alpha仅利用至少7个核苷酸长的引物这一事实。
