Ledwith B J, Manam S, Van Tuyle G C
J Biol Chem. 1986 May 15;261(14):6571-7.
A DNA primase was partially purified from rat liver mitochondria and separated from the bulk of DNA polymerase gamma and mtRNA polymerase by heparin-agarose chromatography. The primase was distinguished from mtRNA polymerase by its response to pH, monoand divalent cations, and ATP concentrations. In the absence of an active DNA polymerase and using poly(dT) as template, primase synthesized mixed polynucleotide products consisting of units of oligo(A) 1-12 alternating with units of oligo(dA)25-40. Contributions to these products by contaminating DNA polymerase gamma were eliminated by the addition of dideoxy-ATP. Addition of 50 microM dATP to the primase reaction caused a 50% inhibition of AMP incorporation as compared to reactions containing low levels of dATP present only as a contaminant of the ATP added. The inhibition was due primarily to a reduction of new chain initiations. The dATP did not "lock" the primase reaction into the DNA mode of synthesis since the proportion of internal and 3'-terminal RNA segments was little affected. However, the addition of both 50 microM dATP and exogenous DNA polymerase to the primase reaction greatly reduced the amount of internal and 3'-terminal RNA segments, presumably due to the displacement of primase by DNA polymerase. Our data are consistent with the hypothesis (Hu, S.-Z., Wang, T.S.-F., and Korn, D. (1984) J. Biol. Chem. 259, 2602-2609) that the physiologically significant primer is a mixed 5'-oligoribonucleotide-3'-oligodeoxyribonucleotide and that the formation of the RNA to DNA junction is inherently a primase function.
从大鼠肝脏线粒体中部分纯化了一种DNA引发酶，并通过肝素 - 琼脂糖层析将其与大部分DNA聚合酶γ和线粒体RNA聚合酶分离。该引发酶通过其对pH、单价和二价阳离子以及ATP浓度的反应与线粒体RNA聚合酶区分开来。在没有活性DNA聚合酶且以聚（dT）为模板的情况下，引发酶合成了由1 - 12个寡聚（A）单元与25 - 40个寡聚（dA）单元交替组成的混合多核苷酸产物。通过添加双脱氧ATP消除了污染的DNA聚合酶γ对这些产物的影响。与仅含有作为添加ATP污染物存在的低水平dATP的反应相比，向引发酶反应中添加50μM dATP导致AMP掺入受到50%的抑制。这种抑制主要是由于新链起始的减少。dATP并没有将引发酶反应“锁定”到DNA合成模式，因为内部和3' - 末端RNA片段的比例受影响较小。然而，向引发酶反应中同时添加50μM dATP和外源DNA聚合酶大大减少了内部和3' - 末端RNA片段的量，这可能是由于DNA聚合酶取代了引发酶。我们的数据与以下假设一致（Hu，S.-Z.，Wang，T.S.-F.，和Korn，D.（1984）J. Biol. Chem. 259，2602 - 2609），即生理上重要的引物是一种混合的5' - 寡核糖核苷酸 - 3' - 寡脱氧核糖核苷酸，并且RNA与DNA连接的形成本质上是引发酶的功能。
