Thompson H C, Kuchta R D
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Biochemistry. 1995 Sep 5;34(35):11198-203. doi: 10.1021/bi00035a027.
Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase-synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication.
研究了在DNA聚合酶α催化引发酶合成引物的延伸过程中NTPs和阿拉伯呋喃糖基三磷酸腺苷（araATP）的聚合情况。引发酶合成引物后，polα通常会将多个dNTPs聚合到该引物上。然而，在缺少必需的dNTP时，引物仍可通过相应NTP替代缺失的dNTP进行聚合而延伸多达35个核苷酸。然而，在外源添加引物/模板的延伸过程中，NTPs不易聚合。在引发酶合成引物的延伸过程中，araATP很容易掺入产物中。重要的是，araATP的聚合不会导致链终止；相反，会添加下一个正确的核苷酸，使得araATP只是一种替代底物。相比之下，在外源添加引物/模板的延伸过程中araATP的聚合会导致强烈的链终止。因此，就与dNTP类似物的相互作用而言，polα-引发酶对引发酶合成引物的延伸与外源添加引物/模板的延伸有根本不同。此外，这些数据为araNMPs如何有效掺入全细胞DNA的核苷酸间连接提供了理论依据，并表明polα-引发酶引发新的DNA链可能是抑制复制的一个独特靶点。
