Mikhailov V S, Bogenhagen D F
Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
J Biol Chem. 1996 Nov 29;271(48):30774-80. doi: 10.1074/jbc.271.48.30774.
Xenopus laevis DNA polymerase gamma (pol gamma) exhibits low activity on a poly(dT)-oligo(dA) primer-template. We prepared a single-stranded phagemid template containing a dT41 sequence to test the ability of pol gamma to extend a primer through a defined oligo(dT) tract. pol gamma terminates in the center of this dT41 sequence. This replication arrest is abrogated by addition of single-stranded DNA-binding protein or by substitution of 7-deaza-dATP for dATP. These features are consistent with the formation of a T.A*T DNA triplex involving the primer stem. Replication arrest occurs under conditions that permit highly processive DNA synthesis by pol gamma. A similar replication arrest occurs for T7 DNA polymerase, which is also a highly processive DNA polymerase. These results suggest the possibility that DNA triplex formation can occur prior to dissociation of DNA polymerase. Primers with 3'-oligo(dA) termini annealed to a template with a longer oligo(dT) tract are not efficiently extended by pol gamma unless single-stranded DNA-binding protein is added. Thus, one of the functions of single-stranded DNA-binding protein in mtDNA maintenance may be to enable pol gamma to successfully replicate through dT-rich sequences.
非洲爪蟾DNA聚合酶γ（polγ）在聚（dT）-寡聚（dA）引物模板上表现出低活性。我们制备了一个包含dT41序列的单链噬菌粒模板，以测试polγ通过特定的寡聚（dT）序列延伸引物的能力。polγ在这个dT41序列的中心终止。通过添加单链DNA结合蛋白或用7-脱氮-dATP替代dATP，这种复制停滞被消除。这些特征与涉及引物茎的T.A*T DNA三链体的形成一致。复制停滞发生在允许polγ进行高度连续DNA合成的条件下。对于同样是高度连续DNA聚合酶的T7 DNA聚合酶，也会发生类似的复制停滞。这些结果表明DNA三链体形成可能在DNA聚合酶解离之前发生。除非添加单链DNA结合蛋白，否则与具有更长寡聚（dT）序列的模板退火的具有3'-寡聚（dA）末端的引物不能被polγ有效地延伸。因此，单链DNA结合蛋白在线粒体DNA维持中的功能之一可能是使polγ能够成功地通过富含dT的序列进行复制。
