Burgers P M, Gerik K J
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1998 Jul 31;273(31):19756-62. doi: 10.1074/jbc.273.31.19756.
Yeast DNA polymerase delta (Poldelta) consists of three subunits encoded by the POL3, POL31, and POL32 genes. Each of these genes was cloned under control of the galactose-inducible GAL1-10 promoter and overexpressed in various combinations. Overexpression of all three genes resulted in a 30-fold overproduction of Poldelta, which was identical in enzymatic properties to Poldelta isolated from a wild-type yeast strain. Whereas overproduction of POL3 together with POL32 did not lead to an identifiable Pol3p.Pol32p complex, a chromatographically distinct and novel complex was identified upon overproduction of POL3 and POL31. This two-subunit complex, designated Poldelta*, is structurally and functionally analogous to mammalian Poldelta. The properties of Poldelta* and Poldelta were compared. A gel filtration analysis showed that Poldelta* is a heterodimer (Pol3p.Pol31p) and Poldelta a dimer of a heterotrimer, (Pol3p.Pol31p.Pol32p)2. In the absence of proliferating cell nuclear antigen (PCNA), Poldelta* showed a processivity of 2-3 on poly(dA). oligo(dT) compared with 5-10 for Poldelta. In the presence of PCNA, both enzymes were fully processive on this template. DNA replication by Poldelta* on a natural DNA template was dependent on PCNA and on replication factor C. However, Poldelta*-mediated DNA synthesis proceeded inefficiently and was characterized by frequent pause sites. Reconstitution of Poldelta was achieved upon addition of Pol32p to Poldelta*.
酵母DNA聚合酶δ（Poldelta）由POL3、POL31和POL32基因编码的三个亚基组成。这些基因中的每一个都在半乳糖诱导型GAL1-10启动子的控制下进行克隆，并以各种组合方式过表达。所有三个基因的过表达导致Poldelta产量增加30倍，其酶学性质与从野生型酵母菌株中分离出的Poldelta相同。虽然POL3与POL32一起过表达不会产生可识别的Pol3p.Pol32p复合物，但在POL3和POL31过表达时鉴定出一种色谱上不同的新型复合物。这种二亚基复合物，称为Poldelta*，在结构和功能上类似于哺乳动物的Poldelta。对Poldelta和Poldelta的性质进行了比较。凝胶过滤分析表明，Poldelta是一种异二聚体（Pol3p.Pol31p），而Poldelta是一种异三聚体的二聚体，即（Pol3p.Pol31p.Pol32p）2。在没有增殖细胞核抗原（PCNA）的情况下，Poldelta在聚（dA）·寡聚（dT）上的持续合成能力为2-3，而Poldelta为5-10。在有PCNA的情况下，两种酶在该模板上都具有完全的持续合成能力。Poldelta在天然DNA模板上的DNA复制依赖于PCNA和复制因子C。然而，Poldelta介导的DNA合成效率低下，其特征是频繁出现停顿位点。通过向Poldelta中添加Pol32p实现了Poldelta的重组。
