Karthikeyan R, Vonarx E J, Straffon A F, Simon M, Faye G, Kunz B A
School of Biological and Chemical Sciences, Deakin University, Geelong, Victoria, 3217, Australia.
J Mol Biol. 2000 Jun 2;299(2):405-19. doi: 10.1006/jmbi.2000.3744.
Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.
尽管δ聚合酶和ε聚合酶是真核细胞DNA复制所必需的，但每种聚合酶是否在单独的模板链上发挥作用仍是一个悬而未决的问题。为了开始研究这两种聚合酶在细胞内的相对作用，我们使用了一个质粒携带的酵母tRNA基因和由于DNAδ聚合酶或ε聚合酶校对功能缺失而成为突变体的酵母菌株。将tRNA基因倒置以改变前导链和滞后链模板的序列，改变了两种突变体聚合酶的特异性，但方向相反。也就是说，tRNA基因处于一种方向时的δ聚合酶突变体的特异性与tRNA基因处于另一种方向时的ε聚合酶突变体的特异性相似，反之亦然。我们还获得了与基因方向对野生型菌株中发生的复制错误的错配校正有轻微影响一致的结果。然而，数据表明，无论是这种效应还是复制保真度的差异都不是导致在基因倒置时校对缺陷型突变体中观察到的突变特异性变化的原因。总体而言，数据表明，tRNA基因倒置时，δ聚合酶和ε聚合酶各自遇到不同的模板序列，因此在质粒DNA复制叉处复制相反的链。
