Darmawan Hariyanto, Harrison Melissa, Reha-Krantz Linda J
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9.
DNA Repair (Amst). 2015 May;29:36-46. doi: 10.1016/j.dnarep.2015.02.014. Epub 2015 Feb 23.
Proofreading by the bacteriophage T4 and RB69 DNA polymerases requires a β hairpin structure that resides in the exonuclease domain. Genetic, biochemical and structural studies demonstrate that the phage β hairpin acts as a wedge to separate the primer-end from the template strand in exonuclease complexes. Single amino acid substitutions in the tip of the hairpin or deletion of the hairpin prevent proofreading and create "mutator" DNA polymerases. There is little known, however, about the function of similar hairpin structures in other family B DNA polymerases. We present mutational analysis of the yeast (Saccharomyces cerevisiae) DNA polymerase δ hairpin. Deletion of the DNA polymerase δ hairpin (hpΔ) did not significantly reduce DNA replication fidelity; thus, the β hairpin structure in yeast DNA polymerase δ is not essential for proofreading. However, replication efficiency was reduced as indicated by a slow growth phenotype. In contrast, the G447D amino acid substitution in the tip of the hairpin increased frameshift mutations and sensitivity to hydroxyurea (HU). A chimeric yeast DNA polymerase δ was constructed in which the T4 DNA polymerase hairpin (T4hp) replaced the yeast DNA polymerase δ hairpin; a strong increase in frameshift mutations was observed and the mutant strain was sensitive to HU and to the pyrophosphate analog, phosphonoacetic acid (PAA). But all phenotypes - slow growth, HU-sensitivity, PAA-sensitivity, and reduced fidelity, were observed only in the absence of mismatch repair (MMR), which implicates a role for MMR in mediating DNA polymerase δ replication problems. In comparison, another family B DNA polymerase, DNA polymerase ɛ, has only an atrophied hairpin with no apparent function. Thus, while family B DNA polymerases share conserved motifs and general structural features, the β hairpin has evolved to meet specific needs.
噬菌体T4和RB69 DNA聚合酶的校对功能需要一个位于核酸外切酶结构域的β发夹结构。遗传学、生物化学和结构研究表明，噬菌体β发夹在核酸外切酶复合物中充当楔子，将引物末端与模板链分开。发夹顶端的单个氨基酸取代或发夹缺失会阻止校对功能，并产生“突变型”DNA聚合酶。然而，对于其他B族DNA聚合酶中类似发夹结构的功能却知之甚少。我们对酵母（酿酒酵母）DNA聚合酶δ的发夹进行了突变分析。删除DNA聚合酶δ发夹（hpΔ）并没有显著降低DNA复制保真度；因此，酵母DNA聚合酶δ中的β发夹结构对于校对功能并非必不可少。然而，如生长缓慢的表型所示，复制效率降低了。相比之下，发夹顶端的G447D氨基酸取代增加了移码突变以及对羟基脲（HU）的敏感性。构建了一种嵌合酵母DNA聚合酶δ，其中T4 DNA聚合酶发夹（T4hp）取代了酵母DNA聚合酶δ发夹；观察到移码突变大幅增加，并且突变菌株对HU和焦磷酸类似物膦甲酸（PAA）敏感。但所有这些表型——生长缓慢、对HU敏感、对PAA敏感以及保真度降低，仅在错配修复（MMR）缺失的情况下才会出现，这暗示了MMR在介导DNA聚合酶δ复制问题中所起的作用。相比之下，另一种B族DNA聚合酶，DNA聚合酶ɛ，只有一个萎缩的发夹，没有明显功能。因此，虽然B族DNA聚合酶具有保守的基序和一般的结构特征，但β发夹已经进化以满足特定需求。
