Murphy Kelly, Darmawan Hariyanto, Schultz Amy, Fidalgo da Silva Elizabeth, Reha-Krantz Linda J
Department of Biological Sciences, CW405 BioSciences Building, University of Alberta, Edmonton, AB T6G 2E9, Canada.
Genome. 2006 Apr;49(4):403-10. doi: 10.1139/g05-106.
Proofreading DNA polymerases share common short peptide motifs that bind Mg(2+) in the exonuclease active center; however, hydrolysis rates are not the same for all of the enzymes, which indicates that there are functional and likely structural differences outside of the conserved residues. Since structural information is available for only a few proofreading DNA polymerases, we developed a genetic selection method to identify mutant alleles of the POL3 gene in Saccharomyces cerevisiae, which encode DNA polymerase delta mutants that replicate DNA with reduced fidelity. The selection procedure is based on genetic methods used to identify "mutator" DNA polymerases in bacteriophage T4. New yeast DNA polymerase delta mutants were identified, but some mutants expected from studies of the phage T4 DNA polymerase were not detected. This would indicate that there may be important differences in the proofreading pathways catalyzed by the two DNA polymerases.
校对DNA聚合酶具有共同的短肽基序，这些基序在外切核酸酶活性中心结合Mg(2+)；然而，并非所有酶的水解速率都相同，这表明在保守残基之外存在功能和可能的结构差异。由于仅少数校对DNA聚合酶有结构信息，我们开发了一种遗传筛选方法来鉴定酿酒酵母中POL3基因的突变等位基因，该基因编码保真度降低的DNA聚合酶δ突变体。筛选程序基于用于鉴定噬菌体T4中“诱变”DNA聚合酶的遗传方法。鉴定出了新的酵母DNA聚合酶δ突变体，但未检测到一些从噬菌体T4 DNA聚合酶研究中预期的突变体。这表明两种DNA聚合酶催化的校对途径可能存在重要差异。
