Ye F, Carrodeguas J A, Bogenhagen D F
Department of Pharmacological Sciences, State University of New York at Stony Brook 11794-8651, USA.
Nucleic Acids Res. 1996 Apr 15;24(8):1481-8. doi: 10.1093/nar/24.8.1481.
We used the known sequence of the Saccharomyces cerevisiae DNA polymerase gamma to clone the genes or cDNAs encoding this enzyme in two other yeasts, Pychia pastoris and Schizosaccharomyces pombe, and one higher eukaryote, Xenopus laevis. To confirm the identity of the final X.laevis clone, two antisera raised against peptide sequences were shown to react with DNA polymerase gamma purified from X.laevis oocyte mitochondria. A developmentally regulated 4.6 kb mRNA is recognized on Northern blots of oocyte RNA using the X.laevis cDNA. Comparison of the four DNA polymerase gamma gene sequences revealed several highly conserved sequence blocks, comprising an N-terminal 3'-->5'exonuclease domain and a C-terminal polymerase active center interspersed with gamma-specific gene sequences. The consensus sequences for the DNA polymerase gamma exonuclease and polymerase domains show extensive sequence similarity to DNA polymerase I from Escherichia coli. Sequence conservation is greatest for residues located near the active centers of the exo and pol domains of the E.coli DNA polymerase I structure. The domain separating the exonuclease and polymerase active sites is larger in DNA polymerase gamma than in other members of family A (DNA polymerase I-like) polymerases. The S.cerevisiae DNA polymerase gamma is atypical in that it includes a 240 residue C-terminal extension that is not found in the other members of the DNA polymerase gamma family, or in other family A DNA polymerases.
我们利用酿酒酵母DNA聚合酶γ的已知序列，在另外两种酵母（巴斯德毕赤酵母和粟酒裂殖酵母）以及一种高等真核生物（非洲爪蟾）中克隆编码该酶的基因或cDNA。为了确认最终的非洲爪蟾克隆的身份，针对肽序列产生的两种抗血清被证明可与从非洲爪蟾卵母细胞线粒体中纯化的DNA聚合酶γ发生反应。使用非洲爪蟾cDNA在卵母细胞RNA的Northern印迹上可识别出一种受发育调控的4.6 kb mRNA。对四种DNA聚合酶γ基因序列的比较揭示了几个高度保守的序列块，包括一个N端3'→5'核酸外切酶结构域和一个C端聚合酶活性中心，其间散布着γ特异性基因序列。DNA聚合酶γ核酸外切酶和聚合酶结构域的共有序列与大肠杆菌的DNA聚合酶I显示出广泛的序列相似性。对于位于大肠杆菌DNA聚合酶I结构的外切酶和聚合酶结构域活性中心附近的残基，序列保守性最大。DNA聚合酶γ中分隔核酸外切酶和聚合酶活性位点的结构域比A家族（类DNA聚合酶I）聚合酶的其他成员更大。酿酒酵母DNA聚合酶γ是非典型的，因为它包含一个240个残基的C端延伸，该延伸在DNA聚合酶γ家族的其他成员或其他A家族DNA聚合酶中未发现。
