Zhong Xuejun, Pedersen Lars C, Kunkel Thomas A
Laboratory of Molecular Genetics and Laboratory of Structural Biology National Institute of Environmental Health Sciences, NIH, DHHS Research Triangle Park, NC 27709, USA.
Nucleic Acids Res. 2008 Jul;36(12):3892-904. doi: 10.1093/nar/gkn312. Epub 2008 May 24.
Changing a highly conserved amino acid in motif A of any of the four yeast family B DNA polymerases, DNA polymerase alpha, delta, epsilon or zeta, results in yeast strains with elevated mutation rates. In order to better understand this phenotype, we have performed structure-function studies of homologous mutants of RB69 DNA polymerase (RB69 pol), a structural model for family B members. When Leu415 in RB69 pol is replaced with phenylalanine or glycine, the mutant polymerases retain high-catalytic efficiency for correct nucleotide incorporation, yet have increased error rates due to increased misinsertion, increased mismatch extension and inefficient proofreading. The Leu415Phe mutant also has increased dNTP insertion efficiency opposite a template 8-oxoG and opposite an abasic site. The 2.5 A crystal structure of a ternary complex of RB69 L415F pol with a correctly base-paired incoming dTTP reveals that the phenylalanine ring is accommodated within a cavity seen in the wild-type enzyme, without steric clash or major change in active site geometry, consistent with retention of high-catalytic efficiency for correct incorporation. In addition, slight structural differences were observed that could be relevant to the reduced fidelity of L415F RB69 pol.
改变四种酵母B家族DNA聚合酶（DNA聚合酶α、δ、ε或ζ）中任何一种的基序A中的一个高度保守氨基酸，会导致酵母菌株的突变率升高。为了更好地理解这种表型，我们对RB69 DNA聚合酶（RB69 pol）的同源突变体进行了结构-功能研究，RB69 pol是B家族成员的结构模型。当RB69 pol中的Leu415被苯丙氨酸或甘氨酸取代时，突变聚合酶在正确掺入核苷酸方面保留了高催化效率，但由于错掺入增加、错配延伸增加和校对效率低下，错误率增加。Leu415Phe突变体在模板8-氧代鸟嘌呤（8-oxoG）和无碱基位点对面的dNTP插入效率也有所提高。RB69 L415F pol与正确碱基配对的进入dTTP的三元复合物的2.5埃晶体结构表明，苯丙氨酸环容纳在野生型酶中可见的一个腔内，没有空间冲突或活性位点几何形状的重大变化，这与正确掺入的高催化效率的保留一致。此外，还观察到一些轻微的结构差异，这些差异可能与L415F RB69 pol保真度降低有关。
