The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations.
The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations.
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DNA 聚合酶-β 在体外 DNA 合成过程中的突变特异性。
DOI：
10.1016/s0021-9258(18)89090-1
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
T. Kunkel
中科院分区：
文献类型：
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作者：
T. Kunkel
利用M13mp2 DNA中250个碱基的靶序列进行正向突变试验，确定了真核DNA聚合酶-β体外产生突变的频率和特异性。均相DNA聚合酶-β来自四个不同的来源，每一轮填补缺口的DNA合成产生突变的频率为4-6%。对这种容易出错的DNA合成产生的460个独立突变体的DNA序列分析表明，发生了各种各样的突变事件。移码和碱基替换的频率大致相同，加在一起约占所有突变的90%。观察到移码突变和碱基替换突变的两个突变“热点”。这些位点的突变特征表明，某些碱基替换错误是模板碱基错位造成的，而不是DNA聚合酶-β直接形成错对造成的。当考虑整个靶序列时，单碱基移码突变主要发生在相同碱基的序列中，通常是嘧啶。失去一个碱基的频率是单一碱基添加的20-80倍，比失去两个或更多碱基的频率要高得多。碱基替换发生在整个靶标的许多位置，代表了广泛的错对形成。在大量表型可检测的位点上平均，碱基替换错误频率大于每聚合5000个碱基一个错误。还观察到大的缺失突变，频率是背景的10倍以上，这表明仅纯化的DNA聚合酶就能够产生这种缺失。这些数据与纯化的酶的物理和动力学性质有关，并与DNA聚合酶在体内的作用有关。
The frequency and specificity of mutations produced in vitro by eucaryotic DNA polymerase-beta have been determined in a forward mutation assay using a 250-base target sequence in M13mp2 DNA. Homogeneous DNA polymerase-beta, isolated from four different sources, produces mutations at a frequency of 4-6%/single round of gap-filling DNA synthesis. DNA sequence analyses of 460 independent mutants resulting from this error-prone DNA synthesis demonstrate a wide variety of mutational events. Frameshift and base substitutions are made at approximately equal frequency and together comprise about 90% of all mutations. Two mutational “hot spots” for frameshift and base substitution mutations were observed. The characteristics of the mutations at these sites suggest that certain base substitution errors result from dislocation of template bases rather than from direct mispair formation by DNA polymerase-beta. When considering the entire target sequence, single-base frameshift mutations occur primarily in runs of identical bases, usually pyrimidines. The loss of a single base occurs 20-80 times more frequently than single-base additions and much more frequently than the loss of two or more bases. Base substitutions occur at many sites throughout the target, representing a wide spectrum of mispair formations. Averaged over a large number of phenotypically detectable sites, the base substitution error frequency is greater than one mistake for every 5000 bases polymerized. Large deletion mutations are also observed, at a frequency more than 10-fold over background, indicating that purified DNA polymerases alone are capable of producing such deletions. These data are discussed in relation to the physical and kinetic properties of the purified enzymes and with respect to the proposed role for this DNA polymerase in vivo.