ANALYSIS OF MUTAGENESIS INDUCED BY A THERMOLABILE-T4 PHAGE DEOXYCYTIDYLATE HYDROXYMETHYLASE SUGGESTS LOCALIZED DEOXYRIBONUCLEOTIDE POOL IMBALANCE
ANALYSIS OF MUTAGENESIS INDUCED BY A THERMOLABILE-T4 PHAGE DEOXYCYTIDYLATE HYDROXYMETHYLASE SUGGESTS LOCALIZED DEOXYRIBONUCLEOTIDE POOL IMBALANCE
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DOI：
10.1007/bf00273611
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发表时间：
1991-04-01
期刊：
影响因子：
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通讯作者：
MATHEWS, CK
中科院分区：
文献类型：
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作者：
JI, JP;MATHEWS, CK
为了了解脱氧核糖核苷酸库失衡引起的突变的分子基础，我们研究了温度敏感的 T4 噬菌体基因 42 突变体 (LB3)，它指定了一种不耐热的脱氧胞苷酸羟甲基酶。 对 rII 突变的分析显示，在半允许温度（34 摄氏度）下，GC 至 AT 突变可被刺激 8 至 80 倍，这些突变可沿 GC 至 AT 或 AT 至 GC 转换途径恢复为野生型。 其中一种标记物 rII SN103 在 34 摄氏度下表现出最高的刺激性，在通过聚合酶链式反应扩增模板后进行了测序。 rII SN103 中的突变位点在 rII B 翻译起始点的核苷酸位置 265 处被鉴定为 AT 至 GC 转换，这将 TCA 更改为 CCA。 对 34 摄氏度下产生的回复突变体和假回复突变体的序列分析表明，该三联体中的两个胞嘧啶均可转变为胸腺嘧啶或腺嘌呤，这与羟甲基脱氧胞苷三磷酸库在复制位点耗尽的假设一致。 然而，34 摄氏度培养物提取物中的 dNTP 库测量结果显示，与 30 摄氏度下获得的值没有显着偏差，这表明库不平衡仅发生在复制叉附近的局部。 我们的研究支持这样的假设：ts LB3 在半允许温度下表现出的突变表型是核苷酸前体流入 DNA 复制机器的扰动的结果。 hm-dCTP 的假定局部缺失可能会扩大有效 dTTP/hm-dCTP 和 dATP/hm-dCTP 池比率，导致观察到的 C-to-T 转换和 C-to-A 颠换突变。
To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34-degrees-C). One such marker, rII SN103, which showed the highest stimulation at 34-degrees-C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34-degrees-C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34-degrees-C cultures showed no significant deviations from values obtained at 30-degrees-C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the mutator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.