Wang F J, Ripley L S
Department of Microbiology and Molecular Biology, UMD-New Jersey Medical School, Newark 07103.
Genetics. 1994 Mar;136(3):709-19. doi: 10.1093/genetics/136.3.709.
Most single base deletions detected after DNA polymerization in vitro directed by either Escherichia coli DNA polymerase I or its Klenow fragment are opposite Pu in the template. The most frequent mutations were previously found to be associated with the consensus template context 5'-PyTPu-3'. In this study, the predictive power of the consensus sequence on single base deletion frequencies was directly tested by parallel comparison of mutations arising in four related DNAs differing by a single base. G, a deletion hotspot within the template context 5'-TTGA-3', was substituted by each of the 3 other bases. Previous studies had shown that deletions opposite the G were frequent but that deletions opposite its neighboring A were never detected. Based on the predictions of the consensus, the substitution of T for G should produce frequent deletions opposite the neighboring A due to its new 5'-TTTA-3' template context. This prediction was fulfilled; no deletions of this A were detected in the other templates. The consensus further predicted that deletions opposite template C would be lower than those opposite either A or G at the same site and this prediction was also fulfilled. The C substitution also produced a new hotspot for 1 bp deletions 14 bp away. The new hotspot depends on quasi-palindromic misalignment of the newly synthesized DNA strand during polymerization; accurate, but ectopically templated synthesis is responsible for this mutagenesis. Mutations templated by quasi-palindromic misalignments have previously been recognized when they produced complex sequence changes; here we show that this mechanism can produce frequent single base deletions. The unique stimulation of misalignment mutagenesis by the C substitution in the template is consistent with the singular ability of C at that site to contribute to extended complementary pairing during the DNA misalignment that precedes mutagenesis.
在由大肠杆菌DNA聚合酶I或其Klenow片段指导的体外DNA聚合反应后检测到的大多数单碱基缺失，都位于模板中的嘌呤（Pu）相对位置。先前发现最常见的突变与共有模板序列5'-PyTPu-3'有关。在本研究中，通过对四个仅相差一个碱基的相关DNA中产生的突变进行平行比较，直接测试了共有序列对单碱基缺失频率的预测能力。模板序列5'-TTGA-3'中的缺失热点G，被其他三个碱基中的每一个所取代。先前的研究表明，与G相对的缺失很常见，但从未检测到与其相邻的A相对的缺失。根据共有序列的预测，用T取代G应该会由于新的5'-TTTA-3'模板序列而在相邻的A相对位置产生频繁的缺失。这一预测得到了证实；在其他模板中未检测到该A的缺失。共有序列进一步预测，模板C相对位置的缺失将低于同一位置A或G相对位置的缺失，这一预测也得到了证实。C的取代还在14个碱基以外产生了一个新的1个碱基缺失热点。这个新热点取决于聚合过程中新合成DNA链的准回文错配；准确但异位模板化的合成是这种诱变的原因。当准回文错配产生复杂的序列变化时，先前已经认识到由其模板化的突变；在这里我们表明这种机制可以产生频繁的单碱基缺失。模板中C取代对错配诱变的独特刺激，与该位点的C在诱变前DNA错配期间促进延伸互补配对的独特能力一致。
