Mendelman L V, Petruska J, Goodman M F
Department of Biological Sciences, University of Southern California, Los Angeles 90089-1340.
J Biol Chem. 1990 Feb 5;265(4):2338-46.
A polyacrylamide gel assay is used to measure the kinetics of adding a single deoxyribonucleotide onto either a correctly matched or mismatched primer 3' terminus (on M13 template) for all possible DNA base pairs and mispairs using Drosophila melanogaster DNA polymerase alpha (Pol alpha) and avian myeloblastosis virus reverse transcriptase. The reverse transcriptase catalyzes chain extension from transition mispairs (Pur.Pyr and Pyr.Pur, where Pur is purine and Pyr is pyrimidine) more efficiently than polymerase alpha. Reverse transcriptase extends G(primer).T almost 20% as efficiently as it extends A.T, while Pol alpha's G.T extension efficiency is less than 1%. For transversion mispairs (Pur.Pur and Pyr.Pyr), reverse transcriptase extends C.T and T.T with greater efficiency than polymerase alpha, while polymerase alpha is more efficient at extending A.G and G.G mispairs. Reverse transcriptase and polymerase alpha extend the G.G mispair at an efficiency of only 10(-6) and 10(-5), respectively, compared with G.C extension. The extension data for the two polymerases are compared with previously reported nucleotide misinsertion data for the same enzymes (Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415-14423). While the results obtained with reverse transcriptase and Pol alpha differ in detail, some general rules are indicated: (a) Pur.Pyr and Pyr.Pur mispairs, especially G.T and T.G, are easy to insert and even easier to extend; (b) Pyr.Pyr mispairs, especially C.C, are difficult to insert and slightly easier to extend; (c) Pur.Pur mispairs, notably G.G, are harder to extend than to insert. The comparison also shows that reverse transcriptase extends almost all mismatches more efficiently than it forms them, G.G being the only mismatch having a significantly lower efficiency of extension than insertion. Polymerase alpha inserts A.A mismatches most efficiently, but extends them inefficiently, thereby reducing the probability that such transversion mutations will occur in vivo. We show theoretically that when mispaired primers compete with properly matched primers for extension by polymerase, the relative velocities of extension depend on the concentration of the next correct dNTP substrate. The extension velocities depart from Michaelis-Menten kinetics by exhibiting positive cooperativity with respect to substrate concentration.
使用聚丙烯酰胺凝胶分析法来测量在所有可能的DNA碱基对和错配情况下，将单个脱氧核糖核苷酸添加到正确匹配或错配的引物3'末端（在M13模板上）的动力学，所使用的酶为果蝇DNA聚合酶α（Pol α）和禽成髓细胞瘤病毒逆转录酶。逆转录酶催化从转换错配（嘌呤·嘧啶和嘧啶·嘌呤，其中Pur是嘌呤，Pyr是嘧啶）进行链延伸的效率高于聚合酶α。逆转录酶延伸G（引物）·T的效率几乎是延伸A·T的20%，而Pol α对G·T的延伸效率低于1%。对于颠换错配（嘌呤·嘌呤和嘧啶·嘧啶），逆转录酶延伸C·T和T·T的效率高于聚合酶α，而聚合酶α在延伸A·G和G·G错配时效率更高。与G·C延伸相比，逆转录酶和聚合酶α延伸G·G错配的效率分别仅为10⁻⁶和10⁻⁵。将这两种聚合酶的延伸数据与先前报道的相同酶的核苷酸错插入数据（Mendelman, L. V., Boosalis, M. S., Petruska, J., and Goodman, M. F. (1989) J. Biol. Chem. 264, 14415 - 14423）进行比较。虽然逆转录酶和Pol α获得的结果在细节上有所不同，但表明了一些一般规则：（a）嘌呤·嘧啶和嘧啶·嘌呤错配，特别是G·T和T·G，易于插入且更易于延伸；（b）嘧啶·嘧啶错配，特别是C·C，难以插入且稍易于延伸；（c）嘌呤·嘌呤错配，特别是G·G，延伸比插入更难。比较还表明，逆转录酶延伸几乎所有错配的效率都高于形成错配的效率，G·G是唯一延伸效率明显低于插入效率的错配。聚合酶α最有效地插入A·A错配，但延伸效率低下，从而降低了这种颠换突变在体内发生的概率。我们从理论上表明，当错配引物与正确匹配的引物竞争聚合酶延伸时，延伸的相对速度取决于下一个正确dNTP底物的浓度。延伸速度偏离米氏动力学，表现出对底物浓度的正协同性。
