Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection
Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection
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DOI：
10.1073/pnas.0908463107
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发表时间：
2010-02-02
影响因子：
11.1
通讯作者：
Benner, Steven A.
中科院分区：
文献类型：
--
作者：
Chen, Fei;Gaucher, Eric A.;Benner, Steven A.
任何系统，无论是自然的还是人为的，如果我们分析它的历史和结构，就能更好地理解它。在这里，我们结合联合收割机的结构分析与“重建进化自适应路径”（REAP）的分析，使用DNA聚合酶的进化和功能的历史，以取代氨基酸，使聚合酶接受一类新的三磷酸底物，那些有他们的3 '-OH端封闭为3'-ONH 2组（dNTP-ONH 2）。类似于广泛使用的2 '，3'-双脱氧核苷三磷酸（ddNTPs），dNTP-ONH（2）终止引物延伸。然而，与ddNTP不同，引物延伸可以通过切割O-N键以将-OH基团恢复到引物的3 '端来恢复。REAP结合晶体学分析确定了35个位点，其中置换可能提高Taq接受dNTP-ONH（2）的能力。一个包含93个Taq变体的库，每个变体在其中三个或四个位点上具有替换，其中包含8个变体，其接受dNTP-ONH 2底物的能力有所提高。其中两种（A597 T、L 616 A、F667 Y、E745 H和E520 G、K540 I、L 616 A）表现非常好。第二种变体忠实且有效地掺入dNTP-ONH（2）和ddNTPs两者，通过可逆和不可逆终止子之间的竞争支持适用于平行测序和SNP检测的延伸-切割-延伸循环。对这些结果的分析表明，一个先前未鉴定的替代（L 616 A）允许Taq掺入可逆和不可逆终止子。模型显示了L 616 A如何打开Phe-667后面的空间，使其能够移动以容纳更大的3 '-取代基。这项工作为DNA分析提供了聚合酶，并展示了进化分析如何帮助探索蛋白质结构和功能之间的关系。
Any system, natural or human-made, is better understood if we analyze both its history and its structure. Here we combine structural analyses with a "Reconstructed Evolutionary Adaptive Path" (REAP) analysis that used the evolutionary and functional history of DNA polymerases to replace amino acids to enable polymerases to accept a new class of triphosphate substrates, those having their 3'-OH ends blocked as a 3'-ONH2 group (dNTP-ONH2). Analogous to widely used 2',3'-dideoxynucleoside triphosphates (ddNTPs), dNTP-ONH(2)s terminate primer extension. Unlike ddNTPs, however, primer extension can be resumed by cleaving an O-N bond to restore an -OH group to the 3'-end of the primer. REAP combined with crystallographic analyses identified 35 sites where replacements might improve the ability of Taq to accept dNTP-ONH(2)s. A library of 93 Taq variants, each having replacements at three or four of these sites, held eight variants having improved ability to accept dNTP-ONH2 substrates. Two of these (A597T, L616A, F667Y, E745H, and E520G, K540I, L616A) performed notably well. The second variant incorporated both dNTP-ONH(2)sand ddNTPs faithfully and efficiently, supporting extension-cleavage-extension cycles applicable in parallel sequencing and in SNP detection through competition between reversible and irreversible terminators. Dissecting these results showed that one replacement (L616A), not previously identified, allows Taq to incorporate both reversible and irreversible terminators. Modeling showed how L616A might open space behind Phe-667, allowing it to move to accommodate the larger 3'-substituent. This work provides polymerases for DNA analyses and shows how evolutionary analyses help explore relationships between structure and function in proteins.