A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.
A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase.
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保守的异亮氨酸亮氨酸突变会导致Klentaq1 DNA聚合酶的重新排列和冷敏感性。
DOI：
10.1021/bi501198f
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发表时间：
2015-01-27
期刊：
影响因子：
2.9
通讯作者：
Kermekchiev, Milko B.
中科院分区：
文献类型：
--
作者：
Wu, Eugene Y.;Walsh, Amanda R.;Materne, Emma C.;Hiltner, Emily P.;Zielinski, Bryan;Miller, Bill R., III;Mawby, Lily;Modeste, Erica;Parish, Carol A.;Barnes, Wayne M.;Kermekchiev, Milko B.
Assembly of polymerase chain reactions at room temperature can sometimes lead to low yields or unintentional products due to mispriming. Mutation of isoleucine 707 to leucine in DNA polymerase I from Thermus aquaticus substantially decreases its activity at room temperature without compromising its ability to amplify DNA. To understand why a conservative change to the enzyme over 20 Å from the active site can have a large impact on its activity at low temperature, we solved the X-ray crystal structure of the large (5′-to-3′ exonuclease-deleted) fragment of Taq DNA polymerase containing the cold-sensitive mutation in the ternary (E–DNA–ddNTP) and binary (E–DNA) complexes. The I707L KlenTaq1 ternary complex was identical to the wild-type in the closed conformation except for the mutation and a rotamer change in nearby phenylalanine 749, suggesting that the enzyme should remain active. However, soaking out of the nucleotide substrate at low temperature results in an altered binary complex made possible by the rotamer change at F749 near the tip of the polymerase O-helix. Surprisingly, two adenosines in the 5′-template overhang fill the vacated active site by stacking with the primer strand, thereby blocking the active site at low temperature. Replacement of the two overhanging adenosines with pyrimidines substantially increased activity at room temperature by keeping the template overhang out of the active site, confirming the importance of base stacking. These results explain the cold-sensitive phenotype of the I707L mutation in KlenTaq1 and serve as an example of a large conformational change affected by a conservative mutation.
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DOI：
10.1107/s0907444910045749
发表时间：
2011-04
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Winn MD;Ballard CC;Cowtan KD;Dodson EJ;Emsley P;Evans PR;Keegan RM;Krissinel EB;Leslie AG;McCoy A;McNicholas SJ;Murshudov GN;Pannu NS;Potterton EA;Powell HR;Read RJ;Vagin A;Wilson KS
通讯作者：
Wilson KS
影响因子：
4.8
作者：
Wang, Weina;Wu, Eugene Y.;Beese, Lorena S.
通讯作者：
Beese, Lorena S.
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
5.5
作者：
Roe, Daniel R.;Cheatham, Thomas E., III
通讯作者：
Cheatham, Thomas E., III
影响因子：
5.6
作者：
Astatke, M;Grindley, NDF;Joyce, CM
通讯作者：
Joyce, CM