Structural insights into DNA replication without hydrogen bonds.
Structural insights into DNA replication without hydrogen bonds.
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DOI：
10.1021/ja409609j
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发表时间：
2013-12-11
影响因子：
15
通讯作者：
Marx A
中科院分区：
文献类型：
--
作者：
Betz K;Malyshev DA;Lavergne T;Welte W;Diederichs K;Romesberg FE;Marx A
基因字母表由两个碱基对组成，第三个非自然碱基对的发展将增加DNA的遗传和化学潜力。D5SICS-dNaM是迄今为止发现的复制效率最高的非自然碱基对之一，但它的配对只受疏水和堆积作用力的调节，而且在自由双链DNA中，它形成了一种跨链插层结构，这使得它的有效复制很难理解。最近对KlenTaq聚合酶的研究表明，d5SICSTP与dNaM的插入是通过相互诱导-配对的机制进行的，其中三磷酸的存在诱导聚合酶形成催化活性的闭合结构，进而诱导发育中的非天然碱基对的核苷酸采用平面Watson-Crick结构。为了了解复制的其余步骤，我们现在报告与dNaMTP与d5SICS相对的插入相对应的前化学复合体的表征，以及其中已经形成的非自然碱基对位于插入后位点的多个化学后复合体。与d5SICSTP插入dNaM相反的情况不同，dNaMTP的加入不能完全诱导催化活性闭合状态的形成。数据还显示，一旦合成并转移到插入后位置，不自然的碱基再次插入。根据侧翼核苷酸的性质，观察到两种插层模式，每一种模式都由与聚合酶的不同相互作用稳定，每一种模式似乎都降低了下一个正确的三磷酸结合的亲和力。因此，持续的引物延伸受到与聚合酶活性位点的脱嵌和重排的限制，这是填充催化活性的三磷酸结合构象所必需的。
The genetic alphabet is comprised of two base pairs, and the development of a third, unnatural base pair would increase the genetic and chemical potential of DNA. d5SICS-dNaM is one of the most efficiently replicated unnatural base pairs identified to date, but its pairing is mediated by only hydrophobic and packing forces, and in free duplex DNA it forms a cross-strand intercalated structure that makes its efficient replication difficult to understand. Recent studies of the KlenTaq polymerase revealed that the insertion of d5SICSTP opposite dNaM proceeds via a mutually induced-fit mechanism, where the presence of the triphosphate induces the polymerase to form the catalytically competent closed structure, which in turn induces the pairing nucleotides of the developing unnatural base pair to adopt a planar Watson-Crick-like structure. To understand the remaining steps of replication, we now report the characterization of the pre-chemistry complexes corresponding to the insertion of dNaMTP opposite d5SICS, as well as multiple post-chemistry complexes in which the already formed unnatural base pair is positioned at the post-insertion site. Unlike with the insertion of d5SICSTP opposite dNaM, addition of dNaMTP does not fully induce the formation of the catalytically competent closed state. The data also reveal that once synthesized and translocated to the post-insertion position, the unnatural nucleobases again intercalate. Two modes of intercalation are observed, depending on the nature of the flanking nucleotides, and are each stabilized by different interactions with the polymerase, and each appear to reduce the affinity with which the next correct triphosphate binds. Thus, continued primer extension is limited by de-intercalation and rearrangements with the polymerase active site that are required to populate the catalytically active, triphosphate bound conformation.
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影响因子：
3.2
作者：
Chelliserrykattil, Jijumon;Lu, Haige;Lee, Alex H. F.;Kool, Eric T.
通讯作者：
Kool, Eric T.
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH
DOI：
10.1107/s0907444909042073
发表时间：
2010-01
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Chen VB;Arendall WB 3rd;Headd JJ;Keedy DA;Immormino RM;Kapral GJ;Murray LW;Richardson JS;Richardson DC
通讯作者：
Richardson DC
影响因子：
15
作者：
Berezhna, Svitlana Y.;Gill, Joshua P.;Lamichhane, Rajan;Millar, David P.
通讯作者：
Millar, David P.
DOI：
10.1073/pnas.94.20.10493
发表时间：
1997-09-30
影响因子：
11.1
作者：
Goodman, MF
通讯作者：
Goodman, MF