Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase.
Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase.
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DOI：
10.1093/nar/gkl1015
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发表时间：
2007
影响因子：
14.9
通讯作者：
Evans DH
中科院分区：
文献类型：
--
作者：
Hamilton MD;Nuara AA;Gammon DB;Buller RM;Evans DH
牛痘病毒DNA聚合酶在体外催化双链体DNA连接反应，并且这些重组反应的许多特征在体内重现。这可以解释病毒复制和基因重组之间的密切联系。然而，目前还不清楚为什么这些明显普通的聚合酶表现出这种不寻常的催化能力。在这项研究中，我们已经使用不同的基板进行详细的调查，由双链重组的机制，牛痘DNA聚合酶催化。当同源的平端线性双链体底物与牛痘聚合酶一起孵育时，在Mg 2+和dNTP的存在下，接合分子的出现之前，互补单链序列被校正核酸外切酶暴露。这些中间体退火以形成含有两侧为切口、1-5 nt缺口和/或短突出端的杂交区的接合分子群体。产物对外切核酸酶（和聚合酶）活性具有相对抗性，因此在连接反应中积累。表面等离子体共振（SPR）测量表明，该酶具有相对结合亲和力，有利于平端双链体的分子轴承3′-凹陷的差距。重组双链体是最不受欢迎的配体。这些数据表明，一个特定的组合，否则普通的酶和DNA结合特性，使痘病毒DNA聚合酶，以促进双链连接反应。
Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1–5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3′-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.
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