Rodriguez A C, Park H W, Mao C, Beese L S
Department of Biochemistry, Duke University Medical Center, Durham, NC, 27710, USA.
J Mol Biol. 2000 Jun 2;299(2):447-62. doi: 10.1006/jmbi.2000.3728.
The 2.25 A resolution crystal structure of a pol alpha family (family B) DNA polymerase from the hyperthermophilic marine archaeon Thermococcus sp. 9 degrees N-7 (9 degrees N-7 pol) provides new insight into the mechanism of pol alpha family polymerases that include essentially all of the eukaryotic replicative and viral DNA polymerases. The structure is folded into NH(2)- terminal, editing 3'-5' exonuclease, and polymerase domains that are topologically similar to the two other known pol alpha family structures (bacteriophage RB69 and the recently determined Thermococcus gorgonarius), but differ in their relative orientation and conformation. The 9 degrees N-7 polymerase domain structure is reminiscent of the "closed" conformation characteristic of ternary complexes of the pol I polymerase family obtained in the presence of their dNTP and DNA substrates. In the apo-9 degrees N-7 structure, this conformation appears to be stabilized by an ion pair. Thus far, the other apo-pol alpha structures that have been determined adopt open conformations. These results therefore suggest that the pol alpha polymerases undergo a series of conformational transitions during the catalytic cycle similar to those proposed for the pol I family. Furthermore, comparison of the orientations of the fingers and exonuclease (sub)domains relative to the palm subdomain that contains the pol active site suggests that the exonuclease domain and the fingers subdomain of the polymerase can move as a unit and may do so as part of the catalytic cycle. This provides a possible structural explanation for the interdependence of polymerization and editing exonuclease activities unique to pol alpha family polymerases. We suggest that the NH(2)-terminal domain of 9 degrees N-7 pol may be structurally related to an RNA-binding motif, which appears to be conserved among archaeal polymerases. The presence of such a putative RNA- binding domain suggests a mechanism for the observed autoregulation of bacteriophage T4 DNA polymerase synthesis by binding to its own mRNA. Furthermore, conservation of this domain could indicate that such regulation of pol expression may be a characteristic of archaea. Comparion of the 9 degrees N-7 pol structure to its mesostable homolog from bacteriophage RB69 suggests that thermostability is achieved by shortening loops, forming two disulfide bridges, and increasing electrostatic interactions at subdomain interfaces.
来自嗜热海洋古菌嗜热栖热菌9°N - 7（9°N - 7聚合酶）的α家族（B族）DNA聚合酶的2.25埃分辨率晶体结构，为α家族聚合酶的作用机制提供了新见解，该家族聚合酶基本上包括所有真核生物复制性和病毒DNA聚合酶。其结构折叠成氨基末端、编辑3'-5'外切核酸酶和聚合酶结构域，在拓扑结构上与另外两个已知的α家族结构（噬菌体RB69和最近确定的嗜热栖热菌）相似，但相对方向和构象不同。9°N - 7聚合酶结构域结构让人联想到在存在dNTP和DNA底物时获得的确I型聚合酶家族三元复合物的“封闭”构象特征。在无辅基9°N - 7结构中，这种构象似乎通过离子对得以稳定。到目前为止，已确定的其他无辅基α聚合酶结构采用开放构象。因此，这些结果表明α聚合酶在催化循环中经历一系列构象转变，类似于为I型聚合酶家族提出的转变。此外，将手指结构域和外切核酸酶（亚）结构域相对于包含聚合酶活性位点的手掌亚结构域的方向进行比较表明，外切核酸酶结构域和聚合酶的手指亚结构域可以作为一个单元移动，并且可能作为催化循环的一部分移动。这为α家族聚合酶特有的聚合和编辑外切核酸酶活性的相互依赖性提供了一种可能的结构解释。我们认为9°N - 7聚合酶的氨基末端结构域在结构上可能与RNA结合基序相关，这在古菌聚合酶中似乎是保守的。这种假定的RNA结合结构域的存在表明了一种机制，即噬菌体T4 DNA聚合酶通过与其自身mRNA结合来实现对自身合成进行自动调节。此外，该结构域的保守性可能表明这种对聚合酶表达的调节可能是古菌的一个特征。将9°N - 7聚合酶结构与其来自噬菌体RB69的亚稳态同源物进行比较表明，热稳定性是通过缩短环、形成两个二硫键以及增加亚结构域界面处的静电相互作用来实现的。
