Spectroscopic studies of the structural domains of mammalian DNA beta-polymerase.
Spectroscopic studies of the structural domains of mammalian DNA beta-polymerase.
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哺乳动物 DNA β-聚合酶结构域的光谱研究。
DOI：
10.1016/s0021-9258(18)55038-9
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
R. Karpel
中科院分区：
文献类型：
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作者：
J. Casas;A. Kumar;G. Morris;S. H. Wilson;R. Karpel
通过如所述的受控蛋白水解制备的β-聚合酶的8-和31-kDa片段（Kumar，A.，Widen，S. G.，威廉姆斯，K. R.，Kedar，P.，卡佩尔河L.，Wilson，S. H.（1990）J.Biol.Chem.265，2124 - 2131）构成结构和功能上不同的结构域。有一点破坏的二级结构后，蛋白水解的完整的酶，建议从CD光谱的片段。β-聚合酶能够结合单链和双链核酸：8-kDa片段特异性结合单链晶格，而31-kDa结构域仅对双链多核苷酸显示亲和力。这些结构域通过高度灵活的蛋白酶超敏片段连接，该片段可以允许完整蛋白中的两种结合活性的协调功能。β-聚合酶以更高的亲和力、相似的协同性、但比8-kDa片段更低的盐依赖性结合聚（乙烯腺苷酸）。在生理条件下，完整的酶显示出更大的结合自由能的单链多核苷酸比8-kDa的片段，这表明后者可能携带一个截短的结合位点。双链小牛胸腺DNA的结合带来了一个温和的淬灭的Tyr和色氨酸荧光发射的31-kDa的片段和β-聚合酶，并诱导6 nm的蓝移色氨酸发射最大的完整的酶，但不是在片段。后一种结果可能是由于与双链DNA相互作用后，完整蛋白质中8-和31-kDa结构域的相对方向发生变化;或者，完整蛋白质的结合模式可能与片段的结合模式不同。两个结构域与多核苷酸的同时相互作用很可能不会发生，因为与完整β-聚合酶的31-kDa结构域结合的双链DNA诱导单链多核苷酸从8-kDa结构域置换。根据β-聚合酶在DNA修复中的作用评价这些结果。
The 8- and 31-kDa fragments of beta-polymerase, prepared by controlled proteolysis as described (Kumar, A., Widen, S. G., Williams, K. R., Kedar, P., Karpel, R. L., and Wilson, S. H. (1990) J. Biol. Chem. 265, 2124-2131), constitute domains that are structurally and functionally dissimilar. There is little disruption of secondary structure upon proteolysis of the intact enzyme, as suggested from CD spectra of the fragments. beta-Polymerase is capable of binding both single- and double-stranded nucleic acids: the 8-kDa fragment binds specifically to single-stranded lattices, whereas the 31-kDa domain displays affinity exclusively for double-stranded polynucleotides. These domains are connected by a highly flexible protease-hypersensitive segment that may allow the coordinate functioning of the two binding activities in the intact protein. beta-Polymerase binds to poly(ethenoadenylic acid) with higher affinity, similar cooperativity, but lesser salt dependence than the 8-kDa fragment. Under physiological conditions, the intact enzyme displays greater binding free energy for single-stranded polynucleotides than the 8-kDa fragment, suggesting that the latter may carry a truncated binding site. Binding of double-stranded calf thymus DNA brings about a moderate quenching of the Tyr and Trp fluorescence emission of both the 31-kDa fragment and beta-polymerase and induces a 6-nm blue shift in the Trp emission maximum of the intact enzyme, but not in the fragment. This latter result is likely due to a change in the relative orientation of the 8- and 31-kDa domains in the intact protein upon interaction with double-stranded DNA; alternatively, the binding mode of intact protein may differ from that of the fragment. Simultaneous interaction of both domains with polynucleotides most likely does not occur since double-stranded DNA binding to the 31-kDa domain of intact beta-polymerase induces the displacement of single-stranded polynucleotides from the 8-kDa domain. These results are evaluated in light of the role of beta-polymerase in DNA repair.