Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism.
Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism.
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DOI：
10.1093/nar/gkm506
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发表时间：
2007
影响因子：
14.9
通讯作者：
Coen DM
中科院分区：
文献类型：
--
作者：
Loregian A;Sinigalia E;Mercorelli B;Palù G;Coen DM
疱疹病毒DNA聚合酶的持续合成因子的机制仍然知之甚少。人巨细胞病毒DNA聚合酶的持续合成因子是一种DNA结合蛋白UL44。先前的发现，包括UL44的晶体结构，已经导致了UL44通过赖氨酸残基作为二聚体结合DNA的假设。为了了解UL44如何与DNA相互作用，我们使用了过滤结合和电泳迁移率变动分析以及等温滴定量热法（ITC）分析与寡核苷酸的结合。UL 44直接结合短至12 bp的双链DNA，对于DNA>18 bp，表观解离常数在纳摩尔范围内，表明UL 44相互作用的最小DNA长度。UL44也结合单链DNA，尽管具有较低的亲和力，并且对于单链或双链DNA，没有明显的序列特异性。ITC分析显示，UL44作为二聚体结合双链体DNA。结合是吸热的，表明熵驱动的过程，可能是由于结合离子的释放。与该假设一致，结合和离子强度之间的关系的分析表明，在UL44的每个单体与DNA的相互作用中，平均释放4 ± 1个单价离子。这些结果共同揭示了UL44如何介导持续合成能力的有趣影响。
The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12 bp, with apparent dissociation constants in the nanomolar range for DNAs >18 bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, 4 ± 1 monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity.
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