Wagner Koen, Moolenaar Geri, van Noort John, Goosen Nora
Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, Leiden, the Netherlands.
Nucleic Acids Res. 2009 Apr;37(6):1962-72. doi: 10.1093/nar/gkp071. Epub 2009 Feb 10.
The UvrA protein is the initial damage-recognizing factor in bacterial nucleotide excision repair. Each monomer of the UvrA dimer contains two ATPase sites. Using single-molecule analysis we show that dimerization of UvrA in the presence of ATP is significantly higher than with ADP or nonhydrolyzable ATPgammaS, suggesting that the active UvrA dimer contains a mixture of ADP and ATP. We also show that the UvrA dimer has a high preference of binding the end of a linear DNA fragment, independent on the presence or type of cofactor. Apparently ATP binding or hydrolysis is not needed to discriminate between DNA ends and internal sites. A significant number of complexes could be detected where one UvrA dimer bridges two DNA ends implying the presence of two separate DNA-binding domains, most likely present in each monomer. On DNA containing a site-specific lesion the damage-specific binding is much higher than DNA-end binding, but only in the absence of cofactor or with ATP. With ATPgammaS no discrimination between a DNA end and a DNA damage could be observed. We present a model where damage recognition of UvrA depends on the ability of both UvrA monomers to interact with the DNA flanking the lesion.
UvrA蛋白是细菌核苷酸切除修复中最初的损伤识别因子。UvrA二聚体的每个单体包含两个ATP酶位点。通过单分子分析，我们发现，在ATP存在的情况下，UvrA的二聚化程度显著高于在ADP或不可水解的ATPγS存在时的情况，这表明活性UvrA二聚体包含ADP和ATP的混合物。我们还表明，UvrA二聚体对线性DNA片段末端具有高度的结合偏好，与辅因子的存在与否或类型无关。显然，区分DNA末端和内部位点不需要ATP结合或水解。可以检测到大量的复合物，其中一个UvrA二聚体桥接两个DNA末端，这意味着存在两个独立的DNA结合结构域，很可能存在于每个单体中。在含有位点特异性损伤的DNA上，损伤特异性结合远高于DNA末端结合，但仅在不存在辅因子或存在ATP的情况下。使用ATPγS时，无法观察到DNA末端与DNA损伤之间的区分。我们提出了一个模型，其中UvrA的损伤识别取决于两个UvrA单体与损伤侧翼DNA相互作用的能力。
