Probing the DNA structural requirements for facilitated diffusion.
Probing the DNA structural requirements for facilitated diffusion.
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DOI：
10.1021/bi5013707
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发表时间：
2015-01-20
期刊：
影响因子：
2.9
通讯作者：
O'Brien, Patrick J.
中科院分区：
文献类型：
--
作者：
Hedglin, Mark;Zhang, Yaru;O'Brien, Patrick J.
DNA 糖基化酶进行全基因组搜索，以在大量未损坏的核苷酸中定位损坏的核苷酸。许多糖基化酶能够促进扩散，从而在单次结合过程中对 DNA 上的多个位点进行采样。带正电的氨基酸和带负电的磷酸盐骨架之间的静电相互作用对于促进扩散至关重要，但扩散蛋白质在多大程度上依赖于双螺旋结构 DNA 尚不清楚。动力学测定用于探测人烷基腺嘌呤 DNA 糖基化酶 (AAG) 的 DNA 搜索机制，并测试扩散需要 B 型双链 DNA 的程度。尽管 AAG 从单链 DNA 中切除 εA 损伤，但它对单链 DNA 不是进行性的，因为解离速度比 N-糖苷键裂解快。然而，AAG 与单链 DNA 的复合物足够稳定，可以在添加互补链时进行 DNA 退火。这一观察结果提供了 AAG 与单链 DNA 非特异性关联的证据。单链间隙、气泡和弯曲结构不会妨碍 AAG 的搜索。相反，这些柔性或弯曲的结构可以捕获附近的损伤部位，这比连续的 B 型双工更有效。 AAG 处理这些螺旋不连续性的能力与扩散的滑动模式不一致，但可以通过涉及微观解离和重新结合的跳跃模式轻松解释。这些实验提供了相对长距离跳跃的证据，这些跳跃允许搜索蛋白质绕过DNA结合蛋白，而DNA结合蛋白将成为滑动蛋白质的障碍。
DNA glycosylases perform a genome-wide search to locate damaged nucleotides among a great excess of undamaged nucleotides. Many glycosylases are capable of facilitated diffusion, whereby multiple sites along the DNA are sampled during a single binding encounter. Electrostatic interactions between positively charged amino acids and the negatively charged phosphate backbone are crucial for facilitated diffusion, but the extent to which diffusing proteins rely on the double-helical structure DNA is not known. Kinetic assays were used to probe the DNA searching mechanism of human alkyladenine DNA glycosylase (AAG) and to test the extent to which diffusion requires B-form duplex DNA. Although AAG excises εA lesions from single-stranded DNA, it is not processive on single-stranded DNA because dissociation is faster than N-glycosidic bond cleavage. However, the AAG complex with single-stranded DNA is sufficiently stable to allow for DNA annealing when a complementary strand is added. This observation provides evidence of nonspecific association of AAG with single-stranded DNA. Single-strand gaps, bubbles, and bent structures do not impede the search by AAG. Instead, these flexible or bent structures lead to the capture of a nearby site of damage that is more efficient than that of a continuous B-form duplex. The ability of AAG to negotiate these helix discontinuities is inconsistent with a sliding mode of diffusion but can be readily explained by a hopping mode that involves microscopic dissociation and reassociation. These experiments provide evidence of relatively long-range hops that allow a searching protein to navigate around DNA binding proteins that would serve as obstacles to a sliding protein.
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影响因子：
16.8
作者：
通讯作者：
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DOI：
10.1073/pnas.0705129104
发表时间：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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影响因子：
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作者：
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