Single-stranded DNA-binding protein in vitro eliminates the orientation-dependent impediment to polymerase passage on CAG/CTG repeats
Single-stranded DNA-binding protein in vitro eliminates the orientation-dependent impediment to polymerase passage on CAG/CTG repeats
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DOI：
10.1074/jbc.m800153200
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发表时间：
2008-05-09
影响因子：
4.8
通讯作者：
McMurray, Cynthia T.
中科院分区：
文献类型：
--
作者：
Delagoutte, Emmanuelle;Goellner, Geoffrey M.;McMurray, Cynthia T.
在复制过程中，通过在模板或新合成的链上形成聚合酶滑移和发夹，可以发生三核苷酸重复序列(TNR)的小插入和小缺失。尽管没有被滑脱模型预测，但当5‘-CTG在滞后链模板中并且在快速复制的细胞中比插入事件更受欢迎时，缺失优先发生。TNR不稳定性的缺失偏向和取向依赖性的机制尚不清楚。我们在这里报道，在5‘-CAG和5’-CTG重复序列上存在方向相关的聚合酶进展障碍，这种障碍可以通过单链DNA结合蛋白的结合而缓解。该序列依赖于TNR的一级序列，但与发夹的热力学稳定性无关。以5‘-CAG为模板时，聚合酶通道的取向依赖性阻断最强。我们提出了一个“模板推送”模型，在该模型中，DNA聚合酶穿过5‘-CAG前导链模板的缓慢速度对解旋酶-聚合酶偶联造成了威胁。为了防止脱钩，TNR模板被推出并绕过。发夹不会导致堵塞，但似乎是聚合酶传递的结果。
Small insertions and deletions of trinucleotide repeats (TNRs) can occur by polymerase slippage and hairpin formation on either template or newly synthesized strands during replication. Although not predicted by a slippage model, deletions occur preferentially when 5 '-CTG is in the lagging strand template and are highly favored over insertion events in rapidly replicating cells. The mechanism for the deletion bias and the orientation dependence of TNR instability is poorly understood. We report here that there is an orientation-dependent impediment to polymerase progression on 5 '-CAG and 5 '-CTG repeats that can be relieved by the binding of single-stranded DNA-binding protein. The block depends on the primary sequence of the TNR but does not correlate with the thermodynamic stability of hairpins. The orientation-dependent block of polymerase passage is the strongest when 5 '-CAG is the template. We propose a "template-push" model in which the slow speed of DNA polymerase across the 5 '-CAG leading strand template creates a threat to helicase-polymerase coupling. To prevent uncoupling, the TNR template is pushed out and by-passed. Hairpins do not cause the block, but appear to occur as a consequence of polymerase pass-over.