Overcoming natural replication barriers: differential helicase requirements
Overcoming natural replication barriers: differential helicase requirements
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DOI：
10.1093/nar/gkr836
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发表时间：
2012-02-01
影响因子：
14.9
通讯作者：
Freudenreich, Catherine H.
中科院分区：
文献类型：
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作者：
Anand, Ranjith P.;Shah, Kartik A.;Freudenreich, Catherine H.
形成二级结构或结合蛋白质复合物的DNA序列是已知的复制障碍和基因组不稳定性的潜在诱导物。为了确定哪些解旋酶促进DNA复制跨越这些障碍，我们分析了叉进展，通过他们在野生型和突变酵母细胞，使用二维凝胶电泳分析的复制中间体。我们表明，Srs 2蛋白有利于复制的发夹形成CGG/CCG重复，并防止染色体脆性的重复，而它不影响复制的G-四链体形成序列或蛋白结合的重复。Srs 2解旋酶活性是发夹解旋和叉进展所必需的。此外，Srs 2的PCNA结合结构域是其通过发夹在体内复制的作用所必需的。与此相反，Sgs 1或Pif 1解旋酶的情况下，没有抑制复制通过结构障碍，虽然Pif 1确实促进端粒蛋白屏障的复制。有趣的是，通过蛋白质屏障而不是DNA结构屏障的复制受到核苷酸库水平的调节，从而阐明了细胞可以通过蛋白质介导的停滞位点调节叉进展的不同机制。我们的分析揭示了DNA结构与蛋白质屏障复制的根本差异，Srs 2解旋酶活性完全是通过发夹结构进行叉进展所需的。
DNA sequences that form secondary structures or bind protein complexes are known barriers to replication and potential inducers of genome instability. In order to determine which helicases facilitate DNA replication across these barriers, we analyzed fork progression through them in wild-type and mutant yeast cells, using 2-dimensional gel-electrophoretic analysis of the replication intermediates. We show that the Srs2 protein facilitates replication of hairpin-forming CGG/CCG repeats and prevents chromosome fragility at the repeat, whereas it does not affect replication of G-quadruplex forming sequences or a protein-bound repeat. Srs2 helicase activity is required for hairpin unwinding and fork progression. Also, the PCNA binding domain of Srs2 is required for its in vivo role of replication through hairpins. In contrast, the absence of Sgs1 or Pif1 helicases did not inhibit replication through structural barriers, though Pif1 did facilitate replication of a telomeric protein barrier. Interestingly, replication through a protein barrier but not a DNA structure barrier was modulated by nucleotide pool levels, illuminating a different mechanism by which cells can regulate fork progression through protein-mediated stall sites. Our analyses reveal fundamental differences in the replication of DNA structural versus protein barriers, with Srs2 helicase activity exclusively required for fork progression through hairpin structures.