Department of Immunology, University of Washington Medical School, Seattle, Washington 98195-7650, USA.
J Biol Chem. 2010 Sep 10;285(37):28514-9. doi: 10.1074/jbc.M110.132738. Epub 2010 Jul 19.
Trinucleotide repeats can form stable secondary structures that promote genomic instability. To determine how such structures are resolved, we have defined biochemical activities of the related RAD2 family nucleases, FEN1 (Flap endonuclease 1) and EXO1 (exonuclease 1), on substrates that recapitulate intermediates in DNA replication. Here, we show that, consistent with its function in lagging strand replication, human (h) FEN1 could cleave 5'-flaps bearing structures formed by CTG or CGG repeats, although less efficiently than unstructured flaps. hEXO1 did not exhibit endonuclease activity on 5'-flaps bearing structures formed by CTG or CGG repeats, although it could excise these substrates. Neither hFEN1 nor hEXO1 was affected by the stem-loops formed by CTG repeats interrupting duplex regions adjacent to 5'-flaps, but both enzymes were inhibited by G4 structures formed by CGG repeats in analogous positions. Hydroxyl radical footprinting showed that hFEN1 binding caused hypersensitivity near the flap/duplex junction, whereas hEXO1 binding caused hypersensitivity very close to the 5'-end, correlating with the predominance of hFEN1 endonucleolytic activity versus hEXO1 exonucleolytic activity on 5'-flap substrates. These results show that FEN1 and EXO1 can eliminate structures formed by trinucleotide repeats in the course of replication, relying on endonucleolytic and exonucleolytic activities, respectively. These results also suggest that unresolved G4 DNA may prevent key steps in normal post-replicative DNA processing.
三核苷酸重复序列可以形成稳定的二级结构，从而促进基因组不稳定。为了确定这些结构是如何被解决的，我们已经定义了相关 RAD2 家族核酸内切酶 FEN1（Flap endonuclease 1）和 EXO1（exonuclease 1）的生化活性，这些酶的底物可以模拟 DNA 复制过程中的中间体。在这里，我们表明，与滞后链复制中的功能一致，人（h）FEN1 可以切割带有由 CTG 或 CGG 重复形成的结构的 5'-flaps，尽管效率低于无结构的 flaps。hEXO1 对带有由 CTG 或 CGG 重复形成的结构的 5'-flaps 没有表现出内切酶活性，尽管它可以切除这些底物。hFEN1 和 hEXO1 都不受 CTG 重复形成的茎环的影响，这些茎环中断了与 5'-flaps 相邻的双链区域，但这两种酶都被 CGG 重复在类似位置形成的 G4 结构所抑制。羟基自由基足迹显示，hFEN1 结合导致在 flap/duplex 连接处附近的超敏反应，而 hEXO1 结合导致在 5'-末端非常接近的位置的超敏反应，这与 hFEN1 在 5'-flap 底物上的内切酶活性与 hEXO1 的外切酶活性的优势相对应。这些结果表明，FEN1 和 EXO1 可以在复制过程中消除由三核苷酸重复形成的结构，分别依赖于内切酶和外切酶活性。这些结果还表明，未解决的 G4 DNA 可能会阻止正常复制后 DNA 处理的关键步骤。
