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微生物遺傳與生物技術(Microbial Genetics and Biotechnology) 金門大學 食品科學系 何國傑 教授 期中考題講解 Stabilization of a stalled replication fork by concerted actions of two helicase PriA helicase plays crucial roles in restoration of arrested replication fork. PriA解旋酶在恢復被停止的複製叉扮演了關鍵角色。 It carries a “3’ terminus binding pocket” in its N- terminal DNA binding domain, which is required for high affinity binding of PriA to a fork carrying a 3’-end of a nascent leading strand at the branch. 在它的N-端的DNA結合區含有一個”3’端結合域”，這個結合 域對PriA能以高親和力結合到一個在分叉處含有新合成之引 導股的3’端之複製叉所必須。 Stabilization of a stalled replication fork by concerted actions of two helicase We show that the abrogation of the 3’ terminus recognition either by a mutation in the 3’ terminus binding pocket or by the bulky modification of the 3’- end leads to unwinding of the unreplicated duplex arm on this fork, causing potential fork destabilization. 我們指出，利用在3’端結合域造成突變，或大量修飾3’端來 廢除(PriA)對3’端的辨認，如此會導致複製叉尚未複製的雙 鏈部分之鏈解，而造成複製叉濳在性不穩定。 This indicates a critical role of the 3’ terminus binding pocket of PriA in its “stable” binding at the fork for primosome assembly. 這也顯示PriA的3’ 端結合域在複製叉穩定的結合對引子酶體 (primosome)的組裝上扮演關鍵角色。 Stabilization of a stalled replication fork by concerted actions of two helicase In contrast, PriA unwinds the unreplicated duplex region on a fork without a 3’-end, potentially destabilizing the fork. 相反地，PriA解鏈了一個沒有3’-端的複製叉之未複製雙鏈區 域，造成了複製叉濳在性不穩定。 However, this process is inhibited by RecG helicase, capable of regressing the fork until the 3’-end of the nascent leading strand reaches the branch. 然而，這個過程會被RecG解鏈酶抑制，它有能力將複製叉 退回，直到新合成的引導股的”3’端到分叉處。 Stabilization of a stalled replication fork by concerted actions of two helicase PriA now stably binds to this regressed fork, stabilizing it. 此時，PriA穩定地結合到這個退回的複製叉，並穩定它。 Using a model arrest-fork-substrate, we reconstitute the above process in vitro with RecG and PriA proteins. 利用被停止-複製叉-受質的模式，我們在試管中利用RecG及 PriA蛋白質重建上述的過程。 Stabilization of a stalled replication fork by conc