微生物遺傳與生物技術 (Microbial Genetics and Biotechnology)

1. 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

2. 期中考題講解

3. 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。

4. 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。

5. 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。

6. 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。

7. Stabilization of a stalled replication fork by concerted actions of two helicase Our results present a novel mechanism by which two helicases function in a highly coordinated manner to generate a structure in which an arrested fork is stabilized for further repair and/or replication restart. 我們的結果呈現一個嶄新的機制，即利用兩個解鏈酶的功能 之高度配合狀況下，產生一個構造，使被停止的複製叉穩定 下來，以進行進一步的修補，及/或重新開始複製工作。

8. A model for stabilization of arrested replication fork by RecG and PriA proteins • A • PriA binds to fork through interaction of the 3’ terminus binding pocket with the 3’ terminus of the nascent leading strand. • This interaction directs the position of the helicase domain in one orientation, which is inert for fork unwinding. • The presence of a 3’ terminus prevents the binding of PriA in other model.

9. A model for stabilization of arrested replication fork by RecG and PriA proteins

10. A model for stabilization of arrested replication fork by RecG and PriA proteins • B • The arrested replication forks carrying the nascent lagging strand at the branch point are recognized either by recG or PriA. • In pathway II, PriA advances the replication fork, resulting in its destabilization. (Note: PriA binds in different orientation) • However, this is normally prevented by RecG protein, which regresses the fork and generates a chicken foot structure that is now recognized and bound by PriA. • Duplex unwinding activity of PriA is suppressed under this condition, and the fork is stabilized (pathway I). • PriA is bound near the 3’-end of the arrested leading strand prior to RecG-mediated fork reversal. • This binding may prevent further unwinding by RecG and may facilitate the coupled action of RecG and PriA on this arrested fork structure.

11. A model for stabilization of arrested replication fork by RecG and PriA proteins