DNA聚合酶λ結構機制探討之忠實度調節

Abstract

研究DNA聚合酶之忠實性機制對於化學催化和生物意義上是個基礎且重要的議題。在2000年以前高忠實性複製型DNA聚合酶已被廣泛的研究，然而對於低忠實性修復型DNA聚合酶則較少被研究。本實驗室藉由X-射線結晶學解出12個DNA聚合酶 λ 蛋白質晶體結構並使用酵素動力學測量DNA聚合酶 λ 如何藉由本身結構特性調節忠實性之功能，亦藉由生物物理之測量方法發現DNA聚合酶 λ 具有無需DNA之存在下可以結合dNTP。本實驗室解出第一個apo-型DNA聚合酶 λ，發現apo-型DNA聚合酶 λ 之dNTP結合位已和催化型活化態之三級結構一樣，總和以上發現指出apo-型DNA聚合酶 λ 可以預先結合dNTP，這可以瞭解低忠實性修復型DNA聚合酶 λ 如何保持較低的忠實性。更進一步，DNA聚合酶 λ 之活化區具有一個疏水性核心，由四個胺基酸所組成(Leu431, Ile492, and the Tyr505/Phe506 motif)。本實驗室藉由突變型Leu431Ala發現Leu431扮演調節忠實性之功能。DNA聚合酶具有結合DNA和dNTP之能力，在過往中心教條上DNA聚合酶先結合上DNA，再結合dNTP，此優點可以藉由DNA序列上之鹼基配對來提高複製忠實性。倘若DNA序列上之鹼基受到氧化壓力(ROS)或是太陽光(UV)照射造成鹼基產生突變，會使得高忠實性複製型DNA聚合酶停在DNA複製叉上，若無法通過這些突變鹼基完成複製，進一步造成雙股螺旋DNA斷裂提升基因組不穩定。本論文證實低忠實性修復型DNA聚合酶 λ 具有預先結合dNTP之能力，提供了一條新的路徑，DNA聚合酶可以先結合dNTP越過突變之鹼基優先完成複製，使基因組穩定。

The mechanism of DNA polymerase (pol) fidelity is of fundamental importance in chemistry and biology. While high-fidelity pols have been well studied, much less is known about how some pols achieve medium or low fidelity with functional importance. Here we examine how human DNA polymerase λ (Pol λ) achieves medium fidelity by determining 12 crystal structures and performing pre-steady-state kinetic analyses. We showed that apo-Pol λ exists in the closed conformation, unprecedentedly with a preformed MgdNTP binding pocket, and binds MgdNTP readily in the active conformation in the absence of DNA. Since prebinding of MgdNTP could lead to very low fidelity as shown previously, it is attenuated in Pol λ by a hydrophobic core including Leu431, Ile492, and the Tyr505/Phe506 motif. We then predicted and demonstrated that L431A mutation enhances MgdNTP prebinding and lowers the fidelity. We also hypothesized that the MgdNTP-prebinding ability could stabilize a mismatched ternary complex and destabilize a matched ternary complex, and provided evidences with structures in both forms. Our results demonstrate that, while high-fidelity pols follow a common paradigm, Pol λ has developed specific conformations and mechanisms for its medium fidelity. Structural comparison with other pols also suggests that different pols likely utilize different conformational changes and microscopic mechanisms to achieve their catalytic functions with varying fidelities.