利用單分子技術探討DNA重組酶Rad51與Dmc1核蛋白絲之成核動力學

Studying the Assembly Kinetics of Saccharomyces cerevisiae Rad51 and Dmc1 Recombinases Using Single Molecule Methods

林聖堯(Sheng-Yao Lin)

指導教授 ： 李弘文

國立臺灣大學/理學院/化學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201702627

摘要

雙股斷裂的DNA損壞，可利用同源重組(homologous recombination)的方式，準確地修復受損的DNA。DNA重組酶催化受損DNA與同源的雙股DNA配對，進行股交換反應，進而利用完整的DNA模板進行修復。真核生物具有兩種同源重組酶Dmc1 與Rad51，其序列及功能大多相似，但兩者在細胞週期出現的時間點卻有不同。在細胞分裂時(mitosis)只需Rad51即可完成DNA修復，然而在減數分裂(meiosis)的過程中，則同時需要Rad51與Dmc1兩者的參與。為何不同的細胞階段對DNA重組酶的需求不同，其機制仍然未明。利用單分子栓球實驗(single molecule tethered particle motion)，本研究直接比較Saccharomyces cerevisiae 酵母菌中Rad51與Dmc1兩個重組酶在形成核蛋白絲階段的動力學。我們觀察到ScRad51與ScDmc1在核蛋白絲的形成動力學明顯不同。在核蛋白絲的形成過程，成核的階段是速率決定步驟，而ScRad51的成核速率比ScDmc1來的快，但兩者在其核蛋白絲的延展及核蛋白絲的長度上，則沒有差別。不同的重組酶濃度實驗中指出，ScRad51與ScDmc1皆以二倍體(dimer)的形式進行成核，因此成核速率的差異是來自於ScRad51對單股DNA (ssDNA)有較高的親和力，這個現象也反應在ScRad51的成核速率隨著不同長度的單股DNA而明顯遞增。相對來說，ScDmc1的成核速率在不同長度的單股DNA中則沒有明顯關聯，而是與單雙股交界處的連接點(ds/ss DNA junction)處的成核速率相似。Rad51與Dmc1核蛋白絲的形成動力學上的差異，及其對於不同DNA構型的偏好性，在建構同源重組在減數分裂過程中進行的分子模型時，可以提供這兩種DNA重組酶角色的重要基礎。

關鍵字

DNA重組? ； Rad51 ； Dmc1 ； 核蛋白絲 ； DNA同源重組修護 ； 單分子栓球實驗

並列摘要

The double-stranded break (DSB) DNA damage can be repaired in high fidelity using homologous recombination (HR) pathway. DNA recombinases form nucleoprotein filaments and catalyze the pairing of the homologous DNA sequence and the exchange of DNA strands. So this allow DNA replication to repair the damaged DNA using the homologous DNA template. In eukaryotes, there exist two recombinases, Rad51 and Dmc1. Both of them share similar amino acid sequences and functions. However, while only Rad51 is required in mitosis, both Rad51 and Dmc1 are essential in meiosis recombination. The mechanism underlying the differential requirement is unknown. Here, we compared the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae ScRad51 and ScDmc1 using single-molecule tethered particle motion experiments (TPM). We found an apparent kinetics difference during nucleoprotein filament assembly for ScRad51 and ScDmc1. Forming recombinase nuclei in single-stranded (ss) DNA is the rate-limited step in the nucleoprotein filament assembly. In our real-time assembly measurement, we found that ScRad51 has much faster nucleation rate than ScDmc1, while the extension time and filament coverage are similar for both ScRad51 and ScDmc1. Study of the nucleation times at different recombinase concentrations showed that ScRad51 and ScDmc1 have the similar power-law dependence of ~ 2, suggesting that both form stable nuclei in dimers. Therefore, the faster nucleation of ScRad51 likely results from the higher ssDNA affinity. This is consistent with the observation that nucleation times of ScRad51 increases with DNA substrates of longer ssDNA lengths. However, nucleation times of ScDmc1 did not show apparent ssDNA length dependence. These kinetic differences in nucleoprotein filament assembly provide important molecular constrains in explaining biochemical roles of Rad51 and Dmc1.

並列關鍵字

DNA Recombinases ； Rad51 ； Dmc1 ； nucleoprotein filament ； DNA homologous recombinational repair ； single molecule Tethered Particle Motion

參考文獻

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