應用質譜儀進行第五型核酸內切酶與第一型DNA聚合酶修復亞黃嘌呤之研究

在一般生理環境下，DNA的腺嘌呤 (adenine; A)會自發性水解脫胺(deamination)產生脫氧肌苷 (deoxyinosine ; dI)。Deoxyinosine如果未修復，在DNA複製時，因為dI偏好與dCTP結合，會造成A:T變G:C的transition mutations。當DNA暴露由輻射、UV、亞硝酸鹽、熱等引起的ROS環境時，會促進adenine的deamination。在Escherichia coli (E. coli)中，dI的修復反應由endonuclease V (Endo V; nfi gene product) nicking作為起始。先前本實驗室進行 in vitro minimum component reconstitution assay時，發現Endo V、DNA polymerase I (Pol I)和E. coli DNA ligase共同反應後成功修復dI，而且發現Pol I的3’-5’ exonuclease對於修復十分重要。先前研究發現Endo V在辨識dI後，會在dI 3’端第二個phosphodiester bond產生一個nick，之後Endo V會結合在DNA上，不會turnover。然而，先前本實驗室進行 in vitro assay時，加入有proofreading能力的Pol I或Klenow fragment (KF)的情況下，Endo V與Pol I turnover增加至少4倍。這結果顯示Endo V與Pol I在修復反應過程中可能有交互作用。為了證明Endo V和Pol I之間在修復dI時可能有交互作用，我們使用MALDI-TOF MS進行分析，當Endo V修復有dI的DNA時會產生nicking product，並且有分子量上的改變，藉由分子量的差異可以辨識出nicking product。使用dI ssDNA substrate、I-T dsDNA substrate和dI與不同正常鹼基配對的DNA substrate分析Endo V nicking活性，發現針對不同substrate具有不同的修復效率。其中，Endo V對含dI ssDNA有較高的活性。使用40 bp I-T substrate進行實驗時，發現Endo V在反應一段時間後幾乎沒有turnover，之後追加等量的Endo V進行反應，發現增加等比例的nicking products。而當在反應中加入KF時，Endo V total product有明顯增加，顯示KF可能是藉由proofreading exonuclease活性移除dI來促進Endo V turnover。為了驗證這個假設，我們使用proofreading exonuclease deficiency KF (KF exo-)和24 bp I-T substrate進行實驗。我們意外發現KF exo-也可以促進Endo V turnover。之後我們使用40 bp I-T和I-C substrate並在反應中加入KF或KF exo-進行實驗，我們推論Endo V和KF之間的interaction是透過Endo V nicking後使dI露出，讓KF辨識後進行修復，並且值得進一步深入研究。為了進一步研究in vitro和in vivo中，Pol I和Endo V在修復過程中的交互作用，我們設計帶有CC marker 的I-T異雙股質體進行實驗。我們已經構築出有selected marker的質體來進行substrate preparation。

關鍵字

脫氧肌苷；第五型核酸內切酶；第一型DNA聚合酶；克列諾片段；基質輔助雷射去吸附離子化飛行時間質譜儀

並列摘要

Deamination of adenine can occur spontaneously under physiological conditions to generate deoxyinosine (hypoxanthine deoxyribonucleotide, dI). Deoxyinosine in DNA is potentially mutagenic since it prefers to pair with dCTP during replication, yielding A:T to G:C transition mutation if not repaired. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli (E. coli), dI repair is initiated by endonuclease V (Endo V; nfi gene product) nicking. Using in vitro minimum component reconstitution assays, we previously showed that Endo V, DNA polymerase I (Pol I), and E. coli DNA ligase were sufficient to repair this dI lesion efficiently and that the 3’-5’ exonuclease of Pol I is essential. Endo V recognizes dI and cleaves the dI containing strand at second phosphodiester bond 3’ to the dI. It is known that, after nicking, Endo V remains tightly bound to dI-containing nicked DNA and does not turnover. However, in the presence of proofreading proficient Pol I or Klenow fragment (KF), an in vitro experiment demonstrated that both Endo V and Pol I turnover at least 4 times. These results strongly suggested interaction between Endo V and Pol I during the repair process. We employed a MALDI-TOF mass spectrometry (MS) analysis to identify possible interactions between Endo V and Pol I in dI processing. The nicking products were identified by mass change of the Endo V processed dI containing DNA. Substrate of single-stranded and double-stranded DNAs with dI and various dI base pair of the substrates were tested for Endo V nicking activity albeit with different efficiencies. Generally, Endo V showed higher activity to single-stranded dI substrate than dI containing duplex. Endo V showed almost no turnover in a prolonged nicking reaction of dI at the center of 40 bp I-T substrate, and chasing with the same amount of Endo V only showed the proportional amount of nicking products increase. In the presence of KF, the Endo V total product was obviously increased suggesting KF promote turnover of Endo V, possibly by proofreading exonuclease removing of dI. To test this possibility, we employed proofreading exonuclease deficiency KF (KF exo-) to nicking reaction of 24 bp I-T substrate. Interestingly, we found that the turnover of Endo V nicking could also be promoted by KF exo-. Next, we employed KF or KF exo- to nicking reaction of 40 bp I-T and I-C substrate. We assume that the dI will expose after Endo V nicking, allowing KF to identify and repair it. It is worthy of further study. To further study the interaction of Pol I and Endo V in the repair process in vitro and in vivo, we also designed a phagemid-based I-T substrate with a CC mismatch as strand discrimination marker. We constructed all the phagemids with selected markers for substrate preparation.

並列關鍵字

deoxyinosine ； endonuclease V ； DNA polymerase I ； Klenow fragment ； matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

參考文獻

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Weiss, B. (2008). Removal of deoxyinosine from the Escherichia coli chromosome as studied by oligonucleotide transformation. DNA Repair (Amst), 7(2), 205-212. doi:10.1016/j.dnarep.2007.09.010

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應用質譜儀進行第五型核酸內切酶與第一型DNA聚合酶修復亞黃嘌呤之研究

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