科研人員報(bào)告了一種在活細(xì)菌中觀察DNA修復(fù)的技術(shù),。
誘變劑導(dǎo)致的DNA核苷酸堿基的損傷被一個(gè)稱(chēng)為切補(bǔ)修復(fù)的過(guò)程所逆轉(zhuǎn),，這個(gè)過(guò)程是由一組酶執(zhí)行的,。其中兩個(gè)酶——取代失蹤的核苷酸的DNA聚合酶I與把DNA鏈連接在一起從而幫助彌合切口的DNA連接酶——在從細(xì)菌到人的一系列生物中執(zhí)行了這個(gè)修復(fù)過(guò)程的最終步驟，但是迄今為止這些酶發(fā)揮作用的過(guò)程還尚未被直接觀察到,。
Stephan Uphoff及其同事使用一種稱(chēng)為光敏定位顯微鏡的技術(shù)把這兩種酶在活的大腸桿菌中的單個(gè)分子的活動(dòng)顯現(xiàn)出來(lái),。這組作者把單個(gè)酶分子與DNA結(jié)合的痕跡解釋為DNA合成與連接的證據(jù)，他們發(fā)現(xiàn)DNA修復(fù)點(diǎn)遍布于整個(gè)細(xì)胞之中,。這組作者還發(fā)現(xiàn),，對(duì)于聚合酶，單個(gè)修復(fù)事件持續(xù)了2.1秒,，而對(duì)于連接酶,，單個(gè)修復(fù)時(shí)間持續(xù)了2.5秒；在實(shí)驗(yàn)誘發(fā)DNA損傷的數(shù)分鐘內(nèi),，這兩種酶的活動(dòng)增加到了基線(xiàn)狀態(tài)的5倍,。
在DNA沒(méi)有損傷的一個(gè)細(xì)菌細(xì)胞內(nèi)，在任何時(shí)間里,，在大約400個(gè)聚合酶的拷貝中只有2.7%的拷貝處于復(fù)制和修復(fù)的活動(dòng)當(dāng)中,，而在大約200個(gè)連接酶的拷貝中大約有3.8%的拷貝處于復(fù)制和修復(fù)的活動(dòng)當(dāng)中。這組作者發(fā)現(xiàn)當(dāng)細(xì)胞受到DNA損傷的時(shí)候,，這兩種酶都把它們壽命的80%以上用于尋找有毒的中間物以進(jìn)行修復(fù)工作,，因此最小化了DNA缺口與切口的生存時(shí)間。
這組作者說(shuō),，直接測(cè)量活細(xì)胞的DNA修復(fù)率可能會(huì)引出一些定量模型,，它們能夠幫助科研人員預(yù)測(cè)生物如何能夠?qū)NA的損傷做出響應(yīng)。（生物谷Bioon.com）
doi:10.1073/pnas.1301804110
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Single-molecule DNA repair in live bacteria
Stephan Uphoff, Rodrigo Reyes-Lamothe, Federico Garza de Leon, David J. Sherratt, and Achillefs N. Kapanidis
Cellular DNA damage is reversed by balanced repair pathways that avoid accumulation of toxic intermediates. Despite their importance, the organization of DNA repair pathways and the function of repair enzymes in vivo have remained unclear because of the inability to directly observe individual reactions in living cells. Here, we used photoactivation, localization, and tracking in live Escherichia coli to directly visualize single fluorescent labeled DNA polymerase I (Pol) and ligase (Lig) molecules searching for DNA gaps and nicks, performing transient reactions, and releasing their products. Our general approach provides enzymatic rates and copy numbers, substrate-search times, diffusion characteristics, and the spatial distribution of reaction sites, at the single-cell level, all in one measurement. Single repair events last 2.1 s (Pol) and 2.5 s (Lig), respectively. Pol and Lig activities increased fivefold over the basal level within minutes of DNA methylation damage; their rates were limited by upstream base excision repair pathway steps. Pol and Lig spent >80% of their time searching for free substrates, thereby minimizing both the number and lifetime of toxic repair intermediates. We integrated these single-molecule observations to generate a quantitative, systems-level description of a model repair pathway in vivo.
