生物谷報(bào)道：在最新一期的Nature Genetics（2007年5月27日）一篇文章中介紹，大腸桿菌（Escherichia coli）的DNA在復(fù)制過(guò)程中發(fā)生斷裂的頻率比之前推測(cè)的要低20－100倍,，而且還描述了一種觀察體內(nèi)鏈斷裂的新技術(shù),。薩塞克斯大學(xué)(University of Sussex)基因組損傷和穩(wěn)定性中心Aidan Doherty（未參與實(shí)驗(yàn)）說(shuō)：我們非常幸運(yùn)，研究人員幾乎能夠得到斷裂的實(shí)際數(shù)目,。
Susan Rosenberg實(shí)驗(yàn)室設(shè)計(jì)出一種新的分析檢測(cè)系統(tǒng),，對(duì)野生型E.coli細(xì)胞進(jìn)行遺傳改造，使細(xì)胞在出現(xiàn)DNA雙鏈斷裂（DSB）時(shí)發(fā)出綠光,。這種技術(shù)提供了原核細(xì)胞中實(shí)際DSB的直接數(shù)據(jù),。之前的研究推測(cè)，染色體每次復(fù)制都會(huì)出現(xiàn)DSB,。Rosenberg與其同事認(rèn)為是染色體每復(fù)制100次約出現(xiàn)一次DSB,。如此低的斷裂頻率提示單個(gè)斷裂在引發(fā)細(xì)胞損傷、突變和癌化時(shí)扮演的角色更重要,。但Rosenberg說(shuō)即便新推測(cè)的頻率很低,，DSB還是很常見(jiàn)。同源重組是E.coli偏愛(ài)的修復(fù)機(jī)制,，是DNA重新結(jié)合的最溫良途徑,，被認(rèn)為是無(wú)差錯(cuò)方式。100次復(fù)制出現(xiàn)一次DSB等同于重組頻率的10％,，這對(duì)于推測(cè)的無(wú)差錯(cuò)方式來(lái)說(shuō)是龐大的,。
為了直接觀測(cè)細(xì)菌細(xì)胞中DSB數(shù)量，Rosenberg與同為Baylor醫(yī)學(xué)院的同事Jeanine Pennington利用病毒,，將編碼綠色熒光蛋白的基因插入細(xì)菌基因組中,。DNA復(fù)制過(guò)程中發(fā)生斷裂時(shí),，細(xì)胞發(fā)動(dòng)一套機(jī)制迅速修復(fù)損傷位點(diǎn)。按照Rosenberg的設(shè)計(jì)方案,，這些機(jī)制被激活后引起細(xì)胞發(fā)熒光,。
利用流式細(xì)胞儀，研究人員通過(guò)計(jì)算綠色細(xì)胞個(gè)數(shù)而記錄DSB量,，還進(jìn)行了多次對(duì)照實(shí)驗(yàn),，消除其它會(huì)引起細(xì)胞變綠的可能因素。比如,，當(dāng)研究人員消除輔助激活修復(fù)機(jī)制的基因后,，沒(méi)有細(xì)胞變綠，進(jìn)一步證實(shí)熒光是由DNA修復(fù)機(jī)制單獨(dú)激發(fā)的,。Doherty說(shuō)如果沒(méi)有這些對(duì)照,，你會(huì)對(duì)驗(yàn)證實(shí)驗(yàn)的可靠性不知所措。
之前估計(jì)DSB數(shù)量利用粗糙,、間接工具,，通過(guò)計(jì)算培養(yǎng)基中死細(xì)胞數(shù)或者分解細(xì)胞然后跑電泳而記錄斷裂率。西南大學(xué)醫(yī)學(xué)院Errol Friedberg（未參與實(shí)驗(yàn)）說(shuō)：“這是一項(xiàng)有趣的研究,，主要來(lái)自于技術(shù)觀點(diǎn),。他們?cè)O(shè)計(jì)了一種溫和的化驗(yàn)，即便只是在E.coli中進(jìn)行的,，但對(duì)真核系統(tǒng)應(yīng)該有‘推斷能力’,。”
研究人員下一步打算在高等細(xì)胞中驗(yàn)證這種方法。真核細(xì)胞中控制損傷修復(fù)的基因數(shù)量更多,，但插入綠色熒光蛋白的基本方法應(yīng)該有效,。Doherty認(rèn)為，DNA發(fā)生斷裂的頻率比想象的要低,，真核細(xì)胞可能消耗巨額能量維持DNA的完整性,。真核細(xì)胞很有可能進(jìn)化出更為復(fù)雜的組蛋白機(jī)制。組蛋白需要緊密陪伴DNA,，防止DNA斷裂,。
原始出處：
Letter abstract
Nature Genetics 39, 797 - 802 (2007)
Published online: 27 May 2007 | doi:10.1038/ng2051
Spontaneous DNA breakage in single living Escherichia coli cells
Jeanine M Pennington1 & Susan M Rosenberg1,2
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Spontaneous DNA breakage is predicted to be a frequent, inevitable consequence of DNA replication and is thought to underlie much of the genomic change that fuels cancer and evolution1, 2, 3. Despite its importance, there has been little direct measurement of the amounts, types, sources and fates of spontaneous DNA lesions in living cells. We present a direct, sensitive flow cytometric assay in single living Escherichia coli cells for DNA lesions capable of inducing the SOS DNA damage response, and we report its use in quantification of spontaneous DNA double-strand breaks (DSBs). We report efficient detection of single chromosomal DSBs and rates of spontaneous breakage 20- to 100-fold lower than predicted. In addition, we implicate DNA replication in the origin of spontaneous DSBs with the finding of fewer spontaneous DSBs in a mutant with altered DNA polymerase III. The data imply that spontaneous DSBs induce genomic changes and instability 20–100 times more potently than previously appreciated. Finally, FACS demonstrated two main cell fates after spontaneous DNA damage: viability with or without resumption of proliferation.
Interdepartmental Program in Cell and Molecular Biology and Department of Molecular and Human Genetics, Houston, Texas 77030-3411, USA.
Department of Biochemistry and Molecular Biology, Department of Molecular Virology and Microbiology and Dan L. Duncan Cancer Center, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030-3411, USA.
Correspondence to: Susan M Rosenberg1,2 e-mail: [email protected]
