直接观测DNA有助于更好地研究遗传物质的复制和修复情况。
文章第一作者、加州州立大学(University of California)博士后学者 Roberto Galletto说:“我们可以直接对复制和修复过程进行监控,获得新的发现。” 研究详细结果刊登在9月20日电子版《Nature》杂志上。
大肠杆菌(E. coli bacteria)的RecA酶,能够延着长链DNA延伸,与DNA形成细丝状结构(filament)。一条完整的DNA延着RecA排列,其它DNA可以与母链的缺口部分进行交换,以填补缺口。在人体中的Rad51蛋白发挥相似功能,有研究显示Rad51与乳腺癌有关。
论文高级作者、加州大学分子和细胞生物学教授、遗传和发育实验室主任Stephen Kowalczykowski说:“RecA和Rad51组装filament的过程决定了DNA修复效果,但目前对于组装的机制还知之甚少。”
Galletto将一小片段DNA附着在微小橡胶粒上,然后将小橡胶粒注入由激光“镊子”控制的流动小室(chamber),橡胶粒上的DNA能够“随波逐流”。Galletto用激光轻轻将这些像水草一样在液体中漂动的DNA推出小室,推到邻近的一个含有荧光标记RecA的通道里。短暂的停留后,再将DNA推回原先的小室。
对同一DNA重复上述浸占过程,结果每一DNA上附着了4-5个RecA。一旦这些RecA在DNA上成串排列,DNA/RecA形成的细丝会迅速向两端延伸。Kowalczykowski认为实验过程中使用的这种技术为将来研究RecA和Rad51提供了很好的参考。
实验中采用的方法是在Kowalczykowski 和分子细胞生物学家Ronald J. Baskin早期研究一种解链酶工作情况时使用的方法上发展起来的。当时的研究结果刊登于2001年Nature杂志上。
研究经费来源于Jeane B. Kempner颁发给Galletto的奖金和美国国立卫生研究院的资助。
英文原文:
Watching DNA Repair In Real Time
"We can monitor the process directly, and that gives us a different perspective," said Roberto Galletto, a postdoctoral scholar at UC Davis and first author on a paper published Sept. 20 on the Web site of the journal Nature.
In E. coli bacteria, molecules of an enzyme called RecA attach themselves along a DNA strand, stretching it out and forming a filament. A piece of complementary DNA lines up along side it, and pieces of DNA can be swapped in to repair gaps in the original strand. A similar protein, called Rad51, does the same job in humans.
"How RecA and Rad51 assemble into filaments determines the outcome of DNA repair, but very little is known about how assembly is controlled," said senior author Stephen Kowalczykowski, professor in the sections of Microbiology and of Molecular and Cellular Biology and director of the Center for Genetics and Development at UC Davis. Genes that control the human gene, Rad51, have been linked to increased risk of breast cancer.
Galletto attached a short piece of DNA to a tiny latex bead and placed it in a flow chamber, held by laser beam "tweezers." Fluid flowing past made the DNA stream out like a banner. Then he nudged it into an adjacent channel containing fluorescently-tagged RecA. After short intervals of time, he moved it back to the first chamber to observe the results.
By repeatedly dipping the same piece of DNA into the fluorescent channel, the researchers could see the RecA form clusters of four to five molecules on the DNA. Once those clusters had formed, the DNA/RecA filament rapidly grew in both directions. The measurements made in those experiments will be the baseline for future studies of both RecA and Rad51, Kowalczykowski said.
The new work adapts an approach developed by Kowalczykowski and Ronald J. Baskin, professor of molecular and cellular biology, to study single enzymes at work unwinding DNA strands. That research was first published in Nature in 2001.
In addition to Galletto, Kowalczykowski and Baskin, the research team included postdoctoral scholar Ichiro Amitani. The work was funded by the National Institutes of Health and a fellowship awarded to Galletto by the Jeane B. Kempner Foundation