人类DNA复制过程被首次记录

For the first time, an elusive¹ step in the process of human DNA² replication has been demystified by scientists at Penn State University. According to senior author Stephen J. Benkovic, an Evan Pugh Professor of Chemistry and Holder³ of the Eberly Family Chair in Chemistry at Penn State, the scientists "discovered how a key step in human DNA replication is performed." The results of the research will be published in the journal eLife on 2 April 2013.

 

Part of the DNA replication process -- in humans and in other life forms -- involves loading of molecular⁴ structures called sliding clamps onto DNA. This crucial step in DNA replication had remained somewhat mysterious and had not been well studied in human DNA replication. Mark Hedglin, a post-doctoral researcher in Penn State's Department of Chemistry and a member of Benkovic's team, explained that the sliding clamp is a ring-shaped protein that acts to encircle the DNA strand⁵, latching⁷（封锁） around it like a watch band. The sliding clamp then serves to anchor special enzymes⁸ called polymerases to the DNA, ensuring efficient copying of the genetic⁹ material. "Without a sliding clamp, polymerases can copy very few bases -- the molecular 'letters' that make up the code of DNA -- at a time. But the clamp helps the polymerase（聚合酶） to stay in place, allowing it to copy thousands of bases before being removed from the strand of DNA," Hedglin said.

 

Hedglin explained that, due to the closed circular structure of sliding clamps, another necessary step in DNA replication is the presence of a "clamp loader," which acts to latch⁶ and unlatch the sliding clamps at key stages during the process. "The big unknown has always been how the sliding clamp and the clamp loader interact and the timing¹⁰ of latching and unlatching of the clamp from the DNA," said Hedglin. "We know that polymerases and clamp loaders can't bind¹¹ the sliding clamp at the same time, so the hypothesis was that clamp loaders latched¹² sliding clamps onto DNA, then left for some time during DNA replication, returning only to unlatch the clamps after the polymerase left so they could be recycled for further use."

 

To test this hypothesis, the team of researchers used a method called Förster resonance¹³ energy transfer (FRET), a technique of attaching fluorescent¹⁴ "tags" to human proteins and sections of DNA in order to monitor the interactions between them. "With these tags in place, we then observed the formation of holoenzymes -- the active form of the polymerase involved in DNA replication, which consists of the polymerase itself along with any accessory factors that optimize¹⁵ its activity," Hedglin said. "We found that whenever a sliding clamp is loaded onto a DNA template in the absence of polymerase, the clamp loader quickly removed the clamp so that free clamps did not build up on the DNA. However, whenever a polymerase was present, it captured the sliding clamp and the clamp loader then dissociated from the DNA strand."

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