April 2, 2013
Clamping Down On The Mystery Of Human DNA Replication
redOrbit Staff & Wire Reports - Your Universe Online
Scientists at Penn State University have discovered how a vital step in the human DNA replication process — the loading of molecular structures known as sliding clamps onto DNA molecules — is performed.
The researchers say their work, the results of which were published in Tuesday´s edition of the journal eLife, will help uncover some of the mystery surrounding this crucial part of the chemical replication process. This step had not previously been closely analyzed in human DNA replication, which is the basis for biological inheritance in all types of living organisms.
According to Mark Hedglin, a post-doctoral researcher in Penn State's Department of Chemistry and one of the investigators behind the discovery, the sliding clamp is a ring-shaped protein which essentially encircles a DNA strand, latching around it much like a watch band. The sliding clamp then anchors polymerases — enzymes involved in the synthesis of nucleic acids — to ensure more efficient copying of the genetic 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 to stay in place, allowing it to copy thousands of bases before being removed from the strand of DNA,” Hedglin explained in a statement.
However, because of the closed circular structure of those sliding clamps, another step in DNA replication — the presence of a so-called “clamp loader” to latch and unlatch them at different stages of the process — is required. Previously, researchers did not know exactly how the sliding clamp and the clamp loader interacted with one another, nor did they know exactly when the clamp was attached to or unattached from the DNA.
“We know that polymerases and clamp loaders can't bind the sliding clamp at the same time, so the hypothesis was that clamp loaders latched 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,” Hedglin said.
In order to test their theory, they turned to a method known as FÃ¶rster resonance energy transfer (FRET), which attaches fluorescent “tags” to human proteins and parts of DNA in order to monitor the interactions between them.
With those tags in place, Hedglin said he and his colleagues “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 optimize its activity.”
They discovered that whenever a sliding clamp was loaded onto a DNA template without the presence of a polymerase, the clamp loader almost immediately removed it so free clamps did not build up on the genetic molecule. When a polymerase was there, however, it captured both the sliding clamp and the clamp loader, and then “dissociated” from the DNA strand, the Penn State researcher noted.
Furthermore, he and his associates discovered when both the clamp and the clamp loader were attached to the DNA, they were “not intimately engaged” with one-another. Instead, the clamp loader released the closed sliding clamp onto the genetic molecule, giving the polymerase a chance to capture a clamp and finish assembling the holoenzyme. The clamp loader then dissociated from the DNA molecule.
The senior author of the study was Stephen J. Benkovic, an Evan Pugh Professor of Chemistry and Holder of the Eberly Family Chair in Chemistry at Penn State. In addition to Benkovic and Hedglin, Penn State researchers Senthil K Perumal and Zhenxin Hu contributed to the National Institutes of Health (NIH)-funded study.
Image 2 (below): Stephen J. Benkovic, Mark Hedglin, and other members of Professor Benkovic's research team have studied the importance of "clamp loader" enzymes and their activities during DNA replication. In this image, the clamp loader is represented, for illustrative purposes, by a hand, which is loading the sliding clamp ring onto DNA. Credit: Benkovic lab, Penn State University