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New DNA replication mechanism

Curator: Larry H. Bernstein, MD, FCAP

 

 

Structural Study Points to New DNA Replication Mechanism

 

GEN  http://www.genengnews.com/gen-news-highlights/structural-study-points-to-new-dna-replication-mechanism/81252345/

https://youtu.be/EbXlPUfajCk

This movie shows the helicase protein complex from all angles, and reveals how its shape changes back and forth between two forms. The research team hypothesizes that the rocking action of this conformational change could help split the DNA double helix and move the helicase along one strand so it can be copied by DNA polymerase.

http://www.genengnews.com/Media/images/GENHighlight/108310_web1851822472.jpg

These are two images showing the structure of the helicase protein complex from above. (a) A surface-rendered three-dimensional electron density map as obtained by cryo-EM. (b) A computer-generated ‘ribbon diagram’ of the atomic model built based on the density map. The helicase has three major components: the Mcm2-7 hexamer ring in green, which encircles the DNA strand; the Cdc45 protein in magenta; and the GINS 4-protein complex in marine blue. Cdc45 and GINS recruit and tether other replisome components to the helicase, including the DNA polymerases that copy each strand of the DNA. [Brookhaven National Laboratory]

 

A collaborative team of researchers from the U.S. Department of Energy’s Brookhaven National Laboratory, Stony Brook University, Rockefeller University, and the University of Texas have just released detailed structural images of DNA helicase from yeast and are proposing a novel mechanism for how the molecular machinery functions. The scientists believe their new data could provide valuable insight into ways that DNA replication can go askew.

“DNA replication is a major source of errors that can lead to cancer,” explained senior study author Huilin Li, Ph.D., a professor with a joint appointment at Brookhaven Lab and Stony Brook University. “The entire genome, all 46 chromosomes, gets replicated every few hours in dividing human cells, so studying the details of how this process works may help us understand how errors occur.”

The findings from this study were published recently in Nature Structural & Molecular Biology through an article entitled “Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation.”

This current study builds upon previous work that produced the first-ever images of the complete DNA-copying protein complex, called the replisome. That study provided a surprising revelation about the location of the DNA-copying enzymes, DNA polymerases. This new study focuses on the atomic-level details for the helicase portion of the protein complex—the part that encircles and splits the DNA double helix so the polymerases can synthesize two new daughter strands.

As they had done in their previous work, Dr. Li and his colleagues produced high-resolution images of the helicase using cryo-electron microscopy (cryo-EM). This technique holds an advantage over other EM methods in that proteins can be studied in solution, closely replicating intracellular conditions.

“You don’t have to produce crystals that would lock the proteins in one position,” Dr. Li remarked. It’s an important point because helicase is a molecular machine made of 11 associated proteins that must be flexible to work. “You have to be able to see how the molecule moves to understand its function,” Dr. Li said.

Once the images of the replication machinery were assembled, the investigators were able to map out the locations of the individual amino acids that make up the helicase complex in each conformation. Then, combining those maps with existing biochemical knowledge, they came up with a mechanism for how the helicase works.

“One part binds and releases energy from a molecule called ATP. It converts the chemical energy into a mechanical force that changes the shape of the helicase,” Dr. Li stated. The molecule subsequently ejects the drained ATP and the helicase complex reverts to its original shape so a new ATP molecule can come in and begin the process again.

“It looks and operates similar to an old-style pumpjack oil rig, with one part of the protein complex forming a stable platform, and another part rocking back and forth,” Dr. Li noted. The researchers postulate that the rocking motion would nudge the DNA strands apart and move the helicase along the double helix in a linear fashion.

This direct translocation mechanism appears to be quite different from the way helicases are thought to operate in more primitive organisms such as bacteria, where the entire complex is believed to rotate around the DNA. However, there is biochemical evidence to support the idea of linear motion, including the fact that the helicase can still function even when the ATP hydrolysis activity of some, but not all, of the components is knocked out by mutation.

“We acknowledge that this proposal may be controversial, and it is not really proven at this point, but the structure gives an indication of how this protein complex works, and we are trying to make sense of it,” Dr. Li stated.

 

Decades Old DNA Replication Models Called into Question

GEN  http://www.genengnews.com/gen-news-highlights/decades-old-dna-replication-models-called-into-question/81251929/?kwrd=Replisome

http://www.genengnews.com/media/images/GENHighlight/102252_web8123122217.jpg

A series of electron micrographs show the barrel-shaped helicase, which is the enzyme that separates the two DNA strands, along with other components of the replisome, including polymerase-epsilon (green).[Brookhaven National Laboratory]

 

Previously (left), the replisome’s two polymerases (green) were assumed to be below the helicase (tan), the enzyme that splits the DNA strands. The new images reveal one polymerase is located at the front of the helicase, causing one strand to loop backward as it is copied (right). [Brookhaven National Laboratory]

 

It may be time to update biology texts to reflect newly published data from a collaborative team of scientists at Rockefeller University, Stony Brook University, and the U.S. Department of Energy’s Brookhaven National Laboratory. Using cutting-edge electron microscopy (EM) techniques, the investigators gathered the first ever images of the fully assembled replisome, providing new insight into the molecular mechanisms of replication.

“Our finding goes against decades of textbook drawings of what people thought the replisome should look like,” remarked co-senior author Michael O’Donnell, Ph.D., professor and head of Rockefeller’s Laboratory of DNA Replication. “However, it’s a recurring theme in science that nature does not always turn out to work the way you thought it did.”

The researcher’s findings focused on the replisome found in eukaryotic organisms, a category that includes a broad swath of living things, including humans and other multicellular organisms. Over the past several decades, there has been an array of data describing the individual components comprising the complex nature of replisome. Yet, until now no pictures existed to show just how everything fit together.

“This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell,” explained co-senior author Huilin Li, Ph.D., biologist with joint appointments at Brookhaven Lab and Stony Brook University. “These new images show the fully assembled and fully activated ‘helicase’ protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two ‘polymerase’ enzymes that duplicate each strand to copy the genome.”

The image and implications from this study were described in a paper entitled “The architecture of a eukaryotic replisome,” published recently through Nature Structural & Molecular Biology.

Traditional models of DNA replication show the helicase enzyme moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands.

However, the images that the researchers collected of intact replisomes revealed that only one of the polymerases is located at the back of the helicase. The other is on the front side of the helicase, where the helicase first encounters the double-stranded helix. This means that while one of the two split DNA strands is acted on by the polymerase at the back end, the other has to thread itself back through or around the helicase to reach the front-side polymerase before having its new complementary strand assembled.

“DNA replication is one of the most fundamental processes of life, so it is every biochemist’s dream to see what a replisome looks like,” stated lead author Jingchuan Sun, EM biologist in Dr. Li’s laboratory. “Our lab has expertise and a decade of experience using electron microscopy to study DNA replication, which has prepared us well to tackle the highly mobile therefore very challenging replisome structure. Working together with the O’Donnell lab, which has done beautiful, functional studies on the yeast replisome, our two groups brought perfectly complementary expertise to this project.”

The positioning of one polymerase at the front of the helicase suggests that it may have an unforeseen function—the possibilities of which the collaborative group of scientists is continuing to study. Whatever the function the offset polymerase ends up having, Drs. Li and O’Donnell hope that it will not only provide them better insight into the replication machinery but that they may uncover useful information that can be exploited for disease intervention.

“Clearly, further studies will be required to understand the functional implications of the unexpected replisome architecture reported here,” the scientists concluded.

 

The architecture of a eukaryotic replisome

Jingchuan Sun, Yi Shi, Roxana E Georgescu, Zuanning Yuan, Brian T Chait, Huilin Li & Michael E O’Donnell

Nature Structural & Molecular Biology 2015; 22:976–982     http://dx.doi.org:/10.1038/nsmb.3113

At the eukaryotic DNA replication fork, it is widely believed that the Cdc45–Mcm2–7–GINS (CMG) helicase is positioned in front to unwind DNA and that DNA polymerases trail behind the helicase. Here we used single-particle EM to directly image a Saccharomyces cerevisiae replisome. Contrary to expectations, the leading strand Pol ε is positioned ahead of CMG helicase, whereas Ctf4 and the lagging-strand polymerase (Pol) α–primase are behind the helicase. This unexpected architecture indicates that the leading-strand DNA travels a long distance before reaching Pol ε, first threading through the Mcm2–7 ring and then making a U-turn at the bottom and reaching Pol εat the top of CMG. Our work reveals an unexpected configuration of the eukaryotic replisome, suggests possible reasons for this architecture and provides a basis for further structural and biochemical replisome studies.

 

Figure 4: Subunit proximities within CMGE determined by chemical cross-linking with mass spectrometry readout (CX-MS).close

 

Structure of the eukaryotic replicative CMG helicase suggests a pumpjack motion for translocation

Zuanning Yuan, Lin Bai, Jingchuan Sun, Roxana Georgescu, Jun Liu, Michael E O’Donnell & Huilin Li

Nature Structural & Molecular Biology 8 Feb 2016    http://dx.doi.org:/10.1038/nsmb.3170

The CMG helicase is composed of Cdc45, Mcm2–7 and GINS. Here we report the structure of theSaccharomyces cerevisiae CMG, determined by cryo-EM at a resolution of 3.7–4.8 Å. The structure reveals that GINS and Cdc45 scaffold the N tier of the helicase while enabling motion of the AAA+ C tier. CMG exists in two alternating conformations, compact and extended, thus suggesting that the helicase moves like an inchworm. The N-terminal regions of Mcm2–7, braced by Cdc45–GINS, form a rigid platform upon which the AAA+ C domains make longitudinal motions, nodding up and down like an oil-rig pumpjack attached to a stable platform. The Mcm ring is remodeled in CMG relative to the inactive Mcm2–7 double hexamer. The Mcm5 winged-helix domain is inserted into the central channel, thus blocking entry of double-stranded DNA and supporting a steric-exclusion DNA-unwinding model.

 

Figure 1: Cryo-EM and overall structure of theS. cerevisiae CMG complex.

http://www.nature.com/nsmb/journal/vaop/ncurrent/carousel/nsmb.3170-F1.jpg

 

Figure 2: Structure and interactions of yeast GINS and Cdc45.

 

Figure 3: Side-by-side comparison of conformer I and conformer II in the Mcm2–7 region of CMG helicase.

 

Figure 6: Pol2 footprint on the atomic model of CMG helicase.