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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

Decades Old DNA Replication Models Called into Question

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 SunYi ShiRoxana E GeorgescuZuanning YuanBrian T ChaitHuilin 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

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

CMGE was cross-linked with a lysine-specific bifunctional cross-linker, then fragmented by proteolysis, and cross-linked peptides were identified by mass spectrometry. (a) Overview of cross-links observed within the region of Pol2 …

 

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

Zuanning YuanLin BaiJingchuan SunRoxana GeorgescuJun LiuMichael 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.

Cryo-EM and overall structure of the S. cerevisiae CMG complex.

(a) A typical motion-corrected raw image from ~8,000 images of frozen CMG particles recorded on a direct detector. (b) Six selected 2D averages representing the particles in different views. (c) 3D cryo-EM map of CMG, color-coded accord…

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

 

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

Structure and interactions of yeast GINS and Cdc45.

(a) The full-length GINS structure in top and side views. Domain A is shown in cartoon and domain B in surface. Top, schematic showing that all four subunits have a similar two-domain architecture, but domains A and B in Psf2 and Psf3 a…

http://www.nature.com/nsmb/journal/vaop/ncurrent/carousel/nsmb.3170-F2.jpg

 

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

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

(a,b) Comparison of the two conformations, shown in cartoon representation and viewed from the right side, from Cdc45 and GINS (which are both removed for clarity) with the CTD motor ring on top and the NTD ring at the bottom. The two b…

http://www.nature.com/nsmb/journal/vaop/ncurrent/carousel/nsmb.3170-F3.jpg

 

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

Pol2 footprint on the atomic model of CMG helicase.

(a) The two-domain architecture of Pol2, the catalytic subunit of the Pol ε complex. The N-terminal half contains the polymerase and exonuclease activities. The C-terminal half is homologous to a B-family polymerase but lacks enzymatic…

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DNA Replication

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Decades Old DNA Replication Models Called into Question

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

 

Decades Old DNA Replication Models Called into Question

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]

  • 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.”

    “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.”

http://www.genengnews.com/Media/images/GENHighlight/102254_web2322915422.jpg

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]

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.

 

RELATED CONTENT

 

Fifth Histone Found to Recruit Proteins for DNA Repair   

http://www.genengnews.com/gen-news-highlights/fifth-histone-found-to-recruit-proteins-for-dna-repair/81251895/

Scientists at the University of Copenhagen say they have located a previously unknown function for histones, which allows for an improved understanding of how cells protect and repair DNA damages. This new discovery may be of great importance to the treatment of diseases caused by cellular changes such as cancer and immune deficiency syndrome.

The study (“Histone H1 couples initiation and amplification of ubiquitin signaling after DNA damage”) is published in Nature.

“I believe that there’s a lot of work ahead. It’s like opening a door onto a previously undiscovered territory filled with lots of exciting knowledge. The histones are incredibly important to many of the cells’ processes as well as their overall wellbeing,” said Niels Mailand, Ph.D., from the Novo Nordisk Foundation Center for Protein Research at the Faculty of Health and Medical Science.

Histones enable the tight packaging of DNA strands within cells. The strands are two meters in length and the cells usually about 100,000 times smaller. Generally speaking, there are five types of histones. Four of them are core histones and they are placed like beads on the DNA strands, which are curled up like a ball of wool within the cells. The role of the histones is already well described in research, and in addition to enabling the packaging of the DNA strands they also play a central part in practically every process related to the DNA-code, including repairing possibly damaged DNA.

The four core histones have tails and, among other things, they signal damage to the DNA and thus attract the proteins that help repair the damage. Between the histone “yarn balls” we find the fifth histone, Histone H1, but up until now its function has not been thoroughly examined.

Using a mass spectrometer, Dr. Mailand and his team have discovered that, surprisingly, the H1 histone also helps summon repair proteins.

“In international research, the primary focus has been on the core histones and their functionality, whereas little attention has been paid to the H1 histone, simply because we weren’t aware that it too influenced the repair process. Having discovered this function in the H1 constitutes an important piece of the puzzle of how cells protect their DNA, and it opens a door onto hitherto unknown and highly interesting territory,” noted Dr. Mailand.

He expects the discovery to lead to increased research into Histone H1 worldwide, which will lead to increased knowledge of cells’ abilities to repair possible damage to their DNA and thus increase our knowledge of the basis for diseases caused by cellular changes. It will also generate more knowledge about the treatment of these diseases.

“By mapping the function of the H1 histone, we will also learn more about the repair of DNA damages on a molecular level. In order to provide the most efficient treatment, we need to know how the cells prevent and repair these damages,” point out Dr. Mailand.

 

Cover All the Bases for Oligonucleotide Analysis

Stephen Luke

Synthetic oligonucleotides have emerged as promising therapeutic agents for the treatment of a variety of diseases, including viral infections and cancer. Researchers are looking at several classes of nucleic acids, such as antisense oligonucleotides, small interfering RNAs (siRNAs), and aptamers, for therapeutic applications.

However, various impurities – product-related, in the starting materials, and arising from incomplete capping of coupling reactions – must be identified and removed and postsynthesis processing must be monitored. Thus, a key challenge in the development and manufacture of oligonucleotide therapeutics is to establish analytical methods that are capable of separating and identifying impurities.

Exploring Better Options for Oligonucleotide LC Separations

 

 

Table 1. Options for oligonucleotide LC separations

Ion-pair, reversed-phase separation of the trityl-on oligos and is relatively simple to perform. This method separates the full-length target oligo, which still has the dMT group attached, from the deprotected failure sequences. The analytical information obtained is limited, so this is generally considered a purification method.

An alternate method, ion-exchange separations of the trityl-off, deprotected oligos uses the negative charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high concentrations of salt are needed to achieve elution from the column, making the technique unsuitable for use with LC/MS.

Finally, ion-pair, reversed-phase separation of the trityl-off, deprotected oligos makes use of organic solvents and mobile phase additives such as TEAA (triethylammonium acetate) or TEA-HFIP (triethylamine and hexafluoroisopropanol) to ion-pair with the negatively charged phosphodiester backbone of the oligonucleotide. High-performance columns deliver excellent resolution. What’s more, methods with volatile mobile phase constituents such as TEA-HFIP are suitable for use with LC/MS, providing useful information to help characterize oligonucleotide structures and sequences.

In Table 1 we summarize some of the options for oligonucleotide analysis by liquid chromatography.

Designed for ion-pair, reversed-phase separation of the trityl-off, deprotected oligos using either TEAA or TEA-HFIP mobile phases –Agilent AdvanceBio Oligonucleotide columns meet these challenges.

more….   http://www.genengnews.com/gen-articles/cover-all-the-bases-for-oligonucleotide-analysis/5594/

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