Clock that measures aging found hidden in our DNA

Subodh Varma, TNN | Oct 21, 2013, 11.57 AM IST

Theoretically, it is possible to reverse aging if one understands how it is taking place.

NEW DELHI: In a step towards unraveling the mystery of aging, a US scientist has discovered a human body clock tucked away in body's DNA. It measures the age of cells, tissues and organs.

Steve Hovarth of the University of California, Los Angeles found that different parts of the body were aging at different speeds - some faster, others slower. Significantly, diseased organs showed ages many years in ahead of the rest of the normal body. Hovarth's study is reported in the scientific journal Genome Biology on Monday.

"The big question is whether the biological clock controls a process that leads to aging," Horvath said. "If so, the clock will become an important biomarker for studying new therapeutic approaches to keeping us young."

Theoretically, it is possible to reverse aging if one understands how it is taking place. Horvath's work is a clear identification of a biochemical process linked to aging. By understanding how Hovarth's clock works, it may be possible to get the key to aging, and perhaps develop ways of stopping or slowing it down.

While earlier clocks have been linked to saliva, hormones and telomeres, the new research is the first to identify an internal timepiece able to accurately gauge the age of diverse human organs, tissues and cell types. Unexpectedly, the clock also found that some parts of the anatomy, like a woman's breast tissue, age faster than the rest of the body.

To create the clock, Horvath focused on methylation, a naturally occurring process that chemically alters DNA. Horvath sifted through 121 sets of data collected previously by researchers who had studied methylation in both healthy and cancerous human tissue. Analysing 8,000 samples of 51 types of tissue and cells taken from throughout the body, Horvath charted how age affects DNA methylation levels from pre-birth through 101 years. To create the clock, he zeroed in on 353 markers that change with age and are present throughout the body. He tested the clock's effectiveness by comparing a tissue's biological age to its chronological age. Repeated tests showed that the clock was accurate.

"It's surprising that one could develop a clock that reliably keeps time across the human anatomy," he admitted. "My approach really compared apples and oranges, or in this case, very different parts of the body: the brain, heart, lungs, liver, kidney and cartilage."

While most samples' biological ages matched their chronological ages, others diverged significantly. For example, Horvath discovered that a woman's breast tissue ages faster than the rest of her body.

"Healthy breast tissue is about two to three years older than the rest of a woman's body," said Horvath. "If a woman has breast cancer, the healthy tissue next to the tumor is an average of 12 years older than the rest of her body."

The results may explain why breast cancer is the most common cancer in women. Given that the clock ranked tumor tissue an average of 36 years older than healthy tissue, it could also explain why age is a major risk factor for many cancers in both genders.

Horvath next looked at pluripotent stem cells, adult cells that have been reprogrammed to an embryonic stem cell-like state, enabling them to form any type of cell in the body and continue dividing indefinitely.

"My research shows that all stem cells are newborns," he said. "More importantly, the process of transforming a person's cells into pluripotent stem cells resets the cells' clock to zero."

Horvath also found that the clock's rate speeds up or slows down depending on a person's age. At birth, the clock is faster and continues like that till adolescence. But after about 20 years age it slows down to a constant rate for the rest of the life.

Posted by:

 Rajni Jaiswal

Faculty of Bioinformatics

BII,Noida

The Discovery of the Molecular Structure of DNA -The Double Helix

A Scientific Breakthrough

The sentence "This structure has novel features which are of considerable biological interest" may be one of science's most famous understatements. It appeared in April 1953 in the scientific paper where James Watson and Francis Crick presented the structure of the DNA-helix, the molecule that carries genetic information from one generation to the other.

Nine years later, in 1962, they shared the Nobel Prize in Physiology or Medicine with Maurice Wilkins, for solving one of the most important of all biological riddles. Half a century later, important new implications of this contribution to science are still coming to light.

What is DNA?

The work of many scientists paved the way for the exploration of DNA. Way back in 1868, almost a century before the Nobel Prize was awarded to Watson, Crick and Wilkins, a young Swiss physician named Friedrich Miescher, isolated something no one had ever seen before from the nuclei of cells. He called the compound "nuclein." This is today called nucleic acid, the "NA" in DNA (deoxyribo-nucleic-acid) and RNA (ribo-nucleic-acid).

Two years earlier, the Czech monk Gregor Mendel, had finished a series of experiments with peas. His observations turned out to be closely connected to the finding of nuclein. Mendel was able to show that certain traits in the peas, such as their shape or color, were inherited in different packages. These packages are what we now call genes.

For a long time the connection between nucleic acid and genes was not known. But in 1944 the American scientist Oswald Avery managed to transfer the ability to cause disease from one strain of bacteria to another. But not only that: the previously harmless bacteria could also pass the trait along to the next generation. What Avery had moved was nucleic acid. This proved that genes were made up of nucleic acid. 15

Solving the Puzzle

In the late 1940's, the members of the scientific community were aware that DNA was most likely the molecule of life, even though many were skeptical since it was so "simple." They also knew that DNA included different amounts of the four bases adenine, thymine, guanine and cytosine (usually abbreviated A, T, G and C), but nobody had the slightest idea of what the molecule might look like.

In order to solve the elusive structure of DNA, a couple of distinct pieces of information needed to be put together. One was that the phosphate backbone was on the outside with bases on the inside; another that the molecule was a double helix. It was also important to figure out that the two strands run in opposite directions and that the molecule had a specific base pairing.

As in the solving of other complex problems, the work of many people was needed to establish the full picture.

Using X-rays to See Through DNA

Watson and Crick used stick-and-ball models to test their ideas on the possible structure of DNA. Other scientists used experimental methods instead. Among them were Rosalind Franklin and Maurice Wilkins, who were using X-ray diffraction to understand the physical structure of the DNA molecule.

When you shine X-rays on any kind of crystal – and some biological molecules, such as DNA, can form crystals if treated in certain ways – the invisible rays bounce off the sample. The rays then create complex patterns on photographic film. By looking at the patterns, it is possible to figure out important clues about the structures that make up the crystal.

A Three-Helical Structure?

The scientist Linus Pauling was eager to solve the mystery of the shape of DNA. In 1954 he became a Nobel Laureate in Chemistry for his ground-breaking work on chemical bonds and the structure of molecules and crystals. In early 1953 he had published a paper where he proposed a triple-16

helical structure for DNA. Watson and Crick had also previously worked out a three-helical model, in 1951. But their theory was wrong.

Their mistake was partly based on Watson having misremembered a talk by Rosalind Franklin where she reported that she had established the water content of DNA by using X-ray crystallographic methods. But Watson did not take notes, and remembered the numbers incorrectly.

Instead, it was Franklin's famous "photograph 51" that finally revealed the helical structure of DNA to Watson and Crick in 1953. This picture of DNA that had been crystallized under moist conditions shows a fuzzy X in the middle of the molecule, a pattern indicating a helical structure.

Specific Base-Pairing

The base-pairing mystery had been partly solved by the biochemist Erwin Chargoff some years earlier. In 1949 he showed that even though different organisms have different amounts of DNA, the amount of adenine always equals the amount of thymine. The same goes for the pair guanine and cytosine. For example, human DNA contains about 30 percent each of adenine and thymine, and 20 percent each of guanine and cytosine.

With this information at hand Watson was able to figure out the pairing rules. On the 21st of February 1953 he had the key insight, when he saw that the adenine-thymine bond was exactly as long as the cytosine-guanine bond. If the bases were paired in this way, each rung of the twisted ladder in the helix would be of equal length, and the sugar-phosphate backbone would be smooth.

Structure Shows Action

"It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material" wrote Watson and Crick in the scientific paper that was published in Nature, April 25, 1953.

This was indeed a breakthrough in the study of how genetic material passes from generation to generation. Once the model was established, its mere structure hinted that DNA was indeed the carrier of the genetic code and thus the key molecule of heredity, developmental biology and evolution.

The specific base pairing underlies the perfect copying of the molecule, which is essential for heredity. During cell division, the DNA molecule is able to "unzip" into two pieces. One new molecule is formed from each half-17

ladder, and due to the specific pairing this gives rise to two identical daughter copies from each parent molecule.

We All Share the Same Building Blocks

DNA is a winning formula for packaging genetic material. Therefore almost all organisms – bacteria, plants, yeast and animals – carry genetic information encapsulated as DNA. One exception is some viruses that use RNA instead.

Different species need different amounts of DNA. Therefore the copying of the DNA that precedes cell division differs between organisms. For example, the DNA in E. coli bacteria is made up of 4 million base pairs and the whole genome is thus one millimeter long. The single-cell bacterium can copy its genome and divide into two cells once every 20 minutes.

The DNA of humans, on the other hand, is composed of approximately 3 billion base pairs, making up a total of almost a meter-long stretch of DNA in every cell in our bodies.

In order to fit, the DNA must be packaged in a very compact form. In E. coli the single circular DNA molecule is curled up in a condensed fashion, whereas the human DNA is packaged in 23 distinct chromosome pairs. Here the genetic material is tightly rolled up on structures called histones.

A New Biological Era

This knowledge of how genetic material is stored and copied has given rise to a new way of looking at and manipulating biological processes, called molecular biology. With the help of so-called restriction enzymes, molecules that cut the DNA at particular stretches, pieces of DNA can be cut out or inserted at different places.

In basic science, where you want to understand the role of all the different genes in humans and animals, new techniques have been developed. For one thing, it is now possible to make mice that are genetically modified and lack particular genes. By studying these animals scientists try to figure out what that gene may be used for in normal mice. This is called the knockout technique, since stretches of DNA have been taken away, or knocked out.

Scientists have also been able to insert new bits of DNA into cells that lack particular pieces of genes or whole genes. With this new DNA, the cell 18

becomes capable of producing gene products it could not make before. The hope is that, in the future, diseases that arise due to the lack of a particular protein could be treated by this kind of gene therapy.

Was Franklin Nominated?

Many voices have argued that the Nobel Prize should also have been awarded to Rosalind Franklin, since her experimental data provided a very important piece of evidence leading to the solving of the DNA structure. In a recent interview in the magazine Scientific American, Watson himself suggested that it might have been a good idea to give Wilkins and Franklin the Nobel Prize in Chemistry, and him and Crick the Nobel Prize in Physiology or Medicine – in that way all four would have been honored.

Rosalind Franklin died in 1958. As a rule only living persons can be nominated for the Nobel Prize, so the 1962 Nobel Prize was out of the question. The Nobel archives, at the Nobel Prize-awarding institutions, that among other things contain the nominations connected to the prizes, are held closed. But 50 years after a particular prize had been awarded, the archives concerning the nominees are released. Therefore, in 2008 it was possible to see whether Rosalind Franklin ever was a nominee for the Nobel Prize concerning the DNA helix. The answer is that no one ever nominated her - neither for the Nobel Prize in Physiology or Medicine nor in Chemistry.

The DNA-Helix

The two strands of the double helix are anti-parallel, which means that they run in opposite directions.

The sugar-phosphate backbone is on the outside of the helix, and the bases are on the inside. The backbone can be thought of as the sides of a ladder, whereas the bases in the middle form the rungs of the ladder.

Each rung is composed of two base pairs. Either an adenine-thymine pair that form a two-hydrogen bond together, or a cytosine-guanine pair that form a three-hydrogen bond. The base pairing is thus restricted.

This restriction is essential when the DNA is being copied: the DNA-helix is first "unzipped" in two long stretches of sugar-phosphate backbone with a line of free bases sticking up from it, like the teeth of a comb. Each half will then be the template for a new, complementary strand. Biological machines 19

inside the cell put the corresponding free bases onto the split molecule and also "proof-read" the result to find and correct any mistakes. After the doubling, this gives rise to two exact copies of the original DNA molecule.

The coding regions in the DNA strand, the genes, make up only a fraction of the total amount of DNA. The stretches that flank the coding regions are called introns, and consist of non-coding DNA. Introns were looked upon as junk in the early days. Today, biologists and geneticists believe that this non-coding DNA may be essential in order to expose the coding regions and to regulate how the genes are expressed.

By Lotta Fredholm, Science Journalist

First published 30 September 2003

These changes are associated with the enzyme's fidelity-enhancing mechanisms and translocation.

We have also determined the structures of another B-family DNA polymerase, φ29 DNA polymerase, which initiates replication by attaching the first nucleotide of the phage genome to a serine side chain of protein primer called terminal protein, as well as its binary and ternary substrate complexes. The basis of DNA strand displacement activity exhibited by this enzyme is explained by the template strand passing through a tunnel that is too small to accommodate the nontemplate strand that is displaced. The extreme processivity of this polymerase is explained by its topological encirclement of the substrate and product DNA at the active site. The structure of the φ29 DNA polymerase bound to terminal protein provides the first structural insights into the mechanism of protein-primed DNA replication, suggesting that a four-helix domain containing the priming serine must back out of the duplex DNA product-binding site as DNA synthesis proceeds.

Toward our goal of understanding eubacterial replication, we determined the structure of the T. aquaticus DNA polymerase III, which we discovered exhibits no similarity to that of the archaeal or eukaryotic replicating polymerases, but rather possesses a catalytic domain that is homologous to that of repair DNA polymerase β. Furthermore, our structure of a ternary complex of Pol III with substrates shows that the DNA and nucleoside triphosphate (NTP) substrates bind identically in these two polymerases. The possibility that the last common ancestor had a ribozyme-replicating polymerase is thus raised. Our structures of the hexomeric DnaB helicase and its complex with the helicase-binding domain of primase are beginning to illuminate the structural bases of primosome function.

HHMI Scientist Abstract: Thomas A. Steitz, Ph.D.

Structural Basis of Replication and Gene Expression

Summary: Thomas Steitz uses the methods of x-ray crystallography and molecular biology to establish the structures and mechanisms of the proteins and nucleic acids involved in gene expression, replication, and recombination.

Our general long-term goal is to determine the detailed molecular mechanisms by which the proteins and nucleic acids involved in the central dogma of molecular biology (DNA replication, transcription, and translation) achieve their biological functions. Virtually all aspects of the maintenance and expression of information stored in the genome involve interactions between proteins and nucleic acids. Over the past three decades we have obtained detailed structural insights into the mechanisms by which specific proteins and nucleic acids catalyze and control the fundamental processes of DNA replication, mRNA synthesis, and protein synthesis, as well as DNA recombination.

DNA Replication

To establish the structural basis of DNA replication, we have been studying DNA polymerases and associated proteins involved in replication. Following on our earlier structures of Escherichia coli DNA polymerase I Klenow fragment and Thermus aquaticus DNA polymerase and their DNA substrate complexes, we established the crystal structure of a replicative DNA polymerase (from phage RB69) that is homologous to the eukaryotic B-family polymerases. The structures of the RB69 polymerase complexed with duplex DNA substrate, bound both at the editing site and the polymerase site, and of the sliding clamp complexed with a polymerase carboxyl-terminal peptide allowed construction of a replisome core structure. These structures showed that this macromolecular machine, charged with the responsibility of faithfully copying the DNA genome, undergoes large conformational changes throughout its catalytic cycle. 11

Site-Specific Recombination

Transposable elements encode recombination proteins that catalyze recombination of DNA at specific sequences. Our structure of a γδ resolvase synaptic tetramer bound to two DNA duplexes captured in an intermediate state of the recombination process shows a cleaved DNA substrate covalently linked to the protein, with the ends to be recombined separated by 50 Å. The very flat interface between the protein dimers linked to the DNAs to be recombined suggests that recombination is achieved by an unprecedented 180° rotation of one dimer relative to the other. Our recent structure of a synaptic tetramer of the homologous Gin recombinase exhibits a state in which one dimer is in a rotated position relative to the other dimer when compared with their orientation in γδ resolvase, consistent with the rotation hypothesis. 12

Transcription Genes encoded in DNA are transcribed into mRNA by DNA-dependent RNA polymerases that can initiate RNA synthesis at a specific promoter sequence. To understand this process and its regulation and to explain how RNA polymerases differ from DNA polymerases, we have determined the crystal structures of several T7 RNA polymerase complexes with promoter DNAs, mRNAs, and incoming NTP. These structures show how portions of the RNA polymerase recognize the bases in the duplex DNA promoter and denature part of the promoter to form a transcription initiation bubble. In an initiation complex, three nucleotides of transcript are seen base-paired to the template strand. We have also captured this polymerase in a transcription elongation phase as a complex with 30 base pairs of DNA and a 17-nucleotide RNA transcript. The transition from the initiation to the elongation phases of transcription is accompanied by a massive structural rearrangement of the amino-terminal domain, which eliminates the promoter DNA-binding site on the enzyme and creates a tunnel through which the transcript exits the enzyme, thus explaining the high processivity of the elongation phase. Our recent structures of initiation complexes with either a 7- or 8-nucleotide transcript show intermediates in this structural transition in which the promoter binding domain rotates by 45° to accommodate the growing transcript.

The structures of T7 RNA polymerase elongation complexes captured at each step of nucleotide incorporation show a 22° rotation of a five-helix subdomain upon NTP binding and upon pyrophosphate release. The conformational change that accompanies pyrophosphate release produces both the translocation of the product heteroduplex and the strand separation of downstream duplex DNA.

Translation Our structural studies of the proteins and nucleic acids involved in translating the gene sequence carried in the messenger RNA into the protein products are providing insights into the translation of the genetic code. This includes our earlier structural studies of aminoacyl-tRNA synthetases, as well as more recent structural studies explaining how the CCA-adding enzyme is able to mature or repair the 3' CCA end of tRNA without using a nucleic acid template. We have established the structures of the CCA-adding enzyme captured in the steps of adding penultimate C and final A as well as the product tRNA.

We have been pursuing high-resolution structural studies of the machine that synthesizes proteins, the ribosome, and have determined the atomic structure of the 1.6-mDa ribosomal subunit that catalyzes the formation of the peptide bond. The structures of the large subunit with either substrate or product analogs bound to the active site of peptide synthesis show a 13

peptidyltransferase center that is made entirely of RNA. Ribosomal RNA positions the substrate α-amino group appropriately for attack of the peptidyl-tRNA, and it also interacts with the latter's A76 2'-OH group, which may function as a proton shuttle between the α-amino group and the A76 3'-OH. More recently, we have obtained the structure of the 70S ribosome complexed with fMet-tRNA in the P site and an essential protein factor EF-P that is seen to be interacting with the tRNA and a rearranged L1 stalk, suggesting that it may be stimulating the first step of protein synthesis by correctly positioning the fMet-tRNA in the P site.

We have also established the structures of nearly two dozen different antibiotics that target the large ribosomal subunit in complex with the large subunit as well as complexes with a T. thermophilus 70S ribosome, including two members of the tuberactinomycin family of antibiotics that are used to treat tuberculosis. These structures not only establish how these antibiotics stop peptide synthesis but also are providing the basis for structure-based design of new antibiotics (by Rib-X Pharmaceuticals, Inc.) that are effective against ribosomes containing antibiotic-resistance mutations.

Portions of this work were supported in part by grants from the National Institutes of Health and the Agouron Institute.

As of February 09, 2011