Telomeres

Telomeres, together with the enzyme Telomerase are mentioned a lot lately in association with both aging and with cancer. It seems that their gradual shortening during the lifetime of a cell eventually results in damage to DNA and thus the genes of which they are a part. This has led to a great deal of research into Telomeres and Telomerase, some of which will be outlined in this section of the site. First of all however, let us get some of the basic definitions out of the way.

Definition of a Telomere

A telomere is a region of highly repetitive DNA at the end of a linear chromosome that functions as a disposable buffer. Every time linear eukaryotic chromosomes are replicated during late S-phase the DNA polymerase complex is incapable of replicating all the way to the end of the chromosome; if it were not for telomeres, this would quickly result in the loss of useful genetic information, which is needed to sustain a cell’s activities. http://en.wikipedia.org/wiki/Telomere

Definition of Telomerase

Telomerase is the reverse transcriptase responsible for the extension of telomeric repeat sequences in most species studied. If telomerase activity is diminished or absent, telomeres will shorten. Shortened telomeres appear to lead to cell senescence. Eventually telomeric sequences can shorten to the point where they are not long enough to support the telomere-protein complex protecting the ends and the chromosomes become unstable. These shortened ends become 'sticky' and promote chromosome rearrangements. Some rearrangements may contribute to the development of cancers. http://www.genlink.wustl.edu/teldb/tel.html

Role, Function and Compesition of Telomeres

In the majority of Prokaryotes, the chromosomes are circular. This means that they do not have ends that are prone to damage or ‘premature replication termination.’ Note that a small number of bacteria, Borrelia and Streptomyces for example, do possess linear chromosomes (together with circular plasmids). These chromosomes are very different however, from those which are found in Eukaryotic cells (such as those in humans.

A telomere consists of repeating base sequences (in humans this is a repeating string of TTAGGG bases between 3 to 20 kilobases in length). In addition to the telomere itself, there is a 100-300 kilobase sequence associated with the telomere, which is located between the telomere and the rest of the chromosome.

As replication of DNA occurs during the lifetime of an organism, the telomeres gradually shorten. This shortening eventually results in damage to the chromosomes themselves and to the genes that they contain. The results of this are either a reduction in the cells ability to express its genes and a descent into cellular senescence (cellular dormancy) or in some cases the over or under-expression of specific genes. When this chromosomal damage leads to either an over or under-expression of certain genes, then cellular functionality can be compromised. In some cases the organisms survival can also be compromised as cellular replication can become unchecked. These cells thus become cancerous and can lead to the death of the organism.

Role, Function and Compesition of Telomeres

During DNA replication, The DNA unzips and a complimentary strand is formed against the unzipped sections. Telomeres shorten during this process due to the ‘lagging strand’ phenomenon.

Basically DNA replication does not begin at the end of the DNA, but in the centre. DNA Helicase unzips the DNA forming Replication bubbles. An RNA Primer or Primase then attaches to each DNA strand and replication begins in the 3-5 direction, thus forming a new strand in the 5 -3 direction.

DNA polymerases move and replicate the DNA in the 3 to the 5 direction (thus making a replica strand in the 5 to 3 direction). Note that 3 refers to the 3 OH group of the sugar and the 5 refers to the 5 phosphate group of the nucleotide.

The leading strand of DNA is the strand that is oriented in the 3-5 direction. The primer of complementary molecules that binds to the first few of the exposed bases ends with a 3 sugar group. The phosphate of a new nucleotide can be attached here by DNA polymerase. DNA polymerase then continues along the strand, synthesising a new strand as it goes. An animation of this process can be seen below, provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/replication_ani.html.

The lagging strand however, faces more problems when it comes to replication. Because DNA polymerase can only attach to the 3 sugar group and thus move in the 3 to the 5 direction, it needs therefore to move in small jumps (in the opposite direction to overall replication) to replicate the lagging strand. See below animation. This is again provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/lagging_ani.html

Note that the small segments of DNA and primers which are produced on the lagging strand are called Okazaki fragments. Another DNA polymerase enzyme is then recruited to remove the primers and to replace them with DNA. Finally DNA Ligase seals the gaps between the Okazaki fragments.

DNA Replication

The problem with Telomere shortening occurs because, in order to change the RNA primers into DNA, there must be another DNA segment in front of the primer. There is a DNA segment ahead of the primer at every section of the strand, except where the last primer attaches (the end of the telomere). This means that this final primer cannot be replaced with DNA. It does get removed by various enzymes however, but in the process, the telomere shortens.

In human blood cells, the range of lengths of the telomeres varies between 8000 base pairs at birth, to 1,500 base pairs in the elderly. During cellular division, an average of 30 to 200 base pairs are removed from the ends of the telomeres.

In normal cases, the cells of a human can divide between 50 to 70 times, with the telomeres shortening with each division, until the cells either commit suicide through a process known as Apoptosis, become Senescent (dormant) or transform to cancerous cells due to genetic damage.

The process of Cellular senescence due to Telomere Shortening

As a cells telomeres shorten during multiple cellular divisions, DNA damage occurs and the cell, recognising this damage shuts itself down. Below is a simplified diagram of how this occurs.

Telomere shortening and aging

DNA damage results in an activation of the p53 gene. p53 then activates p21, which blocks the actions of a number of CDK’s (Cycline Dependent Kinases). Note that CDK’s are involved in the regulation of the cell cycle, transcription and mRNA processing. The blocking of certain CDK’s prevents the phosphorylation of pRb. This lack of hyperphosphorylated pRb results in a failure of expression of several critical genes, which are involved in cellular division. Cellular division then stops.

Telomerase and Telomere extension

A study was conducted (as so many aging studies are) using the nematode Caenorhabditis elegans (Joeng KS, Song EJ, Lee KJ, Lee J (2004). "Long lifespan in worms with long telomeric DNA". Nature Genetics 36 (6): 607-11.). This study indicates that by lengthening the Telomere, longevity can be increased.

Two distinct groups of Nematodes were engineered. The only difference between the two groups, was the length of the Telomere. The group with the longer telomere’s lived approximately 20 percent longer than the group with the shorter telomeres. Also, it was observed that the Nematode’s with the longer telomeres possessed a greater resistance to the effects of heat exposure.

Telomerase is the natural enzyme which promotes telomere repair. It is however not active in most cells. It certainly is active though in stem cells, germ cells, hair follicles and (worryingly) in 90 percent of cancer cells. Telomerase functions by adding bases to the ends of the telomeres. As a result of this telomerase activity, these cells seem to possess a kind of immortality.

In 1990 a team at Geron Corp in Menlo Park, California led by Serge Lichtsteiner and Andrea Bodnar,in association with the University of texas Southwestern Medical center, managed to activate the production of telomerase in cells that do not usually produce this enzyme. The results were that the telomeres started to lengthen. By the time that they released the results of their study, their cells had divided 20 or more times than would normally be expected before senescence would set in. These cells also seemed to retain their normal gene expression and showed no signs of becoming cancerous.

I will now try to explain in more detail the details and results of the teams study.

Basically, telomerase is present in all cell types, however the human gene for the catalytic protein telomerase transcriptase (hTRT) is present only in immortal cells (such as stem cells or cancerous cells). Scientists from the teams observed that lengthening of telomeres in retinal pigment epithelial cells, foreskin fibroblasts and vascular endothelial cells by introducing the hTRT gene into these cells, results in a resumption of telomerase activity. This resumption resulted in the extended longevity of the cultured cells, as discussed previously.

Varying Telomere decline

One interesting study by Peter Lansdorp of the Terry Fox Laboratory, Canada, observed in human fibroblasts that the rate of telomere shortening as cells divide varies between different telomeres. This variation is between 50 to 150 base pairs per cell division. It is worth noting that the telomeres that are shorter initially. For example in humans the 17p telomere, are not necessarily the ones to be destroyed first! It does seem that it is the shortening of specific telomeres that are linked to a cells decline into senescence, apoptosis or its transformation into a cancer. Martens et al, 1998, observed that the shortening of telomeres 1p, 5p and 22p, but not that of 17p, showed a statistical correlation with the descent into cellular senescence.

Despite all of the above outlined observations, it is a statement of fact that the mean telomere length of a species does not always relate to the longevity of that species. For example Katuo et al, 1999, noted that of all studied primates, humans seem to have both the shortest telomeres and the longest lifespan! Bassham et al, 1998, also observed that the long lived frog Xenopus Laevis displayed a great variation in telomere length and that telomere length could even diminish between parent and offspring, with no detectable consequences.

To summarise, it seems that it has not yet been determined whether the instability of a chromosome and its eventual deterioration is a result of general telomere shortening or the shortening of specific telomeres. It has however been found that by artificially lengthening the telomeres of cells, which do not normally have a mechanism to lengthen their own telomeres, the cells longevity and ability to function normally for longer, does increase. More research therefore needs to be conducted in order to determine the exact significance of telomere length in the aging of an organism and the exact consequences of artificially stimulating telomere repair.

http://www.whyweage.com/node/11

DNA Quadruplex structures

Structure of a DNA quadruplex formed by telomere repeats. The conformation of the DNA backbone diverges significantly from the typical helical structure^[31]

Quadruplex structures

Further information: G-quadruplex

At the ends of the linear chromosomes are specialized regions of DNA called telomeres. The main function of these regions is to allow the cell to replicate chromosome ends using the enzyme telomerase, as the enzymes that normally replicate DNA cannot copy the extreme 3′ ends of chromosomes.^[32] These specialized chromosome caps also help protect the DNA ends, and stop the DNA repair systems in the cell from treating them as damage to be corrected.^[33] In human cells, telomeres are usually lengths of single-stranded DNA containing several thousand repeats of a simple TTAGGG sequence.^[34]

These guanine-rich sequences may stabilize chromosome ends by forming structures of stacked sets of four-base units, rather than the usual base pairs found in other DNA molecules. Here, four guanine bases form a flat plate and these flat four-base units then stack on top of each other, to form a stable G-quadruplex structure.^[35] These structures are stabilized by hydrogen bonding between the edges of the bases and chelation of a metal ion in the centre of each four-base unit.^[36] Other structures can also be formed, with the central set of four bases coming from either a single strand folded around the bases, or several different parallel strands, each contributing one base to the central structure.

In addition to these stacked structures, telomeres also form large loop structures called telomere loops, or T-loops. Here, the single-stranded DNA curls around in a long circle stabilized by telomere-binding proteins.^[37] At the very end of the T-loop, the single-stranded telomere DNA is held onto a region of double-stranded DNA by the telomere strand disrupting the double-helical DNA and base pairing to one of the two strands. This triple-stranded structure is called a displacement loop or D-loop.^[35]

http://en.wikipedia.org/wiki/DNA

DNA sequence and Supercoiling

Sense and antisense

Further information: Sense (molecular biology)

A DNA sequence is called "sense" if its sequence is the same as that of a messenger RNA copy that is translated into protein. The sequence on the opposite strand is called the "antisense" sequence. Both sense and antisense sequences can exist on different parts of the same strand of DNA (i.e. both strands contain both sense and antisense sequences). In both prokaryotes and eukaryotes, antisense RNA sequences are produced, but the functions of these RNAs are not entirely clear.^[17] One proposal is that antisense RNAs are involved in regulating gene expression through RNA-RNA base pairing.^[18]

A few DNA sequences in prokaryotes and eukaryotes, and more in plasmids and viruses, blur the distinction between sense and antisense strands by having overlapping genes.^[19] In these cases, some DNA sequences do double duty, encoding one protein when read along one strand, and a second protein when read in the opposite direction along the other strand. In bacteria, this overlap may be involved in the regulation of gene transcription,^[20] while in viruses, overlapping genes increase the amount of information that can be encoded within the small viral genome.^[21]

Supercoiling

Further information: DNA supercoil

DNA can be twisted like a rope in a process called DNA supercoiling. With DNA in its "relaxed" state, a strand usually circles the axis of the double helix once every 10.4 base pairs, but if the DNA is twisted the strands become more tightly or more loosely wound.^[22] If the DNA is twisted in the direction of the helix, this is positive supercoiling, and the bases are held more tightly together. If they are twisted in the opposite direction, this is negative supercoiling, and the bases come apart more easily. In nature, most DNA has slight negative supercoiling that is introduced by enzymes called topoisomerases.^[23] These enzymes are also needed to relieve the twisting stresses introduced into DNA strands during processes such as transcription and DNA replication.^[24]

http://en.wikipedia.org/wiki/DNA

Enzymes Built from Scratch

Researchers engineer never-before-seen catalysts using a new computational technique

Catalyst creativity: Researchers developed a computational technique to build enzymes from scratch. An enzyme called retro-aldolase, a portion of which is shown above, was designed to break carbon-carbon bonds in a non-natural chemical substrate (yellow and white stick model). The gray mesh is the enzyme's active site, its geometry carefully crafted to hold the substrate in place. The orange and green stick models indicate components of the enzyme that are particularly important in prodding the reaction forward. Credit: Lin Jiang

In a major step forward for computational protein design, scientists have built from scratch a handful of enzymes that successfully catalyze a specific chemical reaction. These proteins have no naturally occurring counterparts, and the reaction--which breaks down a man-made chemical--has no natural catalyst.

"It makes it clear that we can compute a structure that will catalyze a reaction where there was none before," says Frances Arnold, professor of chemical engineering and biochemistry at Caltech, who was not involved in the research. Arnold calls new enzymes the "holy grail" of computational protein design. Designing any protein from scratch is a tall order; engineering a protein that can carry out a given function requires far more sophistication.

David Baker and his colleagues at the University of Washington focused on a reaction that would break certain bonds between carbon atoms. The ability to design enzymes that can break and make carbon-carbon bonds could potentially enable scientists to break down environmental toxins, manufacture drugs, and create new fuels.

As they report in the journal Science, Baker and his group first designed what an ideal active site would look like for the reaction. An active site is a pocket within an enzyme where the catalyzed reaction takes place. In order to do its job, an active site must have precise geometry and chemical makeup, tailored to the reaction it catalyzes. Some components hold the reacting molecules in place, while others participate in the reaction's chemical mechanisms.

Once the researchers computed the active site, they used a newly developed set of algorithms to model proteins that have such a site. Each designed protein was ranked according to its ability to bind the reacting chemicals and hold them in the proper position.

The next step was to actually synthesize the selected proteins. The researchers derived gene sequences for 72 of the designed enzymes, ordered snippets of DNA containing those genes, and used bacteria to turn the genes into proteins. Each protein was then tested for its ability to catalyze the carbon-carbon bond breaking reaction.

Of the 72 proteins selected, 32 successfully helped along the reaction. The most efficient proteins sped up the reaction to 10,000 times the rate without an enzyme.

While that's an impressive feat compared with earlier enzyme design attempts, the synthesized enzymes pale in comparison to naturally occurring ones. "It's not very good at all," says Baker. "Naturally occurring enzymes can increase the rate of reactions by much, much greater amounts"--as much as a quadrillion-fold.

"One of our research problems is to figure out what's missing from our designs that naturally occurring enzymes have figured out," says Baker. In follow-up studies, his group has taken two approaches to this problem: refining its computer algorithms, and asking nature to step in where the researchers left off. By using their minimally functional enzymes as evolutionary starting points, the researchers can use directed evolution to create more efficient catalysts.

In the past, directed evolution has been an alternative approach to creating desirable enzymes. But Baker believes that it can be used as a complement to computational approaches. Computational design gives researchers a way to build proteins from the ground up, allowing them to engineer enzymes for reactions that have no natural counterpart. "That way, we would be free from the tyranny of having to find something in nature to start from," says Arnold, whose work has focused heavily on directed evolution.

But directed evolution provides a means of making those structural tweaks that the computational designs algorithms aren't yet sophisticated enough to handle. "It's actually the wave of the future," says Baker, "because these directed-evolution experiments can capture much more subtle things than we can capture in the calculations."

Baker's is not the first group to tackle computational enzyme design. For example, Caltech biochemist Steve Mayo, a pioneer in computational protein design, reported the creation of enzymes from existing nonenzyme proteins in 2001. But Baker's approach differs in that it doesn't use existing proteins as starting points--it's true de novo design.

Arnold says that Baker's enzymes are also more powerful than Mayo's, but that it's hard to nail down precisely how much more. "It's a different kind of enzyme, so you can't really compare apples and oranges," she says. "But this is a pretty good apple."

http://www.technologyreview.com/Biotech/20389/page2/