Non-B DNA structures and disease

People seem to have started contributing actively to the blog again so I thought it was my turn to try again. And this is an achievement, as facing any science, in particular anything written by me about it, would have been impossible a month ago.

I decided to keep things simple for this one and just give a short introduction on the theme of non-B DNA structures and how they influence disease, the topic of my open essay.

When we think about DNA, we think about its B-DNA structure, the one that Watson and Crick described in that famous Nature paper more than 50 years ago, i.e. a right-handed double helix, 20Å diameter, in which the base pairs are almost perpendicular to the axis of the helix, 10.5bps per turn. Although most DNA will be in this structure, it can also form other structures, the so called non-B DNA structures. Some of these are shown in the figure. These alternative structures have a few common characteristics: firstly, whether or not a structure is formed, and which specific structure, seems to be sequence dependent, as it often involves formation of new pairings of bases. Secondly, most of these structures are in higher energetic state than normal B-DNA, for example, because they require the separation and reformation of hydrogen bonds. Overall, therefore, DNA with a favourable sequence tends to remain in its B-DNA form, requiring events such as DNA replication, transcription or protein binding to be transiently converted to these unusual structures. Sequences with propensity to form unusual structures are quite common in the human genome, and it seems that in some cases they are necessary for the normal functioning of the cell. However, they have also been implicated in disease, and this is what I’ll be focusing till the end of this post.

There is a considerable list of diseases thought to involve, as part of their pathology, the formation of non-B DNA structures, but the mechanisms by which this is though to happen can vary. For example, certain sequences/structures have been thought to cause genomic instability, e.g. by causing chromosomal rearrangements due to the propensity of these structures to promote double strand breaks. Another interesting form of genomic instability associated with these unusual structures is repeat expansion, implicated in motor diseases such as Huntington’s disease. Non-B DNA structures, namely Z-DNA, have also been associated with viral infections.

Now, there isn’t really enough space here to talk about everything, so I’m going to give one examples of a situation in which a non-B DNA structure is though to be involved in disease. Friedreich ataxia (FRDA) is a disease caused by the expansion of GAA•TTC tracts in intron 1 of a gene encoding the protein frataxin, essential for mitochondrial function. In this disease, repeat expansion is associated with loss of protein expression. One of the current models by which frataxin production is thought to be reduced in expanded GAA•TTC repeats is based on their ability to form triplexes (an alternative model suggests that epigenetic changes may also be important). Duplex opening within the repeated region, due to the passage of RNA polymerase, is thought to allow one of the separated single strands to form Hoogsteen hydrogen bonds with the purine strand of a B-DNA duplex within the same repeated sequence. This leads to the formation of a 3-stranded helix. Its formation on the non-template GAA strand in frataxin probably interferes with RNA polymerase progression. The free template strand is then thought to base pair with the newly synthesised RNA transcript, forming stable RNA/DNA dimers and preventing further transcription.

Now, this is only a model, for a specific type of structure within the context of a specific disease. And although different sequences have been shown to form these unusual structures, and these structures to be somehow associated with specific diseases, to neatly demonstrate how exactly one influences the other seems to be quite hard. This is particularly difficult because different groups often use different model organisms or protocols, leading to sometimes contradictory results. Overall, although I liked writing about this topic, since I had never considered how the structure of DNA could have an impact on disease, I got the feeling that a lot of work still needs to be done before convincing evidence is given for how exactly these structures impact on our wellbeing, and what sort of therapeutics can be developed from this knowledge.

Red, GAA repeat strand; blue, GTT complementary strand; orange, RNA transcript; yellow, RNA polymerase. 1, Intramolecular triplex; 2, Stalled RNA polymerase; 3, DNA/RNA hybrid

Hebert, M. (2008). Targeting the gene in Friedreich ataxia Biochimie, 90 (8), 1131-1139 DOI: 10.1016/j.biochi.2007.12.005

Bacolla, A. (2004). Non-B DNA Conformations, Genomic Rearrangements, and Human Disease Journal of Biological Chemistry, 279 (46), 47411-47414 DOI: 10.1074/jbc.R400028200

WELLS, R. (2007). Non-B DNA conformations, mutagenesis and disease Trends in Biochemical Sciences, 32 (6), 271-278 DOI: 10.1016/j.tibs.2007.04.003

Wang, G., & Vasquez, K. (2009). Models for chromosomal replication-independent non-B DNA structure-induced genetic instability Molecular Carcinogenesis, 48 (4), 286-298 DOI: 10.1002/mc.20508

Learning how to tell time with cyanobacteria

Most living things can innately tell the time. If you take a person and put them into continuous darkness for months by themselves they will follow a sleep-wake cycle of about 24-25 hours (as well as going a little mad). Circadian rhythms allow life to predict the raising and setting of the sun to allow us to change our gene expression metabolism and behaviour with the changing of the day. Predicting changes in temperatures and light levels are very important. Circadian clocks have three important features. First of all they are free-running at about 24 hours (in constant conditions like light or darkness they will keep going at ~24 hours). Secondly they are temperature compasatory meaning that changes in temperature do not greatly affect the clock. Finally they must be able to be reset by environmental inputs. The changes of the seasons mean days get longer or shorter so to accurately predict the raising of the sun your clock must be reset. Plus for those who fly great distances we must reset according to the new environment and sadly suffer from jet lag while we are slowly adjusting (Johnson et al., 2008).

These clocks have evolved independently multiple times. In animals, plants, some fungi and recently discovered in cyanobacteria (Johnson et al., 2008). It was once thought because prokaryotes divide so raplidly that a 24 hour rhythm would be useless to them but that is not the case (Johnson et al., 1996). A great deal has been learnt about rhythms in bacteria but there are still many unanswered questions. The fact circadian clocks have evolved multiple times shows it offers a great advantage to those that posses one but was no present in the last common ancestor of life. Changing behaviour to only occur at a specific time of day is a great advantage to organisms, plants to photosynthesize and animals to look for food at the right times (Bell-Pedersen et al., 2005).

Cyanobacteria also rely on the sun for energy so predicting when the sun will rise allows them to start synthesising photosystem proteins before the sun raises while not producing them all night or waiting for the sun to raise. It has been shown when you make a mixed culture of bacteria with a mutant clock (no rhythmic changes in gene expression) and wild-type then wild-type will soon outcompete the mutants. Those that have a different length period (eg 16 hours rather than 24 hours) will always lose to wild-type in 24 hour day conditions but wild-type will lose in conditions that match the internal clock of these mutants (Yan et al., 1998). Interestingly in cyanobacteria the whole genome shows rhythmic expression! (Liu et al., 1995) This is in contrast to most organisms that control 15%-35% of their genome in a circadian dependant manner (McDonald and Rosbach, 2001; Correa et al., 2003; Michael and McClung, 2003). This is controlled by the supercoiled state of the whole genome! (Smith and Williams, 2006) I eagerly await the paper that explains how this achieved.

What is rather fascinating about this system is the oscillator. In most organisms gene expression is negatively regulated by its product in a way it cycles its levels over 24 hours and controls output (Bell-Pedersen et al., 2005). In cyanobacteria, this is not the case. Rhythmic expression of clock proteins occur but they do not repress there own synthesis. Also an in vitro oscillator can be setup. In the test tube the clock protein KiaC will cycle through different phosphorylation states if with KaiA, KaiB and ATP. In the organism this forms the post-translational oscillator (Nakajima et al., 2005). This is temperature compensatory and this is built into the ATPase activity of KaiC (Terauchi et al., 2007). It has been shown even when locked in one phosphorylation state you get a weak rhythm so a transcriptional-translational feedback loop as in eukaryotes is needed for a robust system (Kitayama et al., 2008) posing the question of whether this occurs in eukaryotes.

And to finish off I will add one final oddity about the circadian rhythms of cyanobacteria. Instead of using a photoreceptor to sense light inputs like other organisms they read the state of the metabolism. When light is high photosynthesis is occurring and the redox state of the cell changes and is seen as an input to reset the clock (Ivleva et al., 2005).

References

*Of importance
**Truly significant

Bell-Pedersen, D., Cassone, V.M., Earnest, D.J., Golden, S.S., Hardin, P.E., Thomas, T.L. and Zoran, M.J. (2005) Circadian rhythms from multiple oscillators: Lessons from diverse organisms. Nature Reviews Genetics, 6, 544-556.

Correa, A., Lewis, A.Z., Greene, A.V., March, I.J., Gomer, R.H. and Bell-Pedersen, D. (2003) Multiple oscillators regulate circadian gene expression in Neurospora. Proceedings of the National Academy of Sciences of the United States of America, 100, 13597-13602.

Ivleva, N.B., Bramlett, M.R., Lindahl, P.A. and Golden, S.S. (2005) LdpA: a component of the circadian clock senses redox state of the cell. Embo Journal, 24, 1202-1210.

Johnson, C.H., Golden, S.S., Ishiura, M. and Kondo, T. (1996) Circadian clocks in prokaryotes. Molecular Microbiology, 21, 5-11.

Johnson, C.H., Mori, T. and Xu, Y. (2008) A Cyanobacterial Circadian Clockwork. Current Biology, 18, R816-R825.

**Kitayama, Y., Nishiwaki, T., Terauchi, K. and Kondo, T. (2008) Dual KaiC-based oscillations constitute the circadian system of cyanobacteria. Genes & Development, 22, 1513-1521.

Liu, Y., Tsinoremas, N.F., Johnson, C.H., Lebedeva, N.V., Golden, S.S., Ishiura, M. and Kondo, T. (1995) Circadian Orchestration of Gene-Expression in Cyanobacteria. Genes & Development, 9, 1469-1478.

McDonald, M.J. and Rosbach, M. (2001) Microarray analysis and organization of circadian gene expression Drosophila. Cell, 107, 567-578.

Michael, T.P. and McClung, C.R. (2003) Enhancer trapping reveals widespread circadian clock transcriptional control in Arabidopsis. Plant Physiology, 132, 629-639.

*Nakajima, M., Imai, K., Ito, H., Nishiwaki, T., Murayama, Y., Iwasaki, H., Oyarna, T. and Kondo, T. (2005) Reconstitution of circadian oscillation of cyanobacterial KaiC phosphorylation in vitro. Science, 308, 414-415.

Smith, R.M. and Williams, S.B. (2006) Circadian rhythms in gene transcription imparted by chromosome compaction in the cyanobacterium Synechococcus elongatus. Proceedings of the National Academy of Sciences of the United States of America, 103, 8564-8569.

Terauchi, K., Kitayama, Y., Nishiwaki, T., Miwa, K., Murayama, Y., Oyama, T. and Kondo, T. (2007) ATPase activity of KaiC determines the basic timing for circadian clock of cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 104, 16377-16381.

Yan, O.Y., Andersson, C.R., Kondo, T., Golden, S.S. and Johnson, C.H. (1998) Resonating circadian clocks enhance fitness in cyanobacteria. Proceedings of the National Academy of Sciences of the United States of America, 95, 8660-8664.

The Life and Death of Elysia Chlorotica

Since the end of university I’ve had some trouble letting go of science as a constant occupation. Fortunately I was given the chance to post here about an old journal club presentation rather than go cold turkey, so here is a short piece on one of the most interesting organisms I’ve read about.

The picture on the left is of a sea slug of the genus Elysia which has evolved to blend in with its surroundings by looking almost exactly like a small leaf. The green colour that makes this camouflage so convincing is produced when the slug feeds on algae and steals their chloroplasts, subsequently storing them throughout its body. The use of chloroplast pigments to produce a leaf colour is common among sea slugs but the subject of this post is exceptional among the rest of its family. While in the majority of sea slugs the chloroplasts stop working within days or weeks of incorporation, those taken up by Elysia chlorotica can remain stable and active within the slug for at least ten months. As a result the slug is a rare example of a photosynthetic animal and it can survive without food for a long time, as long as it has light and carbon dioxide.

E. chlorotica has coevolved with a specific species of algae named Vaucheria litorea, which is a source of food and chloroplasts for the slug. The metamorphosis of E. chlorotica larvae into their slug form takes place only if the larvae are attached to filaments of V. litorea and in the absence of this algal species the larvae will die. Immediately after metamorphosis the slugs feed on the algae to which they are attached and take up their chloroplasts, incorporating them into their branched digestive tract.

As mentioned previously, most species of sea slug lose their chloroplast functions soon after obtaining them from algae. This is expected to be because the majority of genes required for chloroplast survival and function are encoded on the algal nucleus. Since animals do not have genes involved in chloroplast maintenance the question of how E. chlorotica manages to sustain its stolen plastids was investigated by several labs. In a recent study, the chloroplast genome of V. litorea was sequenced to determine whether it had greater genetic autonomy than previously sequenced chloroplasts, however, the chloroplast genome was found to be largely normal and was missing many genes essential for photosynthesis.

One of these missing genes, psbO, encodes a component of photosystem II which is vulnerable to damage during photosynthesis and needs to be regularly resynthesised for chloroplast function. Rumpho et al. were able to amplify the whole of this gene from sea slug DNA using primers based on seqeuence databases. The amplified sequence was shown to correspond exactly to the version of psbO from V. litorea, indicating that lateral gene transfer had taken place between the algae and the slug. The same sequence could be amplified from sea slug eggs which had not yet encountered algae, confirming that the sequence had entered the E. chlorotica germline. The authors behind this study speculate that all of the other genes needed for chloroplast survival and function have also been transferred to the slug germline, explaining the unique ability of E. chlorotica to maintain its chloroplasts for its entire life. So it seems that the sea slugs have stolen both DNA and organelles from algae, which is quite an achievement.

Another interesting property of E. chlorotica is that its generations are separated from each other. Whether slugs are collected in the lab or monitored in their natural environment, the entire adult population undergoes a synchronous death every year after they have laid their eggs. Pierce et al. observed that viral particles could be seen in the cytoplasm and nuclei of slugs just before their mass death and suggested that they may be responsible for the mortalities. The morphology of the viruses and the presence of reverse transcriptase activity suggest that they are retroviruses and, since the particles were found even under controlled laboratory conditions, it is likely that the virus is encoded on the genome of the sea slug. The group who made this observation suggested that the viruses may respond to annual environmental changes and kill the sea slugs in the spring, though this hypothesis has not been tested so far.

Unfortunately, the last paper I found on research into the viruses within the sea slugs was in 1999 and so research into this interesting aspect of the organism may not be ongoing. However I'm hoping to find some more about it in the future.

Rumpho, M. (2000). Solar-Powered Sea Slugs. Mollusc/Algal Chloroplast Symbiosis PLANT PHYSIOLOGY, 123 (1), 29-38 DOI: 10.1104/pp.123.1.29

Pierce, S., Maugel, T., Rumpho, M., Hanten, J., & Mondy, W. (1999). Annual Viral Expression in a Sea Slug Population: Life Cycle Control and Symbiotic Chloroplast Maintenance Biological Bulletin, 197 (1) DOI: 10.2307/1542990

Pierce, S., Massey, S., Hanten, J., & Curtis, N. (2003). Horizontal Transfer of Functional Nuclear Genes between Multicellular Organisms Biological Bulletin, 204 (3) DOI: 10.2307/1543594

Rumpho, M., Worful, J., Lee, J., Kannan, K., Tyler, M., Bhattacharya, D., Moustafa, A., & Manhart, J. (2008). From the Cover: Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica Proceedings of the National Academy of Sciences, 105 (46), 17867-17871 DOI: 10.1073/pnas.0804968105

Flu flu go away, come again another day (when I don't have exams)

Right I should be revising for those finals but with my arm hurting I am using it as an excuse not to write practice essays today and typing doesn't put as much strain on it (I think).

So Swine flu/Mexican flu/H1N1 flu has brought Mexico to a halt and scared the rest of the world into a panic. Big mouth Vice-president Biden of the USA has done it again saying he wouldn't trust travelling etc. fair point but you are trying to calm the masses as a leader. From what is humble virologist wannabe can see is this new hybrid strain made from genes of avian, swine and human flu is doing is spreading very quickly among people. For some reason it has killed lots in Mexico (allegedly) but only causing mild cases elsewhere. Today however the researcher at Mill Hill have suggested this strain is not very deadly. This paints a rather paradoxical picture. We see many deaths in Mexico from a fast spreading flu. Yes the world should be worried and has done the right thing by preparing for the worse. But we have only seen mild cases in most people outside Mexico and its genome suggests it is only mild. It doesnt have the make-up that causes cytokine storm that kills young people (like Spanish flu did). That is one thing not to worry about (right now). Plus it only infects cells near the top of the respiratory track so doesn't cause infections deep in the lungs, making it easy to spread but not as deadly as some.

So two things need to be thought about 1) why has it killed so many people in Mexico? and 2) what shall we call it? I am not going to tackle the first but I have some thought. The second I think should be Mexican flu. Swine flu is hurting the pig farming industry (some countries have banned pork imports!) while H1N1 is just rubbish. Loads of flu strains are called H1N1. Some seasonal flu is H1N1 and Spanish flu of 1919 was H1N1!!! I personally think the H and N naming system is out dated and inaccurate. It is not the H and N proteins that make a virus what is is per se but the many other changes with them.

Closing thought though, Flu has a high mutation rate with its RNA genome (no where near HIV but still more than what we would like) so changes may occur soon that could be game changers.

http://news.bbc.co.uk/1/hi/health/8028371.stm

When a bit more fat is not that bad

Nothing is as simple as it looks in Biology. We can’t even rely on gene or protein sequences to tell us what is going to happen. At the chromosomal level, the new field of epigenetics is showing how the nucleotide sequence does not define everything. At protein level we have to consider post-translational modifications. My final year project, now at the final write up stages, concentrated on an aspect of Trypanosoma brucei molecular biology which I very pompously described as a potential drug target and the cure for African sleeping disease. This bit was probably ‘paper talk’ though. In any case, the project focused on protein palmitoylation.

Palmitoylation is part of a group of several lipid modifications that can occur in proteins. Examples include N-myristoylation, prenylation and GPI-anchors. N-myristoylation is the reason why I thought it was not pushing it to much to say that we can kill parasites by targeting these modifications. N-myristoylation corresponds to the co-translational addition of myristate to an N-terminus glycine, and is catalyzed by N-myristoyltransferases (NMT). It so happens that NMTs were shown to be essential for parasite viability, and several antifungal NMT inhibitors apparently work on reducing NMT activity in T.brucei as well(1).

But back to palmitoylation, the post-translational addition of palmitic acid to a cysteine residue (reviewed in 2 and 3). It can occur in any place in the protein, and so far no consensus palmitoylation motif has been identified. Often it occurs in an N-terminus cysteine, just next to a myristoylation site. These proteins are said to be dually acylated. Palmitoylation can also happen in close proximity to prenylation sites. Palmitoylation can perform many functions in the cell, the most obvious being protein tethering to membranes and cellular localization. It has also been shown to interfere with protein-protein interactions and even with protein degradation. For example, the yeast SNARE Tgl1, when is not palmitoylated, can interact with the ubiquitin ligase Tul1(4).

Those paying attention (or still reading) will think that it is a bit strange that palmitoylation can regulate protein degradation like that. In fact, most of these lipid modifications, N-myristoylation for example, last for the life time of the protein. Well, palmitoylation is characterized by a unique feature: it is reversible. This means that it can determine processes that other lipidations cannot. Dynamic trafficking of proteins of proteins, for example. This is the case of Ras protein, which through an deacylation/reacylation cycle is able to exchange between the plasma membrane and the Golgi apparatus (5). Reversibility can also be important for signalling. Regulator of G-protein signalling 2 (RGS2) was shown to increase or decrease its GTPase-activating ability depending on which of its cysteine residues are palmitoylated(6).

The process of palmitoylation in itself is catalyzed by the enzymes palmitoyl-acyltransferases (PATs). These were pretty hard to identify, apparently. Not only they were difficult to purify, but the reaction itself can occur spontaneously with biological significance(7), which made many sceptic on whether these enzymes existed at all. However, they were identified, first in yeast, now in mammals and in my dear T.brucei.

And what was supposed to be a short post if already long enough. I finish by adding that my project was about finding proteins that were palmitoylated and that would change their localization in the absence of this modification, in an attempt to identify substrates for RNAi PAT studies in T.brucei. I must say that the project was not particularly successful, but I developed the important skill of writing long discussions based on few results, which I bet will come handy in the future.

I now challenge Mel to continue in this post-translational modification topic and tell us about his project on glycosylation.


References:

1. Price, H. (2002). Myristoyl-CoA:Protein N-Myristoyltransferase, an Essential Enzyme and Potential Drug Target in Kinetoplastid Parasites Journal of Biological Chemistry, 278 (9), 7206-7214 DOI: 10.1074/jbc.M211391200

2. Nadolski MJ, Linder ME. Protein lipidation. FEBS J 2007;274:5202-10.

3. Greaves J, Chamberlain LH. Palmitoylation-dependent protein sorting. J Cell Biol 2007;176:249-54.

4. Valdez-Taubas J, Pelham H. Swf1-dependent palmitoylation of the SNARE Tlg1 prevents its ubiquitination and degradation. EMBO J 2005;24:2524-32.

5. Rocks O, Peyker A, Kahms M, et al. An acylation cycle regulates localization and activity of palmitoylated Ras isoforms. Science 2005;307:1746-52.

6. Ni J, Qu L, Yang H, et al. Palmitoylation and its effect on the GTPase-activating activity and conformation of RGS2. Int J Biochem Cell Biol 2006;38:2209-18.

7. Kummel D, Heinemann U, Veit M. Unique self-palmitoylation activity of the transport protein particle component Bet3: a mechanism required for protein stability. Proc Natl Acad Sci U S A 2006;103:12701-6.