Mutating into Gold

It’s probably just as well that few us are aware that the bodies we live in are a battlefield – the cells and molecules that make us are in constant strife to ensure our survival. The lid is lifted from time to time – when we get a cold or pick up some other infection and our immune response sorts it out but not without giving us a headache or a runny nose, just to let us know it’s on the job. By and large though, we plough our furrow in glorious ignorance.

Saving our cells

Perhaps the most important of all the running battles is to save our DNA – that is, to repair the damage continuously suffered by our genetic material so we can carry on. It’s an uphill struggle. The DNA in one of our cells can take up to a million hits every day – and the bombardment comes from every direction: from radiation, air pollution and carcinogens in some of the food we eat. And, of course, we don’t need to mention cigarette smoke.

Damaged chromosomes (blue arrows)

Damaged chromosomes    (blue arrows)

On top of all that cells have to make a new DNA copy every time they reproduce – and we do a lot of that: recall that you set sail on the journey of life as one single, fertilized egg cell and now look at you: a clump of ten trillion (1013) cells that, just to stay as you are, has to make one million new cells every second. What’s more some of your cells deliberately break their own DNA in a process called ‘gene shuffling’ that goes to make the finished product of your aforementioned immune system. The biochemical machinery that does these jobs is mighty efficient but nobody’s perfect – except, of course, for John Eales, Australia’s most successful rugby union captain, nicknamed “Nobody” because “Nobody’s perfect”. When the three thousand million base-pairs of DNA are stuck together for a new cell there’s a mistake about once in every million units added – but a kind of quality control check (mismatch repair) then fixes most of these, so that the overall error is about one in a thousand million. That’s one example of the nifty ways evolution has come up with to fix the damage suffered by our genetic material from all this replicating, assaulting and constructing.

Keeping the show on the road

The overall upshot of the repair machinery is that less than one mutation per day becomes fixed in our genomes – and thus passed on to succeeding generations of cells. The range of things that can damage DNA – and hence the different forms that damage can take – tells you that there must be several different repair systems and indeed we now know that about 200 genes and their protein products have a hand in some repair process or another. There’s so much to know that DNA damage and repair has its own data-base called, inevitably, REPAIRtoire. Much of what we know is, to a considerable extent, thanks to the labours of Tomas Lindahl, Paul Modrich and Aziz Sancar who have just been jointly awarded this year’s Nobel Prize in Chemistry. Because damage to DNA – aka mutations – drives the development of cancers you might suppose that in these pages we will have met these gentlemen before – and indeed we have, if not by name.

Tomas Lindahl Paul Modrich Aziz Sancar

Tomas Lindahl                      Paul Modrich                       Aziz Sancar

Winners of the 2015 Nobel Prize in Chemistry

Forty odd years ago much of the above would have bewildered cell biologists. Thirty years before then, in 1944, Oswald Avery, Colin MacLeod and Maclyn McCarty had shown for the first time that genes are composed of DNA, a finding confirmed in 1952 by Alfred Hershey and Martha Chase in a classic experiment using a virus that infects and replicates within a bacterium. But with the acceptance that, however improbable, our genetic material was indeed made of DNA there came the assumption that it must be very stable. After all, if it carried our most valuable possession then surely it had to be made of molecular granite, absolutely resistant to any kind of chemical change or degradation. Had the bewildered boffins been told that in the twenty-first century we would be sequencing woolly mammoth DNA from samples that are millions of years old they would have been confirmed in their view.

It was Tomas Lindahl in the early 1970s who demonstrated that, although DNA is indeed more stable than its close rello RNA (the intermediate in making proteins) it nevertheless decays quite rapidly under normal conditions – it’s only when sealed in permafrost or blobs of amber that it becomes frozen in time. Lindahl realized that for life based on DNA to have evolved there had to be repair systems that could sustain our genetic material in a functional state and he went on to resolve how one of these did it. Aziz Sancar has worked particularly on the circadian clock (discovering that CRY is a clock protein) and how cells repair ultraviolet radiation damage to DNA: people born with defects in this system develop skin cancer if they are exposed to sunlight. Paul Modrich has contributed mainly to our knowledge of mismatch repair.

Lindahl, Modrich, Sancar and their colleagues over many years haven’t come up with the philosopher’s stone – the chemists still can’t transmute base metals into gold without the aid of a particle accelerator. But what they have done is much more useful for mankind. Revealing the detail of how genome maintenance works has already lead to new cancer treatments and from this beginning will come greater benefits as time goes by. They should enjoy the proceeds of turning molecular knowledge if not to gold then into Swedish kronor (8 million of them) – for the rest of the world it’s a bargain.

References

Lindahl, T. (1993). Instability and decay of the primary structure of DNA. Nature 362, 709-715.

Yang YG, Lindahl T, Barnes DE. (2007). Trex1 exonuclease degrades ssDNA to prevent chronic checkpoint activation and autoimmune disease. Cell 131, 873-886.

Shao, H, Baitinger, C, Soderblom, EJ, Burdett, V, and Modrich, P. (2014). Hydrolytic function of Exo1 in mammalian mismatch repair. Nucleic Acids Research 42, 7104-7112.

Tan C, Liu Z, Li J, Guo X, Wang L, Sancar A, Zhong D. (2015). The molecular origin of high DNA-repair efficiency by photolyase. Nat Commun. 6, 7302.

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Twenty more winks

In Episode One we alerted ourselves to the large amount of evidence saying that a good night’s sleep really is essential if you wish to reduce your chances of a wide variety of medical misfortunes. But what do we know about how molecules respond to sleep disruption to produce such nasty effects?

Molecular Clocks

Life on earth depends on energy sent forth by the sun and, in synchrony with the rotation of our planet, many of the inner workings of mammals fluctuate over each period of roughly 24 hours. This pattern is called the circadian clock, its most obvious manifestation being the sleep-wake cycle. Over the years considerable evidence has accumulated that the link between shift-work and cancer is probably due to circadian rhythm disruption and suppression of nocturnal production of a hormone called melatonin. All living things make melatonin (in mammals in the pineal gland of the brain) and it signals through a variety of protein receptors on cells to regulate the sleep-wake cycle but it also plays a role in protecting DNA from damage.

Melatonin production is regulated by the circadian oscillator, itself controlled by two sets of proteins that control each other’s expression in a feedback loop. Thus one pair, CLOCK and BMAL1, activates Cryptochrome and Period. They in turn repress CLOCK and BMAL1 – the upshot being that the activities of both pairs oscillate over a day-night cycle: as one goes up the other comes down. These central regulators are encoded by evolutionarily ancient genes (two for Cryptochromes and three for Period proteins). In plants and insects CRY1 responds to light but in mammals CRY1 and CRY2 work independently of light to inhibit BMAL1-CLOCK.

Two interlocked feedback loops control clock protein expression

CRY-CLOCK

OUTCOME: ≈ 24 hour cycle expression of PER & CRY

BMAL1 & CLOCK 12 hours out of phase

Alarming the Clock

So having sounded the alarm that just one night’s sleep shortage has obvious effects, what do the genes make of it? Well, the short answer is they get upset. A recent study took blood samples from a group of normal people and found that more than 700 genes (about 3% of our total number) significantly changed their level of expression over 1 week of insufficient sleep (5.7 h) by comparison with 1 week of sufficient sleep (8.5 h). About two-thirds were reduced whilst one-third was up-regulated (made more of their protein product). Unsurprisingly, among those that went down were the major clock regulators. It’s worth noting that the sleep perturbation in this experiment was relatively mild – intended to be similar to that experienced by many individuals. The genes most strongly affected play roles in a wide range of biological processes – DNA structure (hence gene expression), metabolism, stress responses and inflammation. The responses of genes to changes in sleep patterns are not the result of mutation (i.e. changes in the sequence of DNA)  but, at least in part, they’re caused by small changes in the structure of DNA. {These are epigenetic modifications – any modification of DNA, other than in the sequence of bases, that affects how an organism develops or functions. They’re brought about by tacking small chemical groups either on to some of the bases in DNA itself or on to the proteins (histones) that act like cotton reels around which DNA wraps itself}. Thus there is evidence for gene silencing by hyper-methylation of CRY2 (adding methyl groups (CH3) to its DNA) and the converse effect of hypo-methylation (removing methyl groups) of CLOCK occurs in women engaged in long-term shift work and is associated with an increased risk of breast cancer.

Inflaming the Problem

The cells that mediate inflammation and immune responses also have circadian clocks – meaning that normally these processes are rhythmically controlled and clock disruption (for example by sleep loss) affects this pattern. Disabling the clock in mice (by knocking out CRY altogether) switches on the release of pro-inflammatory messengers and knocking out one of the Period genes (PER2) makes mice cancer-prone – reflecting the fact that MYC (the key proliferation driver) is directly controlled by circadian regulators and is consistently elevated in the absence of PER2.

Clock Faces

The mass that comprises a tumour is a mixture of cells – cancer cells and normal cells attracted to the locale – so it’s a quite abnormal environment and in particular there may be regions where the supply of oxygen and nutrients is limited. This is sensed as a stress by the cells, one response being to lower protein production until normal conditions are restored. If this doesn’t happen within a given time the response switches to one leading to cell suicide. One way in which overall protein output can be reduced is by activating an enzyme (IRE1α) that breaks down code-carrying messenger RNAs that direct assembly of new proteins. Remarkably, it has emerged that one of the mRNAs targetted by IRE1α is the core circadian clock gene, PER1. The degradation of PER1 mRNA means that less PER1 protein is made, which in turn disrupts the clock. However, it seems that PER1 has other roles that include helping the cell suicide response – a major anti-cancer defence. All of which suggests that disruption of the IRE1α/ PER1 balance might have serious consequences. Indeed IRE1α mutations have been found in a variety of cancers including brain tumours in which low levels of PER1 are an indicator of poor prognosis. The IRE1α mechanism coincidentally activates the transcription factor XBP1 (as well as PER1 mRNA decay) and one target of XBP1 is the gene encoding a messenger (CXCL3) that makes blood vessels sprout offshoots. Thus this master regulator suppresses cell death, activates proliferation (lowering PER1 deregulates MYC) and promotes new blood vessel formation.

A Tip for Snoozing

If you’re still wide awake it just goes to prove the utter fascination of biology – but today’s story says that you have to find ways of, if not falling asleep, at least courting insensibility (as Christopher Fry put it). If it’s a real problem for you may I make a really radical suggestion? Turn to our physicist friends and select from their recent literary avalanche. A ‘brief history of …’ something or other will do fine. It’s a knock-out! Sweet dreams!!

References

Möller-Levet, C.S., Archer, S.N., Bucca, G., Laing, E.E., Slak, A., Kabiljo, R., Lo, J.C.Y., Santhi, N., von Schantz, M., Smith, C.P. and Dijk, D.-J. (2013). Effects of insufficient sleep on circadian rhythmicity and expression amplitude of the human blood transcriptome. PNAS 110, E1132-E1141.

Fu, L.N. et al. (2002). The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111, 41-50.

Zhu, Y. et al. (2011). Epigenetic impact of long-term shiftwork: pilot evidence from circadian genes and whole-genome methylation analysis. Chronobiol Int, 28, 852–861.

Pluquet, O. et al. (2013). Posttranscriptional Regulation of PER1 Underlies the Oncogenic Function of IREα. Cancer Res., 73, 4732-4743.