Introductory biophysics A. Y. 2017-18

10. Mutations and carcinogenesis

Edoardo Milotti Dipartimento di Fisica, Università di Trieste “DNA is by no means the inert substance that might be supposed from naive consideration of genome stability.”

D. Voet and J. G. Voet, “Biochemistry, 4th ed.”, Wiley 2011

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Mutations

Sometimes errors in DNA duplication or repair occur, giving rise to new nucleotide sequences: these errors are called mutations.

Mutations can occur both in somatic or germ cells*.

Mutations in somatic cells are not inherited and they can be neglected from an evolutionary point of view. However, mutations in somatic cells are also relevant because they can lead to disease and death.

*Human cells are either somatic cells or germ cells.Germ cells are either a sperm or an egg, all other human cells are called somatic cells.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Estimates of mutation rates (very difficult, only few estimates exist... this one is from Drake et al., “Rates of Spontaneous Mutation”, Genetics 148 (1998) 1667)

mutation rate per mutation rate per base pair per effective genome per replication mutation rate replication per genome mutation rate per per replication effective effective genome genome genome size per sexual size generation

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 • single-base or point mutations affect a single nucleotide

• substitution mutations substitute one base for another • transitions exchange two purines or two pyrimidines • transversions exchange one purine with one pyrimidine

• insertions/deletions insert/delete one base in the sequence

• segmental mutations are similar, but they affect two or more adjacent nucleotides

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Transitions and transversions

Transitions: purine to purine or pyrimidine to pyrimidine

Transversions: purine-pyrimidine exchange

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Hydrolytic deamination of cytosine (net result, C to T: a transition)

Cytosine Uracil

Uracil is analogous to thymine, and this reaction eventually converts the base pair C:G to T:A

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 DNA damage

DNA can be damaged by

• Genotoxic chemicals (exogenous damage) • Reactive species from cell metabolism (endogenous damage) • Viruses • Radiation

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 DNA Damage I: Base Alterations and Single-Strand Breaks

• A base alteration occurs when additional bonds between atoms are formed or broken or new chemical groups attach to the base. All of those situations result in a modified base structure that must be repaired.

• An abasic site occurs when a base separates from the sugar, leaving behind an unpaired base.

• Single strand breaks in the phosphodiester backbone arise largely from hydroxyl radical attack at sugar units comprising the backbone. A gap opens in the normally intact DNA.

All three of these general types of lesions are repaired with only a slight risk of genetic change.

(adapted from R. J. Reynolds and J. A. Schecker, “Radiation, Cell Cycle, and Cancer”, Los Alamos Science, n. 23 (1995) p. 51) Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 DNA Damage II: Double-Strand Breaks

Double-strand breaks result from two single-strand breaks that are induced at closely opposed positions in the complementary strands.

• Simple double strand breaks (upper red box) can often be repaired by a simple end-joining procedure.

• Ionizing radiation often induces a complex lesion (lower red box) with base alterations and base deletions accompanying the breaks.

(adapted from R. J. Reynolds and J. A. Schecker, “Radiation, Cell Cycle, and Cancer”, Los Alamos Science, n. 23 (1995) p. 51)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 from D. Voet and J. G. Voet, “Biochemistry, 4th ed.”, Wiley 2011

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Oxidative attack is activated by metabolites that occur naturally inside cells, or by ions that form in the water environment and that are due either to chemicals or to radiation (ROS = Reactive Oxygen Species).

Globally this is called oxydative stress, and the damage associated to self-produced chemicals is called endogenous damage.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Pyrimidine dimers (thymine dimers) from UV irradiation

these covalent bonds reduce the base stacking distance from 0.34 nm to 0.16 nm

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Pyrimidine dimers block the normal process of DNA copy.

If left unrepaired they kill E. coli.

In humans, DNA Polymerase η skips the defect and performs a copy past the damaged DNA section, albeit with reduced accuracy (10-2 – 10-3).

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 DNA repair Molecular mechanisms sense DNA damage, activate repair mechanisms, and stop the cell cycle progression if the cell is proliferating.

DNA damage is sensed by the MRN complex (double strand breaks) or by RPA (single strand breaks), and this sensing activates a complex molecular network.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 If the cell is proliferating, the network also stops the cell at some specific checkpoints

Cell cycle checkpoints.

(source: Geoffrey M. Cooper e Robert E. Hausman: “The Cell: A Molecular Approach. 5th ed.”, Sinauer Associates 2009.)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 DNA damage, such as that resulting from irradiation, leads to rapid increases in p53 levels. The protein p53 then signals cell cycle arrest at the G1 checkpoint.

(source: Geoffrey M. Cooper e Robert E. Hausman: “The Cell: A Molecular Approach. 5th ed.”, Sinauer Associates 2009.)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Cell “Radiation, Schecker, A. J. and Reynolds J. R. from adapted 51 p. (1995) 23 n. Science, Alamos Los Cancer”, and Cycle, Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Mutations and cancer

Broken mechanisms in tumor cells. From D. Hanahan and R. A. Weinberg, “Hallmarks of Cancer: The Next Generation”, Cell 144 (2011) 646

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 (2011)646 144

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 fromHanahan D. and R. A. Weinberg, “Hallmarks of Cancer: The Next Cell Generation”, Gene modifications linked to cancer (from Vogelstein et al., Nature 408 (2000) 307)

Oncogenes. These are analogous to the accelerators in a car.

Oncogenes stimulate appropriate cell growth under normal conditions, as required for the continued turnover and replenishment of the skin, gastrointestinal tract and blood, for example.

A mutation in an oncogene is tantamount to having a stuck accelerator: even when the driver releases his foot from the accelerator pedal, the car continues to move. Likewise, cells with mutant oncogenes continue to grow (or refuse to die) even when they are receiving no growth signals.

Examples are Ras, activated in pancreatic and colon cancers, and Bcl-2, activated in lymphoid tumours.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Gene modifications linked to cancer/ctd. (from Vogelstein et al., Nature 408 (2000) 307)

Tumour-suppressor genes. When the accelerator is stuck to the floor, the driver can still stop the car by using the brakes.

Cells have brakes, too, called tumour-suppressor genes. These keep cell numbers down, either by inhibiting progress through the cell cycle and thereby preventing cell birth, or by promoting programmed cell death (also called apoptosis).

Just as a car has many brakes (the foot pedal, handbrake and ignition key), so too does each cell. When several of these brakes are rendered non-functional through mutation, the cell becomes malignant.

Examples are the gene encoding the retinoblastoma protein, inactivated in retinoblastomas, p53, and p16INK4a, which inhibits cyclin-dependent kinases and is inactivated in many different tumours.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Gene modifications linked to cancer/ctd. (from Vogelstein et al., Nature 408 (2000) 307)

Repair genes. Unlike oncogenes and tumour-suppressor genes, repair genes do not control cell birth or death directly. They simply control the rate of mutation of all genes.

When repair genes are mutated, cells acquire mutations in oncogenes and tumour-suppressor genes at an accelerated rate, driving the initiation and progression of tumours.

In the car analogy, a defective repair gene is much like having a bad mechanic. Examples are nucleotide-excision- repair genes and mismatch-repair genes, whose inactivation leads to susceptibility to skin and colon tumours, respectively.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Note: homeostasis in cells Homeostasis is the property of a system in which variables are regulated so that internal conditions remain stable and relatively constant.

Examples of homeostasis include the regulation of temperature and the balance between acidity and alkalinity (pH). It is a process that maintains the stability of the human body's internal environment in response to changes in external conditions.

Example: the levels of many enzymes are regulated homeostatically by the equilibrium between production an destruction

destruction rate at equilibrium the total rate of rate of change of proportional to change vanishes and the amount of substance S amount of substance substance is dynamically fixed

dS = α − βS α dt Seq = β fixed production rate

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 OTncologisthe Physician Education

The Molecular Perspective: p53 Tumor Suppressor

DAVID S. GOODSELL

The Scripps Research Institute, Department of Molecular Biology, La Jolla, California, USA

The The p53 tumor suppressor has been termed “the Downloaded from ncologist Guardian of the Cell.” It is not essential for life—mice O that are deficient in this protein are born seemingly nor- —— Activation domain hysician ducation mal—but it is essential in its role of protecting an organ- P E ism from rogue cells. p53 guards two gates: a gate to life and a gate to death. Sensing damage to DNA, p53 can ini-

—— DNA-binding domain www.TheOncologist.com tiate two processes to isolate the damaged cell and prevent The Molecular Perspective: its uncontrolled growth. It can halt cell division, freezing the cell at the G1 checkpoint of the cellular cycle. The cell p53 Tumor Suppressor is unable to reproduce, and its damaged genome is safely —————— Tetramerization isolated. p53 can also initiate a more permanent solution: domain programmed cell death, or apoptosis. DAVID S. GOODSELL p53 tumor suppressor acts as transcriptional activator, The Scripps Research Institute, Department of Molecular Biology, La Jolla, California, USA controlling the expression of a variety of genes important by on April 12, 2010 in cell cycle regulation and apoptosis. p53, composed of four identical subunits, binds to a specific site on the DNA, and interacts with transcription interaction factors, The p53 tumor suppressor has been termed “the leading to the initiationDownloaded from of transcription by RNA poly- Guardian of the Cell.” It is not essential for life—mice merase II. p53 itself is present at extremely low levels in —— Activation domain that are deficient in this protein are born seemingly nor- most cells, and has a life span of mere minutes. These low mal—but it is essential in its role of protecting an organ- levels allow the action of p53 to be genetically controlled. p53ism tumor from suppressor. rogue cells.For those p53 whoguards think two of proteins gates: as a uniformlygate to life Levels may be raised quickly by synthesis of more p53, compactand a andgate globular, to death. the structure Sensing of damagep53 will come to DNA, as a surprise. p53 can It is ini- and high levels are quickly reduced when synthesis

—— DNA-binding domain www.TheOncologist.com composedtiate two of fourprocesses identical to chains, isolate bound the togetherdamaged to formcell anda flexible, prevent abates. The induction of p53 has a hair-trigger: even a sin- four-armedits uncontrolled starfish. Each growth. chain It folds can into halt three cell structured division, domains, freezing connected by long, flexible linkers. At the tip of each arm is an activa- gle double-stranded break of the DNA has been predicted tionthe domain, cell at which the bindsG1 checkpoint to the transcriptional of the machinerycellular cycle. and activates The cell to be enough. geneis unable expression. to reproduce, This domain and also its binds damaged to the regulatory genome protein is safely As one might expect, disruption of a process with —————— Tetramerization MDM2.isolated. At the p53 center can of alsoeach initiatearm is the a largestmore domain,permanent the globular solution: such an important function will have dire consequences. domain DNA-binding domain that binds specifically to the target DNA site. Theseprogrammed DNA-binding cell domains death, are orthe apoptosis.sites of most of the cancer-causing Approximately half of all cases of human cancer may be mutationsp53 observed tumor in suppressor p53. At the center acts ofas the transcriptional tetramer, the four activator, chains attributed to a defective p53 protein. Most of these are interlock,controlling forming the a strong expression Celtic knot of that a variety ties the molecule of genes together. important caused by missense by on April 12, 2010 mutations in the p53 gene, changing in cell cycle regulation and apoptosis. p53, composed of Correspondence:four identical David subunits, S. Goodsell, binds to Ph.D., a specific The Scripps site onResearch the Institute, Department of Molecular Biology, 10550 North TorreyDNA, Pinesand interacts Road, Lawith Jolla, transcription California interaction 92037, factors, USA. Telephone: 619-784-2839; Fax: 619-784-2860; e-mail: [email protected] WorldWideWeb: http://www.scripps.edu/pub/goodsell ©AlphaMed Press 1083-7159/99/$5.00/0 leading to the initiation of transcription by RNA poly- merase II. p53 itself is present at extremely low levels in Themost Oncologist cells, and 1999;4:138-139 has a life span of mere minutes. These low levels allow the action of p53 to be genetically controlled. p53 tumor suppressor. For those who think of proteins as uniformly Levels may be raised quickly by synthesis of more p53, compact and globular, the structure of p53 will come as a surprise. It is and high levels are quickly reduced when synthesis Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 composed of four identical chains, bound together to form a flexible, abates. The induction of p53 has a hair-trigger: even a sin- four-armed starfish. Each chain folds into three structured domains, connected by long, flexible linkers. At the tip of each arm is an activa- gle double-stranded break of the DNA has been predicted tion domain, which binds to the transcriptional machinery and activates to be enough. gene expression. This domain also binds to the regulatory protein As one might expect, disruption of a process with MDM2. At the center of each arm is the largest domain, the globular such an important function will have dire consequences. DNA-binding domain that binds specifically to the target DNA site. These DNA-binding domains are the sites of most of the cancer-causing Approximately half of all cases of human cancer may be mutations observed in p53. At the center of the tetramer, the four chains attributed to a defective p53 protein. Most of these are interlock, forming a strong Celtic knot that ties the molecule together. caused by missense mutations in the p53 gene, changing

Correspondence: David S. Goodsell, Ph.D., The Scripps Research Institute, Department of Molecular Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. Telephone: 619-784-2839; Fax: 619-784-2860; e-mail: [email protected] WorldWideWeb: http://www.scripps.edu/pub/goodsell ©AlphaMed Press 1083-7159/99/$5.00/0

The Oncologist 1999;4:138-139 The p53 network. Activation of the network (by stresses such as DNA damage, ultraviolet light and oncogenes) stimulates enzymatic activities that modify p53 and its negative regulator, MDM2. This results in increased levels of activated p53 protein. The expression of several target genes is then activated by binding of the activated p53 to their regulatory regions. These genes are involved in processes that slow down the development of tumours. For example, some genes inhibit cell-cycle progression or the development of blood vessels to feed a growing tumour; others increase cell death (apoptosis). A negative feedback loop between MDM2 and p53 restrains this network. Many other components of this network, not shown here, have been identified. Similarly, p53 activation results in a variety of other effects, including the maintenance of genetic stability, induction of cellular differentiation, and production of extracellular matrix, cytoskeleton and secreted proteins. The components of the network, and its inputs and outputs, vary according to cell type. p53 is a highly connected ‘node’ in this network. It is therefore unsurprising that the loss of p53 function is so damaging, and that such loss occurs in nearly all human cancers. (adapted from Vogelstein et al., Nature 408 (2000) 307)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 p53 – a tumour suppressor – is an important player in the ATM/ATR network; what happens when it is inactivated?

malfunctioning of the homeostatic regulation of p53

(adapted from Vogelstein et al., Nature 408 (2000) 307)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Ataxia-telangiectasia (also Louis–Bar syndrome)

Ataxia is a neurological sign consisting of lack of voluntary coordination of muscle movements.

Telangiectasia denotes the presence of small dilated blood vessels near the surface of the skin or mucous membranes, measuring between 0.5 and 1 millimeter in diameter.

A-T is caused by a defect in the ATM gene which is responsible for managing the cell’s response to multiple forms of stress including double-strand breaks in DNA.

In simple terms, the protein produced by the ATM gene recognizes that there is a break in DNA, recruits other proteins to fix the break, and stops the cell from making new DNA until the repair is complete

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 A-T matters because it shows us what happens when the DNA repair system is malfunctioning, and therefore it mimics the accumulation of heavy DNA damage in normal individuals.

There is substantial variability in the severity of features of A-T between affected individuals, and at different ages. The following symptoms or problems are either common or important features of A-T:

• Ataxia (difficulty with control of movement) that is apparent early but worsens in school to pre- teen years • Oculomotor apraxia (difficulty with coordination of head and eye movement when shifting gaze from one place to the next) • Involuntary movements • Telangiectasia (dilated blood vessels) over the white (sclera) of the eyes, making them appear bloodshot. Telangiectasia may also appear on sun-exposed areas of skin. • Problems with infections, especially of the ears, sinuses and lungs • Increased incidence of cancer (primarily, but not exclusively, lymphomas and leukemias) • Delayed onset or incomplete pubertal development, and very early menopause • Slowed rate of growth (weight and/or height) • Drooling particularly in young children when they are tired or concentrating on activities • Dysarthria (slurred, slow, or distorted speech sounds) • Diabetes in adolescence or later • Premature changes in hair and skin

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Many aspects of cancer can be modeled analytically and numerically.

Next we consider a simple model of the age dependence of the onset of colorectal cancer.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 (2010)3 10 P .Calabrese and D. Shibata, “A simple algebraic cancer equation: equation: cancer algebraic simple “A Shibata, D. and .Calabrese P calculatinghow cancers may arise with normal mutation rates”, BMCCancer Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 from P .Calabrese and D. Shibata, “A simple algebraic cancer equation: calculating how cancers may arise with normal mutation rates”, BMC Cancer 10 (2010) 3 Normal mutation rate is low, ~ 10-9 per base, per division.

This means that in a 1000 base-long gene, the mutation rate is u ≈ 10-6 per division.

Then the probability that in d divisions the gene is not mutated, is

d p(no mutation in gene) ≈(1− u) and therefore the probability that it is mutated is

p(gene is mutated)

d = p(at least one mutation in gene) ≈1− (1− u)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Then, if there are N compartments with m cells each that are at risk of reaching the critical mutation level, the probability that no cell reaches this critical level is

⎛ no cell in the N compartments ⎞ k Nm p reaches the critical level 1 ⎡1 1 u d ⎤ ⎜ ⎟ = − ⎣ − ( − ) ⎦ ⎝ of mutations ⎠ { } and finally the probability of the onset of illness is

p(onset of illness after d divisions) =

⎛ at least one cell in the N ⎞ k Nm p compartments reaches the 1 1 ⎡1 1 u d ⎤ = ⎜ ⎟ = − − ⎣ − ( − ) ⎦ ⎝ critical level of mutations ⎠ { }

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 k Nm p onset of illness after d divisions 1 1 ⎡1 1 u d ⎤ ( ) = − { − ⎣ − ( − ) ⎦ }

k Nm ≈ 1− {1−[du] }

k ≈ Nm(du)

Since d ≈ a/T (where T is the duplication time)

k k ⎛ a ⎞ ⎛ Nmu ⎞ k k p(onset of illness at age a) ≈ Nm⎜ u⎟ = k a = ba ⎝ T ⎠ ⎝⎜ T ⎠⎟

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 from P .Calabrese and D. Shibata, “A simple algebraic cancer equation: calculating how cancers may arise with normal mutation rates”, BMC Cancer 10 (2010) 3 If the probability of colon cancer risk grows with the number of cells, i.e., with body size, how large is the risk for a whale?

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Assuming all other parameters are equal, larger animals should have a greater lifetime risk of developing cancer compared with smaller organisms. All whales should have CRC by age 80 (2011)175 26 Blue dots for mouse, human and whale indicate the estimated risk of CRC occurring within 90 years of life, given the approximate number of cells in a human colon, 1000 times fewer cells to represent the mouse and 1000 times more cells to represent the whale.

The red dot indicates the lifetime risk of colon cancer according to the American Cancer Society, which is approximately 5.3% for men and women averaged together. A. F. Caulin and C. C. Maley, “Peto’s Paradox: evolution’s prescription for for prescription evolution’s Paradox: “Peto’s Maley, C. C. and Caulin F. A. cancerprevention”, Trends inEcology and Evolution

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 However whales are not more cancer-prone than humans ...

Why cancer is not actually more frequent in large animals?

More cells means higher rate of potentially dangerous mutation events.

The near-constancy of tumor rate in animals is called Peto’s Paradox.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Cancer deaths on total of all deaths

• mice in lab conditions: 46% • dogs: 20% • humans: 25% • beluga whale: 18%

Although the number of cells differs by orders of magnitude and the lifespan also differs, there are no big differences between these values.

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Research article Cell Biology Genomics and Evolutionary Biology

eLife digest As time passes, healthy cells are more likely to become cancerous because more and more damaging mutations accumulate in the cell’s DNA. Assuming that all cells have a similar risk of acquiring mutations, larger and longer-lived animals – like elephants – should have a higher risk of cancer than smaller, shorter-lived animals – like mice. However, there does not appear to be any link between the size of an animal and its risk of developing cancer. Consequently, a key question in cancer biology is how very large animals protect themselves against these diseases. One gene that is often damaged during an animal’s lifetime is called TP53. This gene normally produces a tumor suppressor protein that senses when DNA is damaged or a cell is under stress and either briefly slows the cell’s growth while the damage is repaired or triggers cell death if the stress is overwhelming. One way that large animals could reduce their risk of cancer is to have extra copies of TP53 or other genes that encode tumor suppressor proteins. Here Sulak et al. used an evolutionary genomics approach to study TP53 in 61 animals of various sizes, including several large animals such as African elephants and Minke whales. All of the animals studied had at least one copy of TP53, and several had a few extra copies, known as TP53 retrogenes. African elephants – the largest living land mammal – had more retrogenes than any of the others with 19 in total. To investigate why African elephants have so many TP53 retrogenes, Sulak et al. also analyzed DNA from Asian elephants and several other closely related, but now extinct species, including the woolly mammoth. As expected, as species evolved larger body sizes they also evolved more TP53 retrogenes. Further experiments indicate that several of the TP53 retrogenes in African elephants are likely to be able to produce the tumor suppressor protein and that they contribute to elephant cells being better equipped to deal with DNA damage. The next step following on from this work will be to find out exactly how TP53 retrogenes help to protect animals from cancer. DOI: 10.7554/eLife.11994.002

Figure 1. Body size evolution in vertebrates. (A) Relationship between body mass (g) and lifespan (years) among 2556 vertebrates. Blue line shows the linear regression between log (body mass) and log (lifespan), R2 = 0.32. (B) Body size comparison between living (African and Asian elephants) and extinct (Steppe mammoth) Proboscideans, Cetaceans (Minke whale), and the extinct hornless rhinoceros Paraceratherium (‘Walter’), and humans. DOI: 10.7554/eLife.11994.003

Sulak et al. eLife 2016;5:e11994. DOI: 10.7554/eLife.11994 2 of 30

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 A list of possible answers to Peto’s paradox

• Lower mutation rates in large animals (better error correction mechanisms) • Redundancy of tumor suppressor genes • Lower selective advantage of mutant cells • More efficient immune system • More sensitive or efficient apoptotic processes • Increased contact inhibition • Shorter telomeres • ...

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 At least one solution to Peto's paradox may have been found. Elephants have 20 copies of a gene called p53 (or, more properly, TP53), in their genome, where humans and other mammals have only one. The gene is known as a tumour suppressor, and it snaps to action when cells suffer DNA damage, churning out copies of its associated p53 protein and either repairing the damage or killing off the cell. It was found that elephants produce extra copies of the p53 protein, and that elephant blood cells seem exquisitely sensitive to DNA damage from ionizing radiation. The animals' cells carry out apoptosis in response to DNA damage at much higher rates than do human cells. Instead of repairing the DNA damage, compromised elephant cells seem to have evolved to kill themselves to nip nascent tumours in the bud. (from Nature News, Oct. 8th 2015)

Edoardo Milotti - Introductory biophysics - A.Y. 2017-18 Research article Cell Biology Genomics and Evolutionary Biology and West Indian manatee (Trichechus manatus). The African elephant genome, however, encoded 19 TP53RTG genes (Figure 2A), 14 of which retain potential to encode truncated proteins (Table 1). To trace the expansion of TP53RTG gene family in the Proboscidean lineage with greater phylo- genetic resolution, we used three methods to estimate the minimum (1:1 orthology), average (nor- malized read depth), and maximum (gene tree reconciliation) TP53/TP53RTG copy number in the Asian elephant (Elephas maximus), extinct woolly (Mammuthus primigenius) and Columbian (Mam- muthus columbi) mammoths, and the extinct American mastodon (Mammut americanum) using

Figure 2. Expansion of the TP53RTG gene repertoire in Proboscideans. (A) TP53 copy number in 61 Sarcopterygian (Lobe-finned fish) genomes. Clade names are shown for lineages in which the genome encodes more than one TP53 gene or pseudogene. (B) Estimated TP53/TP53RTG copy number inferred from complete genome sequencing data (WGS, purple), 1:1 orthology (green), gene tree reconciliation (blue), and normalized read depth from genome sequencing data (red). Whiskers on normalized read depth copy number estimates show the 95% confidence interval of the estimate. DOI: 10.7554/eLife.11994.004 The following figure supplement is available for figure 2: Figure supplement 1. Reconciled TP53/TP53RTG gene trees. DOI: 10.7554/eLife.11994.005

Sulak et al. eLife 2016;5:e11994. DOI: 10.7554/eLife.11994 4 of 30