Mutations and Mutagens

One letter can change a life

Sickle cell disease, one of the most common inherited diseases in the world, is caused by a change of one base pair out of about 3 billion. Many skin cancers begin with changes caused by ultraviolet light from the Sun. On the other hand, mutations are also the source of every new allele, and without them there would be no evolution.

In this lesson you will extend what you learned in Grade 11 using everything you now know about DNA, transcription and translation. Expectation D3.4 asks you to explain how mutagens such as radiation and chemicals change the genetic material, and D2.2 asks you to analyse DNA sequences to recognize anomalies.

Point mutations and their effects on proteins

Substitutions replace one base pair with another.

• Silent: the new codon codes for the same amino acid (usually a change in the third base). No effect on the protein.
• Missense: the new codon codes for a different amino acid. The effect depends on where it is and how different the new amino acid is. A change in an enzyme's active site can destroy its function; a change on the surface may do nothing.
• Nonsense: the new codon is a stop codon. The protein is cut short and is usually non-functional.

Substitutions are also classified chemically: a transition swaps a purine for a purine (A ↔ G) or a pyrimidine for a pyrimidine (C ↔ T); a transversion swaps a purine for a pyrimidine or the reverse.

Insertions and deletions of one or two bases cause a frameshift: every codon after the change is read in the wrong frame, so the amino acids are changed and a stop codon usually appears soon. Inserting or deleting three bases (or a multiple of three) adds or removes amino acids without shifting the frame. The most common cystic fibrosis mutation deletes three bases and removes one phenylalanine from the CFTR protein; the protein folds incorrectly.

Other effects. A mutation in a promoter or enhancer can change how much protein is made without changing the protein itself. A mutation at a splice site can make the spliceosome skip an exon or keep an intron.

Most mutations that change a protein reduce its function (loss-of-function). A few give the protein a new or increased activity (gain-of-function), as in some cancer-causing genes.

Chromosome mutations

Some mutations change long segments of a chromosome, often containing many genes. They can happen when chromosomes break (for example, from ionizing radiation) and are rejoined incorrectly, or through errors in crossing over.

• Deletion: a segment is lost.
• Duplication: a segment is repeated.
• Inversion: a segment is flipped end to end.
• Translocation: a segment moves to a different (non-homologous) chromosome.

A famous translocation, the Philadelphia chromosome, joins part of chromosome 9 to chromosome 22 in a blood stem cell. It fuses two genes (BCR and ABL) into one that makes an always-active enzyme that drives cells to divide, causing a type of leukemia. The drug imatinib, designed to block exactly that fused enzyme, was one of the first targeted cancer drugs. (Changes in chromosome number, such as trisomy 21, were in Grade 11.)

Where mutations come from

Spontaneous mutations happen without any outside agent:

• Replication errors that escape proofreading and mismatch repair.
• Chemical instability of DNA itself: for example, cytosine can lose an amino group and become uracil, and some bases fall off the sugar entirely. Most of these are repaired, but not all.

Induced mutations are caused by mutagens, which increase the mutation rate.

Physical mutagens: radiation

• Ultraviolet (UV) light, especially UVB from the Sun and tanning beds, is absorbed by DNA and joins neighbouring pyrimidines (usually two thymines) into pyrimidine dimers. If they are not repaired by nucleotide excision repair, polymerase may insert the wrong bases opposite them. UV mostly damages skin cells because it does not penetrate deeply.
• Ionizing radiation (X-rays, gamma rays, and particles from radioactive materials such as radon gas) has enough energy to knock electrons off molecules. It breaks DNA strands directly and creates reactive free radicals from water that damage bases. Double-strand breaks can lead to deletions and translocations.

Chemical mutagens

• Base analogues, such as 5-bromouracil, resemble normal bases and are built into DNA, but they mispair.
• Intercalating agents are flat molecules that slide between stacked base pairs and distort the helix, so polymerase adds or skips a base: frameshifts. Ethidium bromide, an older DNA stain used in labs, is one example; this is why it is handled with gloves.
• Chemicals that modify bases, for example by adding groups to them, make bases mispair or block replication. Benzo[a]pyrene, formed when things burn (in tobacco smoke, and on charred food), is converted in the body into a compound that attaches to guanine. Aflatoxin, made by a mould that can grow on poorly stored peanuts and grains, damages DNA in liver cells. Nitrosamines, which can form in some processed and charred meats and in tobacco smoke, also modify bases.

Biological mutagens

• Some viruses insert their DNA into host chromosomes, which can disrupt genes or switch them on. Infection with some types of human papillomavirus (HPV) can lead to cervical and other cancers. Ontario offers the HPV vaccine to students in Grade 7.
• Transposons ("jumping genes"), first discovered by Barbara McClintock in maize, can move within the genome and interrupt genes.

Somatic and germ-line mutations; mutations and cancer

A somatic mutation affects only the cell where it happens and that cell's descendants. It is not inherited by children. But if somatic mutations build up in genes that control cell division, a cell can become cancerous. Two kinds of genes matter most:

• Proto-oncogenes promote cell division. A gain-of-function mutation can turn one into an oncogene, like an accelerator pedal stuck down.
• Tumour-suppressor genes, such as TP53 (which codes for the protein p53), stop division or trigger repair when DNA is damaged. Loss-of-function mutations are like brakes that fail.

Cancer usually needs several such mutations in one cell lineage, which is one reason cancer risk rises with age and with exposure to mutagens.

A germ-line mutation, in cells that make eggs or sperm, can be passed to the next generation and will then be in every cell of the child.

Testing for mutagens: the Ames test

In the 1970s, Bruce Ames developed a fast, cheap test. Special strains of Salmonella carry a mutation that stops them from making the amino acid histidine, so they cannot grow on a plate without histidine. The test chemical is added (usually with an extract of rat liver enzymes, because the body converts some harmless chemicals into mutagens). If the chemical is a mutagen, it causes reverse mutations that restore histidine production, and many more colonies grow than on a control plate. Most chemicals that are mutagenic in the Ames test are also carcinogens.

Reducing exposure

Health Canada and the Canadian Cancer Society recommend practical steps: avoid tobacco smoke; use sunscreen, shade and protective clothing and avoid tanning beds; test your home for radon (it seeps from the ground and collects in basements; Health Canada's guideline is to take action above 200 becquerels per cubic metre, and it describes radon as the leading cause of lung cancer in people who have never smoked); and follow medical advice on X-rays, which use low doses only when the benefit outweighs the risk.