MUTATION

Introduction
Mutations can happen at any time and in any cell

The phenotypic effects can range from minor alterations that are detectable only by biochemical methods to drastic changes in essential processes that cause, at one extreme, unrestrained cell proliferation (cancer) or, at the other extreme, the death of the cell or organism
The effect of a mutation is determined by the type of cell containing the mutant allele, by the stage in the life cycle or development of the organism that the mutation affects, and, in diploid organisms, by the dominance or recessiveness of the mutant allele

Definition
A mutation is a change in a DNA base-pair (substitution, deletion, or insertion of a base pair) or a chromosome (deletion, insertion, or rearrangement)
a. Somatic mutations affect only the individual in which they arise b. Germ-line mutations alter gametes (tissues that produces eggs & sperm) and passed the next generation

What can happen when a mutation occurs in the DNA (e.g. sickle cell anemia)

Concept of a mutation in the protein-coding region of a gene. (Note that not all mutations lead to altered proteins and that not all mutations are in protein-coding regions)

Mutations are quantified in two different ways:

a. Mutation rate is the probability of a particular kind of mutation as a function of time (e.g., number per gene per generation) or # mutation per nucleotide pair or gene per generation b. Mutation frequency is number of times a particular mutation occurs in proportion to the number of cells or individuals in a population (e.g., number per 100,000 organisms) or # of a particular mutation per 100,000 organisms

Types of mutations
Two types of point mutations:
1. Base pair substitutions a. Transitions
Convert a purine-pyrimidine to the other purinepyrimidine (e.g., AT to GC or TA to CG) 4 types of transitions; A G and T C Most transitions results in synonymous substitution
Convert a purine-pyrimidine to a pyrimidine-purine (e.g., AT to TA, or AT to CG) 8 types of transversions; A T, G C, A C, and G T Transversion more likely to result in nonsyn substitution

b. Transversions

2. Base pair deletions and insertions

can change the reading frame of the mRNA downstream of the mutation, resulting in a frameshift mutation.
a. When the reading frame is shifted, incorrect amino acids are usually incorporated. b. Frameshifts may bring stop codons into the reading frame, creating a shortened protein. c. Frameshifts may also result in read-through of stop codons, resulting in a longer protein. d. Frameshift mutations result from insertions or deletions when the number of affected base pairs is not divisible by three.

Types of base pair substitutions and mutations

Types of base pair substitutions and mutations

Effect of a nonsense mutation on translation

Types of mutations in ORFs:

Nonsynonymous/missense mutation Base pair substitution results in substitution of a different amino acid.
Nonsense mutation Base pair substitution results in a stop codon (and shorter polypeptide). Neutral nonsynonymous mutation Base pair substitution results in substitution of an amino acid with similar chemical properties (protein function is not altered). Synonymous/silent mutation Base pair substitution results in the same amino acid. Frameshift mutations: Deletions or insertions (not divisible by 3) result in translation of incorrect amino acids, stops codons (shorter polypeptides),or read-through of stop codons (longer polypeptides).

The nine codons that can result from a single base change in the tyrosine codon UAU. Blue arrows indicate transversions, gray arrows, transitions. Tyrosine codons are in boxes. Two possible stop (''nonsense") codons are shown in red. Altogether, the codon UAU allows for six possible missense mutations, two possible nonsense mutations, and one silent mutation

Reverse Mutations and Suppressor Mutations

1. Point mutations are divided into two classes based on their effect on

phenotype:
a. Forward mutations change the genotype from wild type to mutant. b. Reverse mutations (reversions or back mutations) change the genotype from mutant to wild-type or partially wild-type. i. A reversion to the wild-type amino acid in the affected protein is a true reversion. ii. A reversion to some other amino acid that fully or partly restores protein function is a partial reversion.

2. Suppressor mutations occur at sites different from the original mutation, and

mask or compensate for the initial mutation without actually reversing it. Suppressor mutations have different mechanisms depending on the site at which they occur.
a. Intragenic suppressors occur within the same gene as the original mutation, but at a different site. Two different types occur: i. A different nucleotide is altered in the same codon as the original mutation. ii. A nucleotide in a different codon is altered (e.g., an insertion frameshift is suppressed by a nearby deletion event).

b. Intergenic suppressors occur in a different gene (the suppressor gene) from the original mutation. Many work by changing mRNA translation.
i. Each suppressor gene works on only one type of nonsense, missense or frameshift mutation. ii. A given suppressor gene suppresses all mutations for which it is specific. iii. Suppressor genes often encode tRNAs that recognize stop codons and insert an amino acid, preventing premature termination of translation.
(1) Full or partial function of the polypeptide may be restored. (2) The effect depends on how compatible the substituted amino acid is with protein function.

iv. Nonsense suppressors fall into three classes, one for each stop codon (UAG, UAA and UGA) (Figure 19.5). v. Typical tRNA suppressor mutations are in redundant tRNA genes, so the wildtype tRNA activity is not lost. vi. Nonsense suppression occurs by competition between release factors and suppressor tRNAs.
(1) UAG and UGA suppressor tRNAs do well in competition with release factors. (2) UAA suppressor tRNAs are only 15% efficient.

vii. Suppression by a tRNA occurs at all of its specific stop codons (e.g., UGA or UAG), not just the mutant one. This may produce read-through proteins, but they are not as common as expected, possibly due to tandem stop codons (e.g., UAGUGA).

tRNA suppressor gene mechanism for nonsense mutation

New mutations are categorized as induced or spontaneous

Induced mutations are defined as those that arise after purposeful treatment with mutagens, environmental agents that are known to increase the rate of mutations

Spontaneous mutations are those that arise in the absence of known mutagen treatment. They account for the "background rate" of mutation and are presumably the ultimate source of natural genetic variation that is seen in populations.

Spontaneous Mutations
1. All types of point mutations can occur spontaneously, during S, G1 and G2 phases of the cell cycle, or by the movement of transposons. 2. The spontaneous mutation rate in eukaryotes is between 104-to-10-6 per gene per generation, and in bacteria and phages 10-5-to-10-7/gene/generation. a. Genetic constitution of the organism affects its mutation rate. i. In Drosophila, males and females of the same strain have similar mutation rates. ii. Flies of different strains, however, may have different mutation rates. b. Many spontaneous errors are corrected by the cellular repair systems, and so do not become fixed in DNA.

Different types DNA replication errors

Wobble-pairing
T-G, C-A, A-G, T-C Normal pairing typically occurs in the next round of replication; frequency of mutants in F2 is . GT pairs are targets for correction by proofreading and other repair systems

Additions and deletions

DNA loops out on template strand, DNA polymerase skips bases, and deletion occurs DNA loops out on new strand, DNA polymerase adds untemplated bases

Mutation caused by mismatch wobble base pairing

Addition and deletion by DNA looping-out

Spontaneous chemical changes

Depurination
Common; A or G are removed and replaced with a random base.

Deamination
Amino group is removed from a base (C U); if not replaced U pairs with A in next round of replication (CG TA). Prokaryote DNA contains small amounts of 5MC; deamination of 5MC produces T (CG TA). Regions with high levels of 5MC are mutation hot spots.

Deamination

Induced mutations
Radiation (e.g., X-rays, UV)
Ionizing radiation breaks covalent bonds including those in DNA and is the leading cause of chromosome mutations

Ionizing radiation has a cumulative effect and kills cells at high doses
UV (254-260 nm) causes purines and pyrimidines to form abnormal dimer bonds and bulges in the DNA strands

Thymine dimers induced by UV light.

Induced mutations: chemical mutagens

Base analogs
Similar to normal bases, incorporated into DNA during replication Some cause mis-pairing (e.g., 5-bromouracil) Not all are mutagenic

Mutagenic efffects of 5-bromouracil

Mutagenic efffects of 5-bromouracil

Induced mutations:

Chemical mutagens

Base modifying agents, act at any stage of the cell cycle:

Deaminating agents Hydroxylating agents Alkylating agents

Base-modifying agents

10.24

Base-modifying agents (cont.).

10.25

Induced mutations: chemical mutagens

Intercalating agents:
Thin, plate-like hydrophobic molecules insert themselves between adjacent base-pairs,

Mutagenic intercalating agents cause insertions during DNA replication.

Loss of intercalating agent can result in deletion. Examples: proflavin, ethidium bromide

DNA Repair Mechanisms

1. Both prokaryotes and eukaryotes have enzyme-based DNA repair systems that prevent mutations and even death from DNA damage. 2. Repair systems are grouped by their repair mechanisms. Some directly correct, while others excise the damaged area and then repair the gap.

Direct Correction of Mutational Lesions

1. DNA polymerase proofreading corrects most of the incorrect nucleotide

insertions that occur during DNA synthesis, which stalls until the wrong nucleotide is replaced with a correct one.
a. The role of 3-to-5 exonuclease activity is illustrated by mutator mutations in E. coli, which confer a much higher mutation rate on the cells that carry them.
b. The mutD gene, encoding the e subunit of DNA polymerase III, is an example. Cells mutant in mutD are defective in proofreading. 2. UV-induced pyrimidine dimers are repaired using photoreactivation (light

repair).
a. Near UV light (320370 nm) activates photolyase (product of the phr gene) to split the dimer. b. Photolyases are found in prokaryotes and simple eukaryotes, but not in humans.

3. Damage by alkylation (usually methyl or ethyl groups) can be removed by

specific DNA repair enzymes.

a. For example, O6-methylguanine methyltransferase (from the ada gene) recognizes O6-methylguanine in DNA, and removes the methyl group. b. Demethylation restores the base to its original form.

Repair Involving Excision of Base Pairs

1. Another repair system, which does not require light, was discovered in 1964. It is called dark repair, the excision repair system, or the nucleotide excision repair (NER) system.
a. In E. coli, NER corrects pyrimidine dimers and other damage-induced distortions of the DNA helix. b. The proteins required are UvrA, UvrB, UvrC and UvrD (encoded by genes of the same name) (Figure 19.17). c. A complex of two UvrA and one UvrB proteins slides along the DNA. When it encounters a helix distortion, the UvrA subunits dissociate, and a UvrC binds the UvrB at the lesion. d. When UvrBC forms, the UvrC cuts 45 nucleotides from the lesion on the 3 side, and eight nucleotides away on the 5 side. Then UvrB is released and UvrD binds the 5 cut end. e. UvrD is a helicase that unwinds the region between the cuts, releasing the short ssDNA, while DNA polymerase I fills the gap and DNA ligase seals the backbone. f. In yeast and mammalian systems, about 12 genes encode proteins involved in excision repair.

Nucleotide excision repair (NER) of pyrimidine dimmer and other damage-induced distortions of DNA

2.

Methyl-directed mismatch repair recognizes mismatched base pairs, excises the incorrect bases and then carries out repair synthesis.
a. In E. coli, initial stages involve products of the mutS, mutL and mutH genes. i. MutS binds the mismatch, and determines which is the new strand by its lack of methylation. ii. MutL and MutH bind unmethylated GATC sequences (site of methylation in E. coli) and bring the GATC close to the mismatch by binding MutS. iii. MutH then nicks the unmethylated GATC site, the mismatch is removed by an exonuclease and the gap is repaired by DNA polymerase III and ligase. b. Eukaryotes also have mismatch repair, but it is not clear how old and new DNA strands are identified. i. Four genes are involved in humans, hMSH2 (homologous to E. coli mutS), and hMLH1, hPMS1 and hPMS2 (all homologous to mutL). ii. All of these are mutator genes, and mutation in any 1 of them confers hereditary predisposition to hereditary nonpolyposis colon cancer (HNPCC).

Mechanism of mismatch correction repair

3. The SOS response in bacteria results when specific base pairing

cannot occur. It allows the cell to survive otherwise lethal events, but usually at the cost of incurring new mutations.
a. E. coli SOS is controlled by two genes, lexA and recA. (Mutants in either of these genes have their SOS response permanently turned on.) i. When no DNA damage is present, LexA represses transcription of about 17 genes with products involved in various types of DNA repair. ii. Sufficient DNA damage activates the RecA protein, which stimulates LexA to autocleave, removing repression of the DNA repair genes. iii. After damage is repaired, RecA is inactivated, and newly synthesized LexA again represses the DNA repair genes. b. The SOS system is an error-prone bypass synthesis system. i. Some lesions, like T^T dimers, are easily copied to give AA in the new DNA strand. ii. Others, like C^C dimers, stall the SOS repair system, creating a delay during which C can be deaminated to a U (forming a C^U), creating a transition mutation.