Premed · Premed · General Biology 1
Lecture 25: Mutations and DNA Repair
General Biology I — Molecular & Cellular
Learning Objectives
By the end of this lecture, students will be able to:
- Define mutation and distinguish between different types of point mutations and chromosomal mutations
- Explain the causes of spontaneous and induced mutations
- Describe the major DNA repair mechanisms, including mismatch repair, base excision repair, nucleotide excision repair, and double-strand break repair
- Explain the biological consequences of mutations, including their roles in genetic disease and evolution
- Describe the Ames test and its use in identifying mutagens
Lecture Content
I. What Is a Mutation?
A mutation is a permanent, heritable change in the nucleotide sequence of DNA. Mutations are the ultimate source of all genetic variation--without them, there would be no raw material for natural selection, and evolution would grind to a halt. The consequences of any given mutation depend on its nature and location. Beneficial mutations confer a selective advantage and, though rare, are the basis of adaptation. Neutral mutations have no detectable effect on phenotype and are the most common type, particularly in non-coding regions of the genome or when the degeneracy of the genetic code renders a base change silent. Harmful (deleterious) mutations impair protein function and can cause disease or lethality. The biological impact of a mutation also depends on which cells carry it: mutations in somatic cells affect only the individual and cannot be transmitted to offspring (though they can contribute to cancer), while mutations in germ-line cells are heritable and can be passed to the next generation.
II. Types of Mutations
A. Point Mutations (Single Nucleotide Changes)
Base substitutions replace one base pair with another and come in two varieties. Transitions substitute a purine for a purine (A to G or G to A) or a pyrimidine for a pyrimidine (C to T or T to C) and are more common than transversions, which substitute a purine for a pyrimidine or vice versa (for example, A to C or G to T).
The effect of a base substitution on the encoded protein depends on how it changes the codon. A silent (synonymous) mutation produces a new codon that specifies the same amino acid, typically because the change falls at the third (wobble) position of the codon. The protein is unchanged, and there is usually no phenotypic consequence. A missense mutation produces a codon for a different amino acid. The severity depends on the chemical similarity between the original and replacement amino acids: a conservative missense mutation substitutes an amino acid with similar properties (size, charge, hydrophobicity) and may have little or no effect, while a non-conservative missense mutation introduces an amino acid with very different properties and is more likely to disrupt protein function. The classic example is sickle cell disease, in which a single base change (GAG to GTG) in the beta-globin gene replaces glutamic acid (hydrophilic) with valine (hydrophobic) at position 6, causing hemoglobin to polymerize under low-oxygen conditions. A nonsense mutation creates a premature stop codon (UAA, UAG, or UGA), producing a truncated protein that is almost always nonfunctional. The aberrant mRNA may also be eliminated by nonsense-mediated decay (NMD), a quality control pathway.
B. Insertions and Deletions (Indels)
Insertions add one or more nucleotide pairs to the DNA, while deletions remove them. When the number of inserted or deleted nucleotides is not a multiple of three, the mutation causes a frameshift: the reading frame is shifted for all codons downstream of the mutation, typically producing a completely altered amino acid sequence and often encountering a premature stop codon. Frameshifts are almost always severely deleterious. When the insertion or deletion is a multiple of three, the reading frame is preserved, and one or more amino acids are added to or removed from the protein. The functional impact in this case depends on the location and nature of the change.
<image>A multi-panel figure showing the effects of different types of point mutations on a short mRNA and its encoded peptide. The wild-type mRNA sequence is shown at the top with its codons and corresponding amino acids. Panel A (Silent mutation): A base change in the third position of a codon produces a synonymous codon — the amino acid sequence is unchanged. Panel B (Missense mutation): A base change produces a codon for a different amino acid — one amino acid is altered. Panel C (Nonsense mutation): A base change creates a premature stop codon (UAG) — the protein is truncated. Panel D (Frameshift — insertion): A single nucleotide is inserted, shifting the reading frame of all downstream codons — the amino acid sequence is completely altered and a premature stop codon is encountered. Panel E (Frameshift — deletion): A single nucleotide is deleted, similarly shifting the reading frame with a garbled amino acid sequence downstream.</image>
C. Expanding Trinucleotide Repeats
Certain genes contain short sequences of three nucleotides repeated in tandem (for example, CAG, CGG, or CTG). In some individuals, the number of repeats can expand from generation to generation--a phenomenon called trinucleotide repeat expansion. When the repeat count exceeds a critical threshold, disease results. Huntington disease involves CAG repeats in the huntingtin gene; more than 36 repeats produce an abnormally long polyglutamine tract that causes the protein to aggregate in neurons. Fragile X syndrome involves CGG repeats in the FMR1 gene; more than 200 repeats trigger methylation and silencing of the gene. Myotonic dystrophy involves CTG repeats in the DMPK gene. A hallmark of trinucleotide repeat disorders is anticipation--the tendency for the disease to become more severe and appear at an earlier age in successive generations, because the expanded repeats are prone to further expansion during DNA replication.
D. Chromosomal Mutations (Large-Scale)
Mutations can also alter the structure of entire chromosomes. Deletions remove a segment; duplications repeat one; inversions reverse a segment's orientation; and translocations move a segment to a non-homologous chromosome. Reciprocal translocations exchange segments between two chromosomes; the Philadelphia chromosome, a translocation between chromosomes 9 and 22 that creates the BCR-ABL fusion oncogene, is a well-known driver of chronic myelogenous leukemia (CML). Changes in chromosome number include aneuploidy (gain or loss of individual chromosomes, as in trisomy 21 / Down syndrome) and polyploidy (possession of more than two complete chromosome sets, which is common and often beneficial in plants but generally lethal in animals).
III. Causes of Mutations
A. Spontaneous Mutations
Spontaneous mutations arise from normal cellular processes without exposure to external mutagens. Replication errors occur because DNA polymerase, despite its remarkable accuracy, occasionally incorporates the wrong nucleotide--at a rate of approximately 10^-4 to 10^-5 per base per replication. The polymerase's built-in 3' to 5' exonuclease (proofreading) reduces this to roughly 10^-7, and post-replicative mismatch repair lowers it further to approximately 10^-9 to 10^-10 per base per replication.
Tautomeric shifts contribute to replication errors: bases can transiently adopt rare tautomeric forms (such as the imino form of adenine or the enol form of thymine) that form non-standard base pairs, causing a misincorporation that becomes fixed in the next replication cycle. Depurination--the spontaneous loss of a purine base (adenine or guanine) from the sugar-phosphate backbone, creating an apurinic (AP) site--occurs approximately 5,000 times per cell per day in human cells. Deamination removes an amino group from a base: cytosine deaminates to uracil (which, if unrepaired, leads to a C:G to T:A transition), while 5-methylcytosine deaminates to thymine--a particularly insidious event because the product is a normal base, making CpG sites mutational hotspots. Adenine can deaminate to hypoxanthine. Oxidative damage from reactive oxygen species (ROS) generated during aerobic metabolism modifies bases; the most common lesion, 8-oxoguanine (8-oxoG), mispairs with adenine and causes G:C to T:A transversions. Finally, transposable elements (transposons)--mobile DNA sequences that can jump to new locations in the genome--can insert into genes and disrupt their function.
B. Induced Mutations
Induced mutations result from exposure to mutagens, agents that increase the mutation rate above the spontaneous background.
Chemical mutagens include base analogs (such as 5-bromouracil) that are structurally similar to normal bases and are incorporated during replication, causing mispairing; alkylating agents (such as ethyl methanesulfonate and nitrogen mustards) that add alkyl groups to bases, altering their base-pairing properties; deaminating agents (such as nitrous acid) that remove amino groups; and intercalating agents (such as ethidium bromide and acridine orange)--flat molecules that insert between stacked base pairs, distorting the helix and causing insertions or deletions during replication.
Physical mutagens include UV radiation, which causes covalent bonds to form between adjacent pyrimidines on the same DNA strand, creating thymine (pyrimidine) dimers. The most common are cyclobutane pyrimidine dimers (CPDs), while 6-4 photoproducts are less common but more mutagenic. Both distort the helix and block replication and transcription. Ionizing radiation (X-rays, gamma rays) causes single- and double-strand breaks, base modifications, and chromosomal rearrangements, both directly and through the generation of ROS. Biological mutagens include certain viruses that insert their DNA into the host genome (insertional mutagenesis) and transposable elements.
<image>A two-panel figure on common types of DNA damage. Panel A (UV-induced damage): A DNA double helix is shown with two adjacent thymine bases on the same strand. UV light causes a cyclobutane pyrimidine dimer (CPD) to form — a four-membered carbon ring linking the two thymines through covalent bonds between their C5 and C6 carbons. The dimer distorts the DNA backbone and blocks DNA polymerase. Panel B (Spontaneous depurination and deamination): On the left, depurination is shown — the glycosidic bond between a purine (guanine) and its deoxyribose sugar breaks, releasing the free base and leaving an apurinic (AP) site on the DNA. On the right, deamination of cytosine is shown — the amino group on C4 is replaced by a keto group, converting cytosine to uracil. If unrepaired, the uracil pairs with adenine during the next round of replication, resulting in a C:G to T:A transition mutation.</image>
IV. DNA Repair Mechanisms
Cells have evolved multiple overlapping repair systems to protect genome integrity--a testament to the critical importance of faithful DNA maintenance. Human cells sustain an estimated 10,000 to 100,000 DNA lesions per cell per day, and failure to repair them leads to mutations, cancer, aging, and cell death.
A. Direct Reversal (Direct Repair)
Some types of damage can be repaired directly without excising bases. Photolyase (photoreactivation) uses the energy of visible light to break the covalent bonds in pyrimidine dimers, directly restoring the original bases. This enzyme is found in bacteria, fungi, and plants but is absent in placental mammals, including humans. O6-methylguanine-DNA methyltransferase (MGMT/Ada) removes alkyl groups from the O6 position of guanine by transferring them to a cysteine residue on itself--a suicidal mechanism, since each enzyme molecule can act only once.
B. Base Excision Repair (BER)
BER repairs small, non-bulky lesions such as damaged or inappropriate bases (uracil in DNA, 8-oxoguanine, deaminated bases). A specific DNA glycosylase first recognizes and removes the damaged base by cleaving the glycosidic bond, creating an AP site. Different glycosylases handle different lesions; for example, uracil-DNA glycosylase specifically removes uracil from DNA. AP endonuclease then cuts the sugar-phosphate backbone at the AP site. DNA polymerase (Pol beta in eukaryotes; Pol I in prokaryotes) fills the resulting gap using the undamaged strand as a template, and DNA ligase seals the nick.
C. Nucleotide Excision Repair (NER)
NER handles bulky, helix-distorting lesions that BER cannot manage--pyrimidine dimers, bulky chemical adducts, and intrastrand crosslinks. Its versatility lies in recognizing the distortion caused by the damage rather than a specific damaged base. Damage recognition involves the UvrA/UvrB complex in prokaryotes or the XPC and other XP proteins in eukaryotes. Endonucleases then make dual incisions on either side of the lesion: UvrB and UvrC excise a 12-13 nucleotide patch in prokaryotes, while XPG and XPF-ERCC1 excise a 24-32 nucleotide patch in eukaryotes. The damaged oligonucleotide is removed (by UvrD helicase in prokaryotes), DNA polymerase fills the gap, and DNA ligase seals the repair.
Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder caused by mutations in NER genes (XPA through XPG). Patients cannot repair pyrimidine dimers and are extremely sensitive to UV light, with a greater than 1000-fold increase in skin cancer risk. They must avoid all sunlight exposure. Eukaryotic NER operates through two sub-pathways: global genome NER (GG-NER), which repairs damage anywhere in the genome, and transcription-coupled NER (TC-NER), which preferentially repairs the template strand of actively transcribed genes when RNA polymerase stalls at a lesion. Defects in TC-NER cause Cockayne syndrome, characterized by growth failure, neurodegeneration, and photosensitivity but, interestingly, not increased cancer risk.
D. Mismatch Repair (MMR)
MMR corrects base-base mismatches and small insertion/deletion loops that escape the proofreading activity of DNA polymerase. The central challenge for the repair system is distinguishing the newly synthesized strand (which contains the error) from the template strand (which is correct). In E. coli, the solution is Dam methylation: the template strand is methylated at GATC sequences by Dam methyltransferase, while the newly synthesized strand is transiently unmethylated. In eukaryotes, the mechanism of strand discrimination is less clearly understood but may involve recognition of nicks in the new strand.
In the prokaryotic MutHLS system, MutS recognizes the mismatch. MutL is recruited and activates MutH, which nicks the unmethylated (new) strand at a nearby GATC site. Exonuclease and helicase (UvrD) then remove the region containing the mismatch, and DNA Polymerase III and ligase fill and seal the gap. In eukaryotes, the corresponding proteins are MSH2/MSH6 (MutS-alpha) for mismatches and single-base loops, MSH2/MSH3 (MutS-beta) for larger insertion/deletion loops, and MLH1/PMS2 (MutL-alpha) for coordinating excision and repair.
Defects in MMR genes, particularly MLH1 and MSH2, cause Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC), characterized by microsatellite instability (MSI)--expansion or contraction of short tandem repeats throughout the genome that serves as a diagnostic hallmark.
<image>A step-by-step diagram of mismatch repair in E. coli. Step 1: A G-T mismatch is present in newly replicated DNA. The parental (template) strand is methylated at a nearby GATC site (shown with a methyl group); the daughter strand is unmethylated. Step 2: MutS (a homodimer) recognizes and binds to the G-T mismatch, bending the DNA. Step 3: MutL binds to MutS, forming a MutS-MutL complex that activates MutH. Step 4: MutH endonuclease nicks the unmethylated daughter strand at the hemimethylated GATC site. Step 5: A helicase (UvrD) and exonuclease degrade the daughter strand from the nick through and past the mismatch. Step 6: DNA polymerase III resynthesizes the excised strand using the parental strand as template, correcting the mismatch to a proper G-C pair. DNA ligase seals the nick.</image>
E. Double-Strand Break (DSB) Repair
Double-strand breaks are the most dangerous type of DNA damage because both strands of the helix are severed, and if unrepaired they lead to chromosome loss, rearrangements, or cell death. Two major pathways handle DSBs.
Non-Homologous End Joining (NHEJ) directly ligates the two broken ends together without requiring a homologous template, so it can operate in any phase of the cell cycle. The Ku70/Ku80 heterodimer recognizes and binds the broken ends, recruiting DNA-PKcs (DNA-dependent protein kinase) and processing enzymes. The ends are trimmed or filled in as needed and joined by DNA ligase IV. NHEJ is error-prone--it frequently introduces small insertions or deletions at the junction--but it is fast and is the predominant DSB repair pathway in human cells.
Homologous Recombination (HR) uses the sister chromatid as a template, ensuring high-fidelity repair. Because it requires a sister chromatid, HR operates primarily during the S and G2 phases of the cell cycle. The 5' ends at the break are resected to generate 3' single-stranded overhangs, which are coated by RAD51 (the eukaryotic homolog of bacterial RecA) to form a nucleoprotein filament. This filament searches for and invades the homologous sequence on the sister chromatid (strand invasion), DNA synthesis extends the invading strand using the sister as template, and the break is resolved with error-free precision. The tumor suppressors BRCA1 and BRCA2 are essential for HR; mutations in these genes force cells to rely on error-prone NHEJ, dramatically increasing the risk of breast, ovarian, and other cancers.
V. The SOS Response and Translesion Synthesis
When DNA damage is so extensive that it stalls replication forks, bacteria mount the SOS response. RecA protein coats the single-stranded DNA that accumulates at stalled forks and stimulates the self-cleavage of the LexA repressor, derepressing more than 40 SOS genes. Among the most important products are translesion synthesis (TLS) polymerases (such as Pol IV/DinB and Pol V/UmuC-UmuD' in E. coli). These specialized polymerases can replicate past damaged bases that would permanently block the normal replicative polymerase. However, they possess no proofreading activity and are inherently error-prone, introducing mutations at high frequency. TLS represents a "last resort" strategy: it is better for the cell to survive with some mutations than to die from an irreparably stalled replication fork.
Eukaryotes also possess TLS polymerases. Pol eta (polymerase eta) is particularly notable because it can accurately bypass thymine dimers by inserting two adenines opposite the fused thymines. Mutations in the gene encoding Pol eta cause XP variant (XP-V), a form of xeroderma pigmentosum in which NER is intact but UV-induced mutagenesis is elevated because an error-prone polymerase substitutes for the missing Pol eta.
VI. Mutations and Disease
Loss-of-function mutations in DNA repair genes underlie a growing list of cancer predisposition syndromes: xeroderma pigmentosum (NER defects), Lynch syndrome (MMR defects), BRCA1/BRCA2 mutations (HR defects leading to breast and ovarian cancer), ataxia telangiectasia (defective ATM kinase, impairing DNA damage signaling), Fanconi anemia (defects in interstrand crosslink repair), and Li-Fraumeni syndrome (mutations in p53, the "guardian of the genome"). The progressive accumulation of mutations in oncogenes (which gain function and accelerate growth) and tumor suppressor genes (which lose function and remove growth restraints) is a central driver of cancer development. Understanding mutation and repair has also opened therapeutic avenues: PARP inhibitors such as olaparib exploit the dependence of BRCA-deficient tumors on alternative repair pathways, creating a situation of synthetic lethality in which the combination of two repair deficiencies is lethal to cancer cells while sparing normal cells.
VII. The Ames Test
The Ames test, developed by Bruce Ames, is a widely used bioassay for identifying potential mutagens and, by extension, potential carcinogens. It uses a specially engineered strain of Salmonella typhimurium carrying a mutation in a histidine biosynthesis gene (His-) that renders the bacteria unable to grow without exogenous histidine. The bacteria are plated on minimal medium lacking histidine, the suspected mutagen is added along with a rat liver extract (S9 fraction) to simulate mammalian metabolic activation (since some chemicals become mutagenic only after liver metabolism), and the plates are incubated. A significant increase in the number of revertant colonies (His+) compared to a control without the test substance indicates that the substance is mutagenic. Approximately 90% of known carcinogens test positive in the Ames test, strongly supporting the link between mutagenicity and carcinogenicity.
