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Questions and Answers
What sequence does MutL recognize during mismatch repair?
What protein initiates mismatch repair in E. coli?
What is the role of UvrD in nucleotide excision repair?
Which method uses visible light to repair thymine dimers?
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What is the first step in the base excision repair mechanism for modified nucleobases?
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What triggers the activation of SOS repair mechanisms?
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Which complex is involved in scanning DNA for distortions during nucleotide excision repair?
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What is the primary method by which horizontal gene transfer occurs in bacteria?
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What is the final step in the mismatch repair process?
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What is the definition of an auxotroph?
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What is meant by the term 'mutation rate'?
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In what way do spontaneous mutations arise?
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What role do antibiotics play in natural selection among bacterial populations?
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Which of the following characteristics is true of the wild type?
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What is a mutation reversion?
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Which of the following statements about mutations is generally true?
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What defines the mobile gene pool in bacteria?
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How do high-copy-number plasmids manage to replicate within host cells?
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What is a characteristic of low-copy-number plasmids?
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What are resistance plasmids (R plasmids) primarily known for carrying?
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Which factors influence the copy number of plasmids in bacterial cells?
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What can uncontrolled DNA damage lead to in animals?
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Which type of DNA repair pathway is primarily used to prevent mutations?
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What is one characteristic of proofreading by DNA polymerase?
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How does methyl mismatch repair identify incorrect nucleotides?
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Which repair mechanisms are employed when DNA polymerase fails to correct an error?
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What happens to the new DNA strand during methyl mismatch repair?
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What is the primary consequence of activating error-prone repair pathways?
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Which of the following is NOT considered an error-proof repair mechanism?
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What is the primary purpose of indirect selection in mutant selection?
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What does the Ames test primarily measure?
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How do auxotrophs differ from prototrophic strains?
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Which mechanism of horizontal gene transfer involves environmental DNA uptake?
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What is a characteristic of carcinogens in relation to mutagens?
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In horizontal gene transfer, which process involves direct contact between bacterial cells?
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What role does horizontal gene transfer play in prokaryotes?
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What type of mutants can be isolated using the replica plating technique in indirect selection?
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Study Notes
Horizontal Gene Transfer (HGT)
- Movement of DNA from one organism to another
- Often via plasmids
- Can pass onto progeny if the DNA has an origin of replication.
Genetic Change in Bacteria
- Mutations can alter an organism's phenotype
- Ex: A mutation in the gene for tryptophan biosynthesis results in a mutant only able to grow with a tryptophan supply.
- Biosynthesis mutations lead to auxotrophs - organisms that require growth factors
- Prototrophs DO NOT need growth factors.
- Geneticists compare mutants to the wild type.
- Ex: Wild type E.coli is a prototroph.
- Mutant strains are designated with three-letter abbreviations.
- Ex: Trp- indicates a strain that cannot make tryptophan (E.coli Trp-)
Genetic Change in Bacteria - Spontaneous Mutations
- Can be spontaneous or induced.
- Spontaneous mutations are random and occur as a result of normal cellular processes.
- Occur very infrequently.
- Mutation rate is the probability of mutation during each cell division.
- Rates are typically between 10-4 and 10-12 (1/10,000 and 1/1 trillion).
- These rates vary depending on factors like gene size and the organism.
- Mutations are passed on to progeny.
- Occasional reversion is possible.
Genetic Change - Spontaneous Mutations in Populations
- Large populations will always contain mutants (one colony = at least 1 million cells).
- All cells in a colony are not always genetically identical.
- This provides genetic diversity for adaptation to environmental changes.
- The environment selects cells that grow best under current conditions (natural selection).
- Antibiotics, for example, select for resistant bacteria if present.
- Antibiotic kills sensitive cells leaving only resistant mutants.
Genetic Change in Bacteria - Beneficial Mutations
- Single mutations that change phenotype are rare.
- Two beneficial mutations are even rarer.
- Physicians may prescribe two antibiotics to reduce the chance of resistant cells developing.
- Mutations that change phenotype rarely cause a “beneficial” phenotype but often eliminate or reduce pre-existing cellular functions.
- "Beneficial" is context specific.
Genetic Change - Repair of Damaged DNA
- DNA repair is vital to prevent cell death and cancer.
- Examples: Mutations in tumor suppression genes (limit cell growth)
- Mutations are rare because they are repaired before being passed to progeny.
- Many different DNA repair mechanisms exist.
- Eukaryotes and prokaryotes share many repair mechanisms.
DNA Repair - Error Proof and Error Prone
- DNA repair is categorized into two types:
- Error-proof repair pathways - Prevent mutations.
- Include methyl mismatch repair, photoreactivation, nucleotide excision repair, base excision repair, and recombinational repair.
- Error-prone repair pathways - Risk introducing mutations.
- Activated when damage is severe and the cell has no option but to die.
- Error-proof repair pathways - Prevent mutations.
DNA Repair - Errors in Nucleotide Incorporation
- Mispairing slightly distorts the DNA helix.
- This distortion is recognized by enzymes.
- Two repair mechanisms exist:
- Proofreading
- Mismatch repair
DNA Repair - Proofreading
- Proofreading is performed by DNA polymerase during replication.
- DNA polymerase not only synthesizes but also proofreads.
- DNA Polymerases I, II, & III have proofreading ability.
- They can back up, excise mismatched nucleotides, and incorporate the correct base.
- This activity is 3’ → 5’ exonuclease activity.
- Proofreading is very efficient but not flawless.
DNA Repair - Mismatch Repair
- Errors missed by DNA polymerase are fixed by methyl mismatch repair.
- This mechanism determines the incorrect nucleotide (point mutation) by detecting methylation of the parental strand.
- Parental DNA (template strand) is methylated.
- Newly synthesized DNA temporarily lacks methyl groups.
- The repair system cuts out the mismatch from the unmethylated strand.
Mismatch Repair in E.coli
- Mismatch repair is initiated by the protein MutS.
- MutS recognizes the mismatch and recruits two other proteins, MutL and MutH.
- MutL recognizes the unmethylated strand at a GATC sequence (where DNA is methylated).
- Newly synthesized DNA strands are not methylated until after replication is completed.
- The Mut HSL complex causes DNA to loop.
- MutH cleaves the unmethylated strand at the upstream GATC sequence.
- UvrD (a helicase) unwinds and an endonuclease excises the DNA between the strand break and the mismatch.
- The gap is filled by DNA polymerase I and ligase.
Repair of Modified Nucleobases in DNA
- Modified nucleobases lead to base substitutions if not repaired before DNA is replicated.
- Ex: Repair of oxidized guanine is done via base excision repair.
- Glycosylase - Removes oxidized guanine nucleobase.
- Endonuclease VI- Recognizes the missing nucleobase and cuts the DNA backbone at the site.
- DNA polymerase I - Removes the short section and synthesizes a replacement.
- DNA ligase - Seals the gap.
Repair of Thymine Dimers
- UV light can damage DNA.
- Several methods exist to repair this damage.
- Photoreactivation (light repair).
- Photolyase (PhrB) recognizes the distortion, and using energy from visible light, breaks the covalent bonds of the thymine dimer.
- Nucleotide excision repair (dark repair).
- Enzymes detect distorted DNA and remove the damaged section.
Nucleotide Excision Repair
- UvrAB complex scans DNA until UvrA detects a distortion.
- UvrA disassociates while UvrB melts DNA to form a ssDNA bubble.
- UvrA recruits UvrC and UvrC cuts DNA around the primer dimer.
- Helicase UvrD removes the cut single-stranded DNA.
- DNA poly I and ligase fill the gap.
SOS Repair
- SOS Repair is a "last resort" repair mechanism.
- Activated when UV damage is so extensive that photoreactivation and excision repair cannot correct all damage.
- DNA and RNA polymerases stall at damaged sites, halting replication and transcription.
- The cell either dies or activates SOS repair.
Mutant Selection - Indirect Selection
- Used to isolate mutant auxotrophs from wildtype prototrophic strains.
- Ex: Replica plating utilizes differences in growth of auxotrophs on nutrient-rich and -poor media.
- Auxotrophs require growth factors.
Carcinogens and the Ames Test
- Carcinogens are mutagens that cause cancer.
- Animal tests are expensive ($100,000+) and time consuming (2-3 years).
- The Ames test, using bacteria as a model organism, measures the effect of mutation rate on a specific gene.
The Ames Test
- Mutated SalmonellaHis- cells are auxotrophic (require histidine).
- The mutation rate is measured by examining the reversion rate from auxotroph to prototroph.
- If a chemical is mutagenic, it will induce mutations, some of which will revert His- to His+.
- The reversion rate with the potential mutagen is compared to the control.
Horizontal Gene Transfer (HGT) - Mechanisms of Genetic Change
- Genes from one independent, mature organism to another.
- HGT is important for the adaptation of many species.
- Prokaryotes reproduce asexually.
- HGT increases genetic variation, expands ecological niches, and allows genes to be transferred between the same or different species.
- There are three mechanisms of HGT.
- Transformation: Environmental DNA is taken up by a bacterial cell.
- Conjugation: Bacterial DNA is transferred during cell-to-cell contact.
The Mobile Gene Pool
- Genes that vary between strains of a species.
- Often on genetic elements that can move between organisms.
- Examples: Plasmids, transposons, genomic islands, phage DNA.
- They often vastly outnumber the sequences in the core genome (up to 50% of the average E.coli genome).
The Mobile Gene Pool - Plasmids
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Found in most bacteria and archaea.
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Circular, double-stranded DNA with an origin of replication (oriV).
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Usually encode genes nonessential for life.
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Can have many or few genes.
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Exist in two forms:
- Low-copy-number: One or a few plasmids per cell.
- High-copy-number: Many plasmids per cell, up to 500.
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Copy number depends on:
- Origin of replication sequence.
- Size of the plasmid.
- Larger plasmids place a larger metabolic burden to replicate.
Plasmids Ensuring Inheritance
- Plasmids use strategies to maintain themselves in daughter cells.
- High-copy-number plasmids flood the cytoplasm of the host cell.
- Low-copy-number plasmids have partitioning systems involving polymerized filaments to move plasmid copies into both daughter cells.
The Mobile Gene Pool - Resistance Plasmids (R plasmids)
- Carry genes that provide resistance to antibiotics and heavy metals.
- Often have two parts:
- R (resistance) genes (one or many).
- Genes involved in conjugative transfer (allow plasmid to move between cells).
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Description
Test your understanding of horizontal gene transfer and genetic mutations in bacteria. This quiz covers concepts such as plasmids, auxotrophs, prototrophs, and the types of mutations that can occur. Challenge yourself with examples and basic principles of bacterial genetics.
