Lecture 4 - DNA Repair Flashcards
What is a mutation? What is a mutant?
Mutation: A heritable change in genetic material; also refers to the process by which the change occurs
Mutant: an organism that exhibits a novel phenotype resulting from a mutation
What is a spontaneous mutation?
- A mutation that occurs w/o a known cause, rare
- Caused by inherent errors in replication or unknown agents in the environment
- Assuming gene length of 1000 bp => 1 mutation/10,0000 - 10,000,000 genes/generation
What is an induced mutation?
- A mutation that results from a known cause
- Caused by exposure to physical or chemical agents (mutagens)
- Frequency of mutation depends on agent
What role does mutation play in evolution?
What is genetic variation?
When do they occur?
What does effect of the mutation on phenotype depend on?
- Mutations are the raw material of evolution and lead to genetic variation
- Genetic variation: change in genotype that can lead to changes in phenotype, ability to respond to environmental changes (natural selection)
- Delicate balance is required, otherwise lethal b/c compromise faithful transfer of genetic info from gen to gen
- Can occur in any cell at any time, but a mutation in one cell type can only affect that cell type
- Effects of mutation on phenotype depend on cell type, when in life cycle, dominance, and type of mutation
Where do mutations occur in animals?
- Germ-line cells: cells that give rise to gametes
- Somatic cells: all other cells
Only mutations that occur in ________ cells will be passed onto progeny
Germ-line
Example of mutation offering an advantage?
Sickle cell anemia: usually problematic, but in Africa, individuals w/ sickle cell are less affected by malaria causing parasite
Do mutations result from adaptive or nonadaptive processes? What experiment proved this?
Nonadaptive processes; Lederberg experiment of replica plating
Stress simply selects rare pre-existing mutations that result in resistance phenotypes
1. Mutations producing resistance occur
2. Sensitive organisms die
3. Insensitive mutants survive and produce new insensitive populations
Mutations are _____
Reversible
What is a forward mutation?
A change from wild-type to mutant phenotype
What is a reverse mutation? Two types?
Reverse mutation: restoration of wild-type phenotype
Back mutation: second mutation at the same site as the first mutation
Suppressor mutation: second mutation anywhere in genome compensates for effect of first mutation
How to differentiate b/t back and suppressor mutation?
Use backcross (mutant x wild type)
- If back mutation, all progeny will be wild type
- if suppressor, half progeny will be wild type phenotype, half will be recombinant mutant/unknown phenotype
Mutations that effect phenotype are usually ______ and _______
Deleterious and recessive
What are the phenotypic effects of mutation? What can they do to pathways? Example
- Mostly result in an AA change that is deleterious to protein function b/c AA sequence is highly adapted to specific environment/function
- Mutations can disrupt/modulate pathways
- Example: mutation of phenylalanine hydroxylase leads to PKU and accumulation of a toxic byproduct
What are the two molecular mechanisms of the mutation process?
- Point mutation (Transition and Transversion)
- Frameshift
What are point mutations (transitions and transversions)?
- Point mutation: mutation that alters a single nucleotide w/in a gene
- Transition: replacement of purine w/ a purine (A to G) or pyrimidine w/ a pyrimidine (T to C)
- Transversion: substitution of pyrimidine to purine (T to G) or purine to pyrimidine (T to G or A; A to C or T)
What are frameshift mutations?
- Base pair addition or deletion in gene => alter reading frame (codons) => result in nonfunctional protein
Mutations can be permanently incorporated by replication
True
False
True
What chemicals act as mutagenic agents?
- Base pair analogs
- Nitrous Acid
- Acridine Dyes
- Alkylating Agents
- Hydroxylating Agents
What are base analogs? What do they do? 5-bromouracil ketone vs enol form; how does this happen, what does it result in?
- Structures similar to normal bases that are incorporated into DNA during replication => increased mispairing => results in transitions
- Ex: Thymine base analog is 5-Bromouracil, Guanine base analog is 2-Aminopurine
5-BU keto form bps with A
5-BU enol form bps with G
=> transitions
Conversion via tautomeric shift (b/c of Br)
What does nitrous acid do?
- Deaminates amino groups => transitions
What do acridine dyes do? What structures are formed?
- Intercalate into DNA => increased rigidity/bends/kinks => Addition/deletions (frameshift) during replication => nonfunctional proteins
- Adenine -> Hypoxanthine which bp w/ cytosine
-Cytosine -> Uracil which bp w/ adenine - Guanine -> Xanthine which bp w/ cytosine
What do alkylating agents do?
- Transfer alkyl group to base at N or O’s => alter bp potential => induce transitions, transversions, or frameshifts
What do hydroxylating agents do?
- Hydroxylamine (NH2OH) hydroxylates amino group of cytosine => bps with adenine => G/C -> A/T bp
What non-chemical mutagenic agents exist?
- UV radiation
- Ionizing radiation
- Transposons
What does UV radiation do?
It excites bases => pyrimidine dimers and pyrimidine hydrates => interferes w/ DNA replication
What does ionizing radiation do?
X-rays, gamma rays, and cosmic rays
Cause increased reactivity of bases
What are transposons? What do they do?
Transposon: DNA fragments that can move from one site in a genome to another site
- Insertion of transposon into an expressed gene will often render gene non-functional
What kinds of DNA repair mechanisms exist in prokaryotes in nature?
- Light-dependent repair
- Excision repair (base, nucleotide)
- Mismatch repair
- Error-prone repair system (SOS)
- Post-replication repair (w/ homologous recombination)
What kinds of DNA repair mechanisms exist in eukaryotes in nature?
- Excision repair (base, nucleotide)
- Mismatch repair
- Error-prone repair system (SOS)
- Post-replication repair (w/ homologous recombination)
What is light dependent repair by DNA photolyase?
- Prokaryotes only
- Photolyase binds thymine dimers
- Use visible light for energy to cleave crosslink
What is base excision repair? What are glycosylases? What is the general outline?
- Prokaryotes and eukaryotes
- Removes abnormal or chemically modified bases
Glycosylases: enzymes that recognize specific base alterations
Outline:
1. Glycosylases mediate base removal => AP site (A-purine or A-pyrimidine)
2. AP endonuclease removes backbone
3. DNA polymerase I/delta adds new base
4. DNA ligase reforms phosphodiester bond
What is nucleotide excision repair? What is exinuclease? Role of helicase (ATP or no)? What fills in the base gap and missing phosphodiester bond?Difference in bacteria and humans?
- Prokaryotes and eukaryotes
- Unique mechanism that removes large base defects (thymine dimers, large alkylated bases, etc.)
Exinuclease: enzyme that cuts on either side of defect and excises large piece
-Helicase mediates release of nucleotide ssDNA from site of damage (localized unwinding, no ATP)
- Bacteria release 12 nucleotide, human release 29 nucleotide
- Bacteria fill base gap w/ DNA polymerase I and missing phosphodiester bond w/ DNA ligase
- Human fill base gap w/ DNA polymerase delta and missing phosphodiester bond w/ DNA ligase
What is mismatch repair? What removes mismatch? What fills gap? What reforms phosphodiester bonds? Directionality?
- Prokaryotes and eukaryotes
- Checks newly synthesized strand mismatches in addition to 3’-5’ exonuclease proofreading activity of polymerases
Strand detection:
- Bacteria: more methylated A’s => older => template | less methylated => younger => new strand
-Humans: detects nicks prior to DNA ligase sealing of Okazaki fragments
- Bacteria fill base gap w/ DNA polymerase I and missing phosphodiester bond w/ DNA ligase
- Human fill base gap w/ DNA polymerase delta and missing phosphodiester bond w/ DNA ligase
- There is directionality in mismatch repair
What is SOS response: translesion synthesis?
- Prokaryotes and eukaryotes
- Last resort
- Allows replication to proceed across damaged template strands even though accurate replication is not possible
- Increased replication errors, desperate/risky attempt to escape lethal effects of heavily damaged DNA
What is postreplication repair?
- Prokaryotes and eukaryotes
- Doesn’t repair w/ TT dimer, but replicates around dimer => template strand still has dimer
- Restarts replication downstream of dimer
- Gap is filled by homologous recombination from sister chromatid, using recombinase A
What is mechanism of DNA recombination? What is the Holliday model?
Homologous chromosome recombination: crossing over b/t nonsister chromatids during prophase of first meiotic division
Holliday model: a model for homologous recombination - looks like a cross
What is xeroderma pigmentosum? (XP) What is the phenotype?
A defect in DNA repair that is autosomal recessive resulting from defects in 9 different genes (6 involved in nucleotide excision repair/exinuclease activity)
Phenotype: extreme sensitivity to sunlight, intense freckling, skin cancer
