Lecture 7 - DNA repair and recombination Flashcards
Mutation
Heritable change in the DNA
Different types of mutations
Point mutation, insertion/deletion, inversion, reversion
Types of point mutations
Transition and transversion
Transition mutation
Purine –> purine or pyrimidine –> pyrimidine (less traumatic for the cell)
Transversion
Purine <–> pyrimidine
Do mutations always change the protein sequence?
No, but they can give rise to new phenotypes
- depends on where the mutation is and what it is
Information classes of mutations
Silent, missense, nonsense, and frame-shift
Silent mutation
Doesn’t change the aa sequence
Missense mutation
Changes an amino acid sequence to another
- doesn’t always have deleterious effects
- if the change is to the wobble position of a codon, the cell generally can compensate for it
Nonsense mutation
Changes the amino acid sequence to a stop codon (almost always deleterious)
Frame-shift mutation
Changes the open reading frame of the gene
Relationship between the protein sequence and the function of the protein
Proteins are tolerant to certain small changes
- many proteins that have the same function across species will have slightly different sequences
- wobble position changes will either result in no change to the aa placed or a change to an aa with similar properties
Chemical reactions that can induce spontaneous mutations
- tautomeric shifts that alter base-pairing properties (changes the pairing)
- oxidative deamination of bases (can change the identify of a base, most often C to U)
- formation of apurinic sites (no nucleotide present)
Sources of spontaneous mutations in bacteria
- chemical reactions
- DNA replication
How does the tautomeric shift of thymine result in a mutation?
TA pair to TG pair during DNA replication
1. thymine changes to the enol form
2. during replication, enol form of thymine pairs with guanine
3. enol thymine reverts to normal thymine –> TG pairing
Other examples of tautomeric shifts that result in mutations?
- AT –> AC due to rare imino form of adenine
- CG –> CA due to imino form of cytosine
- GC –> GT due to enol form of guanine
What is spontaneous deamination?
Water attacks amine groups on C and converts them to uracil
- result: accumulation of U in the DNA
How does depurination occur?
Water attacks purine and removes the base
- result: abasic site
Purines
Adenine and Guanine
Pyrimidines
Thymine and Cytosine
Chemical mutagens
- base analogs (ex: 2-aminopurine)
- base modifiers and alkylating agents
- intercalators
Electromagnetic mutagens
- ionization radiation (x-rays and gamma rays)
- UV radiation
How does ionization radiation cause mutations?
X-rays and gamma rays can interact with water to form free radical oxygen species
- free radicals cleave the phosphodiester backbone of DNA
How does UV radiation cause mutations?
Forms pyrimidine dimers that put strain on the DNA and prevent progress of the replisome
How does 2-aminopurine (a base analog) cause mutations?
Structure is an intermediate between adenine and guanine, so it can pair with either C or T
How does nitrous acid (HNO2) result in mutations?
Promotes deamination of C and A (accelerates normal oxidative deamination)
How does alkylation (addition of methyl or ethyl groups specifically) induce mutations?
Alkylation stabilizes the enol form of G and T –> conversion of G and T to forms that pair erroneously
How do pyrimidine dimers form?
- Pyrimidine bases absorb UV light
- Electrons are excited and react with adjacent base
- Formation of covalent bonds
Where are pyrimidine dimers most common?
At sites of adjacent pyrimidine bases, mostly at T-T sequences
Outcome of pyrimidine dimer formation
- strain introduced to the double helix
- DNA replication is blocked because replisome can’t move past the dimer
What is the Ames test?
Test that identifies if a material is a chemical mutagen
What does the Ames test use?
Relies on a mutant bacterial strain that is defective in hisG (can’t grow on a medium without histidine)
How do you perform an Ames test?
- Plate histidine-deficient bacteria on agar plate without histidine
- Place a potential mutagen-soaked paper disk on the plate
- If bacteria grow near the suspected mutagen, its mutagenicity is confirmed
Why does bacterial growth signify the presence of a mutagen in the Ames test?
Mutagen causes reversion mutations that repair the defect in hisG
Pattern of colony growth to identify mutagen in the Ames test
High concentration of colonies near the mutagen, no colonies (or very few) far away and very close to the mutagen
- far away, mutagen hasn’t diffused; no reversion mutations
- very close: too much mutation –> death
Examples of error-proof DNA repair
- methyl mismatch repair
- photoreactivation
- nucleotide excision repair
- base excision repair
- recombinational repair
When is error-prone repair used?
Only when DNA damage is so severe that it is potentially lethal
- risk of introducing mutations in order to save the cell from death
Methyl mismatch repair
Uses methylation of the parental strand to discriminate from newly replicated DNA
- new DNA doesn’t have time to be methylated
When does methyl mismatch repair occur?
Occurs right after DNA replication
Steps of methyl mismatch repair
- Mismatch detected by MutS which binds to the site
- MutS recruits other Mut proteins which scan the adjacent DNA for methylation to determine old vs new strand
- DNA is looped and unmethylated strand is cleaved
- Damaged strand removed and replaced with DNA pol I
Methods of pyrimidine dimer repair
Photoreactivation, nucleotide excision repair
Photoreactivation
Photolyase binds to pyrimidine dimer and cleaves the cyclobutane ring using energy from blue light; not present in placental mammals
Nucleotide excision repair
Endonuclease removes a patch of ssDNA containing damage
What proteins are responsible for nucleotide excision repair
Uvr proteins (ultraviolet radiation)
Steps of nucleotide excision repair
- UvrAB binds damaged DNA and bends it
- UvrC cleaves phosphodiester backbone around the area of damage
- damaged ssDNA is removed and replaced by DNA pol I
What causes xeroderma pigmentosum?
Defective nucleotide excision repair –> inability to repair UV damage
What damage does base excision pair correct?
Apurinic sites and cytosine deamination (C–>U)
Steps of base excision repair
- Either an abasic site is already present, or uracil is recognized and cut off to make an abasic site
- Abasic site recovnized and cleaved by a DNA endonuclease
- DNA pol I synthesizes replacement
What does recombinational repair fix?
Pyrimidine dimers
Where does recombinational repair take place?
At the replication fork, binds ssDNA
Steps of recombinational repair
- DNA pol skips over region with dimer
- RecA binds ssDNA of the sister strand to the dimer and scans neighboring daughter DNA for homology
- RecA catalyzes recombination (uses ssDNA from other daughter DNA to fill gap on damaged strand
- Gap in undamaged strand is repaired by DNA pol
What kinds of damage is SOS repair generally used for?
Extensive DNA damage, usually from excessive UV damage
Why does excessive UV damage trigger SOS repair?
Lots of pyrimidine dimers cause extensive stalling of replication forks
What is LexA?
Transcriptional repressor bound to the promoter regions of specific DNA repair protein sequences
How is SOS repair triggered?
- ssDNA accumulates in the cell due to DNA damage
- RecA binds ssDNA –> activates its coprotease activity
- RecA binds LexA and stimulates LexA to cleave itself
- Cleaved LexA has a low affinity for DNA anad falls off –> de-represses synthesis of SOS repair proteins
DinB
“Damage inducible”
- error prone DNA polymerase involved in SOS system
- aka DNA Pol IV
UmuCD
“UV mutation repair”
- error prone DNA polymerase involved in SOS system
What are error-prone DNA polymerases?
Polymerases that lack proofreading ability but can perform translesion synthesis
What proteins are upregulated during the SOS response?
SulA, UmuDC, UvrA, LexA, RecA
SulA
Inhibits cytokinesis by inhibiting FtsZ
- important for preventing cell division before DNA is replicated
Why is UvrA upregulated in the SOS response?
UvrA is involved in nucleotide excision repair, another method of fixing damage from pyrimidine dimers
