Chapter 7 DNA repair and mutations
Excision repair
-Base excision repair -Nucleotide excision repair (NER) -Methyl-directed mismatch repair -Translesion DNA synthesis
Types of mutaions
-Base-pair substitution -Base-pair additions/deletions (Indels) -Duplication -Deletion -Translocation -inversion
Direct reversal or mistake
-DNA Polymerase proofreading -Photoreactivation (photolyase) -Alkylation damage repair
Spontaneous chemical changes (deamination)
-Spontaneous Chemical Change -Amino group is removed from a base -Causes C to turn into a U -If this happens to 5mC (common in bacteria and eukaryotes), 5mC turns into a T -Eventually causes a CG to TA transition mutation (base-pair substitution)
Spontaneous chemical changes (Depurination)
-Spontaneous chemical change -Purine base breaks of phosphate-sugar backbone -"apurinic site" -DNA template now lacks a base -Means further rounds of DNA replication are impossible unless repaired •If repaired incorrectly, may cause base-pair substitution
tautomeric shifts
-Spontaneous during DNA replication -Chemically, each base can exist in an alternative state. -This change is referred to as tautomeric shift. -Normal bases (keto form) sometimes shift into "weird" 3-D shapes (enol form > rare) -Messes up H-bonding/base pairing properties (Watson & Crick base pairing) -Shifted base now base-pairs with the wrong base = base-pair substitution -Wrong base gets incorporated into daughter DNA
Small indels
-Spontaneous during DNA replication -Occur because of displacement. -Usually due to looping trouble -Often in tandem repeat regions •Looping of template strand results in skip by machinery >deletion in new DNA strand •Looping of new DNA strand > insertion in new strand -May cause frameshift mutations!
prokaryotic transposable elements
-insertion (IS) elements first identified in E.coli -Transposons (Tn) -composite -noncomposite
Barbara McClintock
1983 Nobel Prize •Studied transposons in maize (corn) •Discovered Ac/Ds family •Autonomous Ac (activator element) can hop in and out of a gene by itself (unstable mutations) •Nonautonomous Ds (disassociation element) can hop only when near Ac (more stable mutations, although some reversions possible) •Both Ac and Ds Have IRs, but only Ac has transposase
Prokaryotic transposons (composite) Tn10
Contains extra genes, e.g. conferring antibiotic resistance -Extra genes flanked by entire IS elements -IS elements supply transposase and their IRs (inverted repeats) -Transposase recognizes the IRs to begin transposition of the entire composite transposon -Does cut and paste ("conservative"transposition) -Causes target site duplications
spontaneous mutations
Occur during replication, and other stages of cell growth and division. •Can also occur from transposable genetic elements •In humans, mutation rate for individual genes varies between 10^-4 to 4^-6 per gene per generation. •For eukaryotes in general, the spontaneous mutation rate is 10^-4 to 10^-6 6 per gene per generation. •Bacteria and phages, 10^-5 to 10^-7 per gene per generation. •Many are corrected by immediate proofreading during DNA replication or by other cellular repair systems.
translesion DNA synthesis
This is considered the last resort process. •Lesions that block the replication machinery can be lethal if unrepaired. •This repair mechanism allows replication to continue past the lesions. •For this, special DNA polymerases are synthesized for this. •In E. coli, it is called SOS- emergency response. ( two key genes-LexA and recA) •When there is no damage, LexA represses the transcription of proteins involved in the repair •In lieu of damage, RecA is activated and stimulates Lex A to cleave itself, which relieves DNA repair genes
neutral mutation
a base pair change in a gene that changes a codon in the mRNA that results in no detectable change in function of the translated protein
transition
a mutation from one purine-pyrimidine base pair to the other (A-T to G-C)
nonsense point mutation
gene mutation in which a base pair change causes a change mRNA codon for amino acid to a STOP codon -UAA -UAG -UGA
missense point mutation
gene mutation in which base pair change cause a change in mRNA codon that renders a differ amino acids into polypeptide chain
suppressor genes
genes that cause suppression of mutations in other genes
mutagens
interact with DNA and cause a mutation -Occur at a higher frequency than spontaneous -Very useful in genetic studies
mutation rate
probability of a particular mutation over time -ex. # of mutations per nucleotide of occurences of a mutation -or # per gene per generation
true reversion
reversion result back to wild type amino acid
partial reversion
reversion result in another amino acid
errors during DNA replication chemical changes transposable elements
sources of spontaneous muatations
tautomeric shifts and small indels
Give two examples of spontaneous errors during DNA replication.
Mutation rate mutation frequency
How are mutations quantified
nucleotide excision repair
A.k.a as dark repair •A complex of proteins find TT dimers or other DNA distortions •The proteins cut 7 bpupstream and 4 bpdownstream of the mistake (around the mutation) •DNA Polymerase I uses opposing strand as a template •Backbone gaps sealed by DNA Ligase •We have this!
photoreactivation
A.k.a. light repair •Fixes UV-induced TT dimers •Light activtes photolyase enzyme •Photolyase un-crosslinks TT dimers •Found in bacteria and simple eukaryotes, NOT humans
Prokaryotic transposons (noncomposite) Tn3
Also have extra genes -Do not have IS elements -Do have IRs required for transposition -Co-integration transposition mechanism (fusion) -requires transposase and resolvase -Does copy and paste ("replicative"transposition) -Causes target site duplications
alkylation damage repair
An Alkyl group ( usually methyl or ethyl group) is added to a base. •Methyltransferases can remove extra methyl groups off G's or T's •In e.coli, the enzyme is O6- methylguanine methyltransferase. •Removal of the alkyl group changes base back to its original form.
mutation
Any change in in DNA sequence is considered what
transposable elements
Can be a result of spontaneous mutation. •Transposable element- a DNA sequence that can change its position within the genome, sometimes creating or reversing mutations and altering the cell's genome size. Can wreak havoc: •Insert into/disrupt genes •Insert into gene regulatory sequences (up or down regulate cell's /genes' expression) •Transposition mechanism itself can cause chromosomal breakage •Tend to cause unstable phenotype (hallmark) •Thought to be viral relics
Direct reversal of mistake Excision repair-affects one strand and is excised out
DNA repair mechanisms
Eukaryotic tranposons Class 2
DNA transposons -Uses transposase not RNA -First discovered by Barbara McClintock in maize -Cut & paste
Xeoderma Pigmentosum
Genetic disorder •Unable to repair mutations caused by UV or chemicals (no excision repair) •Light causes intense freckling, (malignant) skin cancers •Must live life in darkness
methyl-directed mismatch repair
Fixes DNA replication mismatches (in new DNA strand) not corrected by proofreading •In prokaryotes, DNA is methylated soon after it is synthesized: cell figures mismatched base in unmethylated DNA must be incorrect (daughter strand). •In E. coli- Mut genes are used (Muts S,L,H) •Unclear how eukaryotic cells spot new strand because there is no methylation involved (homologs in humans hMSH genes) •Mutations in human methyl-directed mismatch repair genes cause cancer: Hereditary NonPolyposis Colon cancer
Human retrotransposons E.g. L1 LINE element
Long INterpersed Element -50,000-100,000 copies of L1 element for total of 21% of human genome -May be retrotransposons -Insertion of L1 into factorVIII blood clotting gene causes hemophilia in children.
Base analogs (chemical mutagen)
Most common is 5-bromouracil (+bromine instead of methyl group) -Can shift into unusual tautomers -messes up H-bonding between bps, causing mispairing > Base-pair substitutions
Eukaryotic tranposons Class 1
Most common kind -includes retrotransposons •Transpose through an RNA intermediate •Encode a reverse transcriptase (RNA > dsDNA) •Descendents of retroviruses •Uses integrase to insert into the genome. -Copy & paste
Cockayne syndrome (CS)
Rare autosomal recessive disorders that feature growth deficiency, premature aging, and pigmentary retinal degeneration along with a complement of other clinical findings. •Results in early death •Precise molecular defect is unknown but may involve transcription-coupled repair
Base excision repair
Remove either damaged base or whole nucleotide •Uses a repair glycosylase to remove damaged base •Cleaves bond between base and sugar. •Fixes depurination or deamination mutations •After nucleotide removal, DNA Polymerase I and DNA Ligase use the opposite strand as a template to insert the correct base
Human retrotransposons E.g. Alu family of SINEs
Short INterspersed Element -300 bp repeated 300,000-500,000 times for total of up to 13% of human genome -Each Alu sequence flanked by 7-20 direct repeats -E.g. Alu seq found in NF intron, causing longer mRNA and (nonfunctional protein)
Prokaryotic (IS) elements
Simplest form found in bacteria. -only encode transposase (genes-enzymes required for transposition-mobilization) -carry no "extra" genes -have inverted terminal repeats (IRs) of 9-41 bp (essentially the same sequence is found) -copy and paste mechanism ("replicative transposition") -pasting •causes staggered cut at integration target site •IS inserted •gaps filled with cellular DNA Polymerase and DNA ligase •causes target site repeat
Dna polymerase proof reading
backspace button: 3' to 5' exonuclease activity re-write new DNA with 5" to 3' polymerase activity
point mutations
base-pair substitution base pair insertion and deletions (INDELS) forward and reverse mutations
intra/intergenic
both operates to decrease or eliminate the deleterious effects of the original mutations
autonomous Ac activator element
can hop in and out of a gene by itself (unstable mutations)
nonautonomous Ds disassociation element
can hop only when near Ac (more stable mutations, although some reversions possible)
reverse mutation
change a mutant type --> wild type
forward mutations
change wild type to a mutant gene
silent mutations (synonymous mutation)
changes a base pair in a gene, but the altered codon in the mRNA specifies the same amino acid in the protein. (Different codon=same amino acid) -Most often occurs as a result of wobble position (third base position)
intergenic
different gene
induced mutations
physical mutagens: ionizing radiation (X, alpha, beta, y rays) Uv light Chemical mutagens
base-modifying agents
modifies the chemical structure and properties of bases which affects base pairing) •Hydroxylating agents (add -OH) > on cytosine to pair with adenine •Alkylating agents (add e.g. -CH3) •Deaminating agents (remove -NH3)
suppressor mutation
mutation at a different site from that of original mutation -this mutation will mask or compensate for the effects of the initial mutation Intragenic intergenic
transversion
mutation from one pyrimidine-purine base pair to the other (G-C to C-G)
spontaneous or induced
mutations can be ________ or _________
spontaneously
naturally occurring mutations occurs
point mutation
no mutation silent nonsense missense (conservative and nonconservative) see figure.
mutation frequency
number of occurrences of a mutation, expressed as the number of cells or individuals in a population.
intragenic suppressor mutation
occur at a 2nd codon within same gene -First amino acid change is partly counteracted by a change in a second amino acid codon in the same protein. -More commonly, an insertion frameshift may be suppressed by nearby deletion, or vice versa.
intergenic suppressor mutation
occur in 2nd gene -Usually work by changing mRNA translation -E.g. a nonsense supressor gene is really a tRNA with mutated anticodon -The mutated anticodon recognizes the aberrant stop codon (forward mutation) -Delivers a whatever amino acid is attached to the tRNA, overcoming the forward mutation and allowing translation to continue
somatic
occur in body cells affect individuals only (Not passed on)
germline
occur in gametes (affect the next generation) heritable
frameshift point mutation
occurs when one or more bases are deleted or added to the reading frame causing a shift of the DNA coding sequence. -Usually results in a nonfunctional protein. -Tend to generate new stop codons. -Incorporation of incorrect amino acids (causing missense mutation) -Bring stop codons into reading frame causing too-short protein (causing nonsense mutation) -Read-through of stop codons to make too-long protein
intercalating agents (EtBr) chemical mutagens
tends to cause insertion of 1 or more bases > potential frameshifts -Wedge themselves between base pairs, causing distortion -Can result in the addition of a new base. (base-pair addition) -Interferes with Topoisomerase II action -Leaves a nick > insertion -e.g. proflavin, acridine and ethidium bromide.
base-modifying agents base analogs intercalating agents
these are chemical mutagens
transition and transversion (base-pair substitution)
these mutations can effect proteins if it occurs in the protein coding genes
depurination and deamination
what are two types of spontaneous chemical changes
induced mutations
when one is exposed either deliberately or accidentally to a physical or chemical agent known to be a mutagen
intragenic
within the same gene
UV light
•Formation of abnormal chemical bonds between adjacent pyrimidine molecules in the same strand •Causes chemical crosslinking between neighboring T's •TT dimer formed •Distorts double helix ("scar") •Distorted area cannot serve as template for DNA replication -Cell dies unless TT dimer removed