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