Genetics Ch18 DNA Mutations

mutation

sequence of base pairs in DNA is altered

somatic mutation

effect only seen in individual with mutation, found in somatic cells

germ-line mutations

may be transmitted generation to generation, mutation is in germ cells

Base substitution

one codon changed: GTA to GCA

Base insertion

goes in between 2 codons (extra base), slides sequence to the right: GTA to GTT AGA…

Base deletion

loses one codon (one less) slides over sequence to the left: GTA to GAG

Types of point mutations

base-pair substitutions (in DNA): transition and transversion

missense mutation

nonsense mutation

neutral mutation

silent mutation

frameshift mutation

Transition

pur-pyr bp is replaced by another pur-pyr bp

eg. AT-GC, GC-AT, TA-CG, CG-TA

Transversion

pur-pyr replaced by a pyr-pur bp

8 total; eg. CG-GC, AT-TA

Missense mutation

different aa inserted into protein (1st bp in codon seq): AT to CG (transition)

Nonsense mutation

premature STOP codon in coding sequence (1st bp in codon seq) AT to TA (transversion)

Neutral mutation

changes in aa but no change in protein function (2nd bp in codon sequence): AT to CG (transition)

Silent mutation

changes in DNA and codon, but not protein structure (3rd bp in codon sequence): AT to CG (transition)

code for the same amino acid!

Frameshift mutation

insertion or deletion of one codon causing changes in the mRNA reading frame (and the future codons/aa)

Classification of point mutations

Forward mutations

Reverse mutations (reversions)

Supressor mutations

Forward mutations

wt to mutant

Reverse mutations (reversions)

mutant to wt

true reversion: back to wt aa

partial reversion: change to another aa that may restore partial function

Supressor mutations

A second mutation occurs that masks or counteracts the effect of the first mutation, even if the original sequence isn't restored

intragenic: same gene

intergenic: different gene

Spontaneous mutations

naturally occurring, found on all types of point mutations

All need a subsequent round of replication to pass from gen to gen

Replication errors: tautomeric shift, wobble pairing, indels

Spontaneous chemical changes: depurination and deamination

Tautomeric shift

change in H-bond pairing: pur + pur and pyr + pyr

common (keto/amino) and rare (enol/imino) forms

Wobble pairing

flexible pairing at the third position of the codon (G pairs with U/T/C)

Insertions/deletions (Indels)

looped out strand, replication still proceeds

unequal crossing over causes indels

Depurination

the loss of a purine base (A or G) from a DNA strand

happens through hydrolysis (water-mediated events)

Deamination

removal of an amino group from a nucleotide base, which convert one base to another

e.g. cytosine to uracil or 5-methylcytosine to thymine

happens through hydrolysis (water-mediated events)

Induced mutations

deliberate or accidental exposure to a physical or chemical mutagen (damages DNA, causing mutations)

e.g. ionizing radiation, UV light, base-modifying agents, and intercalating agents

Ionizing radiation

breaks covalent bonds in the sugar-phosphate backbone of DNA (physical breaks in DNA)

linear relationship between dosage and point mutations

e.g. X-rays

UV light

Typically, mutations on adjacent pyrimidines causing cyclopyrimidine dimers (adjacent thymines and cytosines)

T^T (thymine dimers) most common

e.g skin cancer

Base modifying agents

modify chemical structure and properties at bases

e.g alkylating agents

EMS mutagenesis results in CG to TA or TA to CG mutations

Intercalating agents

Insert themselves between DNA pairs and cause frameshift mutations, which:

distorts/causes helix to relax and leads to the addition or deletion of nucleotides during DNA replication

e.g. proflavin, acridine, bromide

DNA damage repair

mismatch repair by DNA polymerase

can be direct or damaged DNA removed and then gap repaired

Direct DNA damage repair

error-free mechanism where a single enzyme directly reverses certain lesions:

repair of UV damage or repair of alkylation damage

Repair of UV damage

Photolyase can directly reverse covalent bond formation caused by UV light

Reverses cyclopyrimidine dimer formation, restoring original bases

Prokaryotic specific mechanism

Repair of alkylation damage

Alkylating agents add alkyl groups (like a methyl group) to guanine at C6

O6-methylguanine methyltransferase removes CH3 groups and transfers cysteine residues in protein (rendering it non-functional)

Methylase “flips” a base outside the DNA helix (suicide enzyme)

Gap repair of DNA damage

filling single-stranded gaps left after lesions block replication through: BER, MDMR, NER, and SOS in prokaryotes,

BER (base excision repair)

removal of base, followed by removal of sugar phosphate backbone

gap repaired by DNA pol and ligase

MDMR (methyl-directed mismatch repair)

can recognize incorrect bases, excise, and replace

mutS, mutL, and mutH are required in E. coli

DNA in E. coli is methylated at adenine in GATC sequences

following replication, DNA is hemimethylated

Me-directed mismatch repair uses this difference

MutS

protein that binds to the mismatch (looking for DNA damage)

forms a complex with mutL and mutH to bring the unmethylated GATC close to the mismatch

MutH

nicks the unmethylated DNA strand, and an exonuclease excises a section of the new DNA strand, including the mismatch

Helicase removes gaps in strand

DNA pol III and ligase repair the gap, producing the correct base-pair

NER (nucleotide excision repair)

removal of segment

identification of uvr genes

uses UvrABC system (uvrA, uvrB, uvrC) and uvrD helicase to fix bulky lesions like UV damage

DNA pol I synthesizes new DNA to fill the gap using the undamaged strand as a template and DNA ligase seals the final nick

uvrA and uvrB

damage recognition, scans DNA and finds thymine dimer

uvrC and uvrD

uvrC acts as an endonuclease making cuts on same side of double helix creating a gap

uvrD (helicase) helps unwind the DNA and release uvrC

SOS response

global stress response (worst case scenario)

translesion DNA synthesis

allows for replication past normal blocks due to damage

recruitment of DNA pol IV (Din B) and V (UmuD’2C), which interact with the β-clamp

controlled primarily by the lexA and recA proteins

lexA

transcriptional repressor (OFF state) that binds to promotor

recA

activator that forms a filament on single-stranded DNA that triggers the cleavage of the lexA repressor