Review of Translation, DNA Damage and DNA Repair

translation

a structurally different biopolymer (protein) is synthesized from RNA

Crick’s Adaptor hypothesis

Can folded RNA act directly as the template for protein synthesis? Seems unlikely: no clear way to discriminate chemically similar amino acids

transfer RNAS (tRNAs)

RNA molecules

Self-folding by complementary base pairs gives a structure with several functional domains

• clover shape

• end with CCA - where amino acid attaches

~10% of cellular RNA abundance

Typically includes several modified, non-standard bases (inosine, ψ, pseudouridine, etc.)

Genetic Code

AUG codes for N-formylmethionine (fMet) in prokaryotes or methionine (Met) in eukaryotes - start code

three stop signals - important for non-natural proteins

redundant

redundancy in genetic code

Trp - special and difficult amino acid → only one codon

Ribosome

50S subunit

• 2 rRNAs

• 34 proteins

30S subunit

• 1 rRNA

• 21 proteins

• binding site for mRNA (read 5' to 3')

Translation elongation

1. binding of specific aa-tRNA to A (amino-acyl) site

2. Peptide bond formation at P (peptidyl) site; chain tranfer from p-tRNA to aa-tRNA

3. translocation of p-tRNA from A to P

Elongation mechanism

Nucleophilic attack on the ester by the incoming amino end with tetrahedral intermediate

Occurs at the peptidyl transferase center (PTC)

codon-anticodon interaction

The first base of codon interacts with the third base of the tRNA anticodon

Synthetase accuracy

essential for fidelity of translation

post-charged modifications can be made - nonnatural amino acids to make aritificial proteins and peptides

Synthetase mechanism

1. amino acid + ATP = activated amino acid (aminoacyladenylate) + PPi

2. 3’ end tRNA + activated amino acid = charged tRNA + AMP

Aminoacyl-tRNA

3’ end of CCA

What can DNA be damaged by?

Temperature

Radical reactions

Nucleophiles

Electrophiles

Pi-stacking

Hydrogen bonding

Ionic interactions

monoclonal antibodies (MABs)

chemotherapeutic - bind only to cancer-cell-specific antigens and induce an immune response against the target cancer cell

Why is uracil only in RNA?

deamination of cytosine makes uracil → if U not removed, it will pair with A in replication → next replication will pair T with A → overall: C to T mutation

Endogenous DNA-damaging reactions

frequency in number of lesions per mammalian cell per day

1. depurination - A to abasic

2. deamination - C to U

3. oxidation - G and T

4. methylation - SAM/AdoMet → abasic site

mutations

Permanent change in DNA sequence

Can affect gene function

Can alter protein-coding sequence

• missense - different amino acid

• nonsense - stop codon

• silent - no change

Repair Mechanisms

1. Recognize the error

2. Remove it

3. Repair the gap with DNA polymerase and DNA ligase

Common Mutations

1. abnormal basepairs

2. chemical adducts

3. base pair mismatches

4. doublestrand breaks

5. single strand breaks

6. abasic site

7. thymine dimers

8. DNA crosslinks

9. nucleotide insertions

10. nucleotide deletions

Direct repair pathway

mistake fixed w/o removing neculeotide

base-excision repair pathway

one nucleotide is removed and fixed

nucleotide excision repair pathway

stretch of DNA removed and fixed

Spontaneous deamination of C to U

Improper complimentary base pairing; Generates an abasic site by glycosylase base removal

UV radiation (photoproducts)

thymine dimer; creates DNA strain

Repair of Thymine dimers

highly reversible reaction due to high strain - fixed by leaving sunlight

Environmental DNA-damaging agents

• radiation

  • ionising

  • ultraviolet

• DNA-methylating reagents

  • N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)

• DNA-cross-linking reagents

  • Cisplatin (chemotherapeutic)

• Bulky electrophilic agents

  • Benzo[a]pyrene (carcinogenic hydrocarbon in tobacco smoke)

Ames test

Several strains of the bacterium Salmonella typhimurium carry mutations in genes involved in histidine synthesis. These strains are auxotrophic mutants, i.e. they require histidine for growth, but cannot produce it.

The method tests the capability of the tested substance in creating mutations so that the cells can grow on a histidine-free medium.

benzopyrene damage

adduct on G-2-exoamino via reactive epoxide

minor groove

Sites of alkylation of DNA

69% of the time adds to G-N7