Mutations & DNA Repair

1. Mutations & DNA Repair • What are mutations? • Mutagenesis: Process of producing a mutation • Repair of mutations

2. Mutations can cause changes in the shape of a protein which alters its function.

3. What are mutations? • Classes of mutations: • Spontaneous mutation - occurs in nature without the addition of a mutagen • Induced mutation – caused by a mutagen • Point mutation – change of 1 nucleotide • Insertion/Deletion – base added or deleted • Frameshift mutation – loss or addition of a nucleotide alters the codon reading frame • Forward mutation – converts wild type to mutant • Reverse mutation – converts mutant back to wild type • Loss of function mutation • Gain of function mutation

4. Base substitutions/Point mutations • Transitions • Purine replaced by a purine… or pyrimidine replaced by a pyrimidine • Transversions • Purimidine replaced by a purine, or vise versa • Less common… why? T & C A & G

5. Additional mutational categories: • Lethal mutation – results in death • Conditional mutation – expression depends on the environment • i.e. temperature sensitive mutations • Somatic mutation – not transmitted to future generations • Germinal/gametic mutation – transmitted to offspring

6. Mutational outcomes • Silent substitution – function of the protein product of gene is unaltered • Missense mutation – alters codon so that it encodes a different amino acid • Nonsense mutation – alters gene so that it creates a nonsense codon (no normal tRNA exists) causing termination of translation

7. Spontaneous mutations • Spontaneous mutations arise from replication errors & base modifications • DNA Replication errors • Replication slippage – one strand loops out and becomes displaced during replication • DNA pol stuttering • Occurs frequently in repeat regions: Hot Spots for DNA mutation • Spontaneous mutation rate various among organisms (table 15.2)

8. Ways Mutations can occur: -Replication Error -Breaks in DNA strands -Damage to nucleic acids -Mobile elements II. Mutagenesis: Process of producing a mutation

9. Induced Mutation Mechanisms • Base replacement • Base alteration • Base damage

10. 1. Base replacement • Base analogs(chemicals that are similar to nucleotides) substitute themselves for the nucleotide • Result = improper base pairing • Examples: a) Tautomeric Shifts - Tautomerization – isomerization of a nitrogen base to an alternative H-bonding condition b) Chemicals: 5-Bromocuracil (T analog), 2-Aminopurine (A analog)

11. Tautomerization – Known as a tautomeric shift “rare” forms result in mispairing, .

12. Mispairing results in replication errors – the wrong bases are incorporated into the daughter strands

13. 5 BU (derivative of uracil) behaves as a thymine analog, if 5 BU is incorporated it will base pair with guanine, after 1 round of replication an A-T to G-C transition results

14. 2. Base alteration • Chemicals cause the shape of the nucleotide to change, resulting in improper base pairing • Depurination (loss of nitrogenous base) & Deamination (amino group converted to keto group) • Alkylation – addition of alkyl group (CH3 or CH3CH2) to bases • EMS (ethylmethane sulfonate) • Intercalation – planer molecules that mimic base pairs and slip themselves between the stacked nitrogen bases at the core of the helix • Ethidium bromide • Proflavin • Acridine orange

15. EMS alkylates the keto groups of G and T, base pairing is altered and a transition results

16. Intercalating agents slip between the nitrogenous bases, which can lead to insertion/deletions. Frameshift mutations result – generated at gaps produced in DNA during replication

17. 3. Base damage • Chemicals, oxidation, radiation cause the nucleotide to become modified in such a way that it can no longer base pair • UV light • Results in pyrimidine dimers • Radiation • Causes ionization of molecules • Creates substitutions • Breaks phosphodiester bonds

18. Spontaneous Mutations– arise due to natrual biological/chemical processes: • DNA replication errors • DNA replication errors = each of the bases can appear in one of several forms called tautomers (isomers) • Spontaneous lesions • Depurination - Apurinic sites can’t specify a base complementary to the original • Deamination = ie deamination of C yields U, which will pair w/A leading to a GC to AT transition • Oxidative damage – superoxide radicals (byproducts of metabolism) alter bases to cause mispairing… 8-oxidG or GO pairs with A • Transposable elements • significant part of the genome consists of “nomadic” DNA sequences that are present at different locations

19. III. Repair of mutations • Direct Reversal of damage • Excision repair • Proofreading • Mismatch repair • Post-replication repair & SOS • Double-strand break repair

20. 1. Direct reversal of damage Photoreactivation repair: reversal of UV damage • Photolyase splits Thymine dimers, restoring DNA to its original condition • Photolyase works with cofactor folic acid • The two bind together in dark to T-dimer • When light shines on cell –folic acid absorbs the light & uses the energy to break the covalent bond between T’s O6-mGua DNA methyltransferase Alkyltransferase – one time repair enzyme that removes ethyl or methyl groups from guanine

22. 2. Excision repair Involved in repair of deamination and depurination Enzymes recognize an abnormal base and cleave the bond between in and the sugar in the DNA backbone. • Uracil N-glycosylase • removes uracil • AP endonuclease • cuts 5’ side of damaged site on apurinic bases • Phosphodiesterase • Removes sugar-phosphate residue

23. deamination AGTGACTTAG TCACTGAATC AGTGACTTAG TCAUTGAATC Ligase Uracil N-clycosylase U AGTGACTTAG TCA TGAATC AGTGACTTAG TCA TGAATC Pol I

24. 3. Proofreading DNA Pol III error rate: 10-5 • Proofreading ability: Pol II can recognize mismatched base pairs, determine which base is the incorrect one excise the wrong base and carry out repair synthesis • 3’ to 5’ exonuclease ability, lowering the error rate to 10-7

25. 4. Mismatch repair • Mismatch repair – after proofreading, mismatches identified, improper base excised and replaced w/correct base • Adenine methylase recognizes parent strand and adds methyl group to A’s • Unmethylated daughter strand recognized by repair enzyme

26. Mismatch repair- Important to recognize difference between old strand & new strand: If mutated base excised, the wild type is restored, but if the original wild type is excised, the mutant sequence becomes fixed.

27. 5. Post-replicational repair & SOS Post-replicational repair (aka recombination repair ): • Damaged DNA cause Pol III to “stutter” and skip past damaged site • Replication restarts downstream and a gap is left • Gap is repaired by retrieving sequence from the normal copy and then the subsequent gap is repaired

29. SOS response • Severe damage due to alkylating agents or cross-linking agents (UV radiationbest studied) triggers this response • Translesional polymerases (POL II, IV, V) can replicate over damaged regions • Has very high error rate: 10-2, however allows for survival under extremely bad conditions

30. 6. Double strand break repair • Repairs DSBs by reannealing the two DNA segments - protein aligns the broken ends of DNA for rejoining • Recombination repair mechanism • Homologous recombination repair – damaged DNA replaced by homologous DNA section from sister chromatid • Nonhomologous recombination repair – uses non-homologous region for replacement • Errors in direct joining may be a cause of translocations