1. 9/21/2010 1 DNA REPAIR AND MUTATION Mutations and mutants Mutation: genetically inheritable change in one or more genes Change in DNA sequence Often leads to change in function of gene product Wild-type (wt): normal (not mutated) Mutant: organism that has a change (mutation) in its genome

2. 9/21/2010 2 Mechanism of Mutation Background (or spontaneous) – Chemical instability of bases in DNA – Errors in DNA replication Induced – Physical (ultraviolet light or ionizing radiation) – Chemical (chemical carcinogens) Spontaneous Mutation Rate Rate differs for different genes – Size dependence – Sequence dependence – Hot spots On average 1 in 100,000 chance of acquiring a mutation in a gene each round of replication. Each individual has multiple new mutations.

3. 3 9/21/2010 Induced Mutations Chemicals and radiation can cause mutations. Chemicals causing mutations are called mutagens. Chemicals causing cancer are called carcinogens. Alkylating agents Acridine dyes Xrays UV radiation remove a base add or remove base break chromosomes delete few nucleotides creates thymidine dimers Point Mutation A point mutation is a change of a single nucleotide to one of the other three possible nucleotides Transition purine replaces purine A G or GA pyrimidine replaces pyrimidine CT or TC Transversion purine replaces pyrimidine or pyrimidine replaces purine A or G T or C A or G

4. 9/21/2010 4 Missense Mutation A point mutation that exchanges one codon for another causing substitution of an amino acid Missense mutations may affect protein function severely, mildly or not at all. Hemoglobin mutation glutamic acid valine causes sickle cell anemia Nonsense Mutation A point mutation changing a codon for an amino acid into a stop codon (UAA, UAG or UGA). Premature stop codons create truncated proteins. Truncated proteins are often nonfunctional. Some truncations have dominant effects due to interference with normal functions.

5. 9/21/2010 5 Insertion or Deletion Mutations The genetic code is read in triplet nucleotides during translation. Addition or subtraction of nucleotides not in multiples of three lead to a change in the reading frame used for translation. Amino acids after that point are different, a phenomenon called a frameshift. Addition or subtraction of nucleotides in multiples of three leads to addition or subtraction of entire amino acids but not a change in the reading frame. Not all mutations impact protein function Silent mutations are mutations that do not alter the amino acid encoded. AAA and AAG both encode the amino acid lysine. A mutation from AAA to AAG in a gene alters the DNA sequence but protein sequence remains unchanged. These codons are called synonymous codons.

6. 9/21/2010 Not all mutations impact protein function Missense mutations are those that alter the encoded amino acid to another amino acid. The alteration creates a nonsynonymous codon. Some nonsynonymous mutations are conservative; chemically similar amino acid may not alter function The impact of a missense mutation is not predictable from protein sequence alone. DNA Repair Errors in DNA replication or damage to DNA create mutations. Most errors and damage are repaired by the cell. The manner in which DNA repair occurs depends upon the type of damage or error. Different organisms vary in their ability to repair DNA. In humans, mutations in DNA replication occur in1 in 100 million bases. 6

7. 9/21/2010 7 DNA Repair There are many different sources and types of DNA damage and the cell contains many different systems to survey for and correct DNA damage. Mismatch repair Excision repair Inducible repair Evidence of DNA Repair No repair DNA damage Repair

8. 9/21/2010 8 Deaminating Agents Hydroxylamine: Removes amino group of cytosine -------  GC to AT Bisulfite: Deaminate only Cytosine --------  Site-directed mutagenesis Nitrous Acid: Deaminates Cytosine Adenine, and Guanine --------  GC to AT AT to GC Base deamination: Potentially Mutagenic DNA damage How to Repair?

9. HypoxanthineCytosine 9/21/2010 Deamination of Adenine to Hypoxanthine leads to mispairing with Cytosine Failure to repair a deaminated base leads to point mutation 9

10. 9/21/2010 10 Failure to repair abasic sites leads to deletions DNA Glycosilase: break glycosyl bond between damaged base and the sugar in the nucleotide AP Endonuclease: cut sugar-phosphate backbone of DNA Apurinic site-----A or G Apyrimidinic site------C or T

11. 9/21/2010 11 Deaminated bases are repaired by a base excision mechanism. Spontaneously occuring abasic sites are repaired by the same mechanism

12. HHHH N NHNH NH 2 HOPHOP HOHO O O- O HHHH HN NO HOHO HOPOHOPO O O- O 9/21/2010 O HHHH HOHO H O P O- O HHHH dCMP dUMP H 2 O NH 3 U O H N glycosylase HHHH HOHO HOPOHOPO O O- O HHHH endonuclease cleavage Deamination O Alkylation Ethyl methanosulfonate (EMS) Methyl Methanosulfonate (MMS) N-methyl-N’-nitro-N-nitrosoguanidine (nitrosoguanidine) Repair: *N-Glycosylase remove alkylated base. *Apurinic/apyrimidinic DNA strand is cut by AP endonuclease *Exonuclease degrade the cut strand *DNA Pol I resynthesized correct DNA strand Also: by methyltransferase----remove alkyl 12

13. N N O NH 2 N O H HH HH O PO O- HO O- CH 3 NH O 6 M eGuanine nucleotide NH N N O NH 2 N O H HH HH OPOP O HO O- 9/21/2010 A lkylating agents O 6 alkyltransferase O 6 alkyltransferase-M e Direct Repair: Methyltransferase O- Adaptive Response Ada : methyltransferase----remove methyl or ethyl group Ada protein regulatory protein of adaptive respons genes alkA: N-glycosylase~remove alkilated base aidB: increases the resistance of the cells to some methylating agents. To detoxyfy alkylating agents alkB: increses the resistance of cells to some alkylating agents. Regulation of Adaptive Response A few copies of the Ada protein normally exist in the cell. DNA damage by alkylation------  Ada protein (Methyl Transferase) transfer alkyl group. -from damaged DNA phosphates to itself: activator -from damaged base, inactivating itself. 13

14. 9/21/2010 Pyrimidine Dimers UV irradiation-----Pyrimidine dimer DNA Damage Thymine dimer---  5-, 6-Carbon link to form a cyclobutane ring 6-Thymine-4 Cytosine DNA Repair: Photoreactivation UV light NH NHNH O O NHNH O O Cyclobutane dimer NH NHNH NH 2 O NH NHNH O O OH 6-4 photoproduct 14

15. 9/21/2010 15 The major form of damage caused by UV irradiation is Thymine dimers: Photoreactivation ~Enzyme: Photolyase~contains FADH2 group. It absorbs light of wavelength between 350~500 nm. ~Bind and then separate the fused bases ~Light absorbsion: Photolyase separate the fused bases ~Other mechanism: N-Glycosilase; AP Endonuclease Resynthesize DNA: DNA Pol Photolyase

16. 9/21/2010 16 Chemical Mutagens Most chemical mutagens cause a modfications of the bases. Often these are small adducts (eg. methyl groups) but in the case of some of the more powerful mutagens, these adducts can be bulky. Also, many mutagens are polycyclic compounds that intercalate into the stacked bases of the double helix. E.g. benzopyrene

17. 9/21/2010 17 Nucleotide Excision Repair *Very efficient *Non-specific *Repair many types of damage *Very important: ability the cell to survive damage to its DNA *Repair all types of damage caused by UV irradiation: Cyclobutane dimers; 6-4 lesion; base-sugar cross-links. Mechanism of Repair?

18. 9/21/2010 18 Mechanism of Repair Enzyme: UvrABC *UvrA protein forms a complex with UvrB protein. *The protein complex bind nonspecifically to the DNA *This complex then migrates up and down the DNA until the DNA damage *UvrB binds to the damage, and UvrA leave. UvrA is replaced by UvrC *UvrC binds UvrB-----  UvrB cuts the DNA 4 nucleotides 3’ of the damage *UvrC cuts the DNA 7 nucleotides 5’ of the damage. *UvrD helicase removes oligonucleotide containing the damage *DNA Pol I resynthesizes the strand that was removed, using complementary strand as a template

19. 9/21/2010 19 Dimeric bases and bulky lesions, eg. large chemical adducts are repaired by Nucleotide excision repair Methyl-Directed Mismatch repair

20. 9/21/2010 Deamination of Cytosine creates a G-U mismatch Easy to tell that U is wrong Deamination of Cytosine creates a G-T mismatch Not easy to tell which base is the mutation. About 50% of the time the G is “corrected” to A resulting in a mutation Damage Due to Reactive O2 Superoxide radicals Hydrogen peroxide Hydroxyl radicals Normal Cellular Reaction UV irradiation Chemicals Hydroxyl radicals Induce genes for: Catalase Superoxide dismutase Peroxide reductase also: Genes to repair the oxidative to DNA caused by the reactive form of O2 Mutagenic lesion in DNA caused By reactive oxygen: 8-oxoG or GO N-Glycosylase Phosphatase 20

21. 9/21/2010 21 Another example: Aflatoxin Natural product produced by the fungus Aspergillus. General Repair Mechanisms Base Analogs Transition Mutation Methyl-directed mismatch repair system

22. 9/21/2010 22 Frameshift Mutagen Acridine dye (9-aminoacridine); Proflavin; Ethidium bromide; Aflatoxins (Produced by fungi) intercalate between bases In the same strand of the DNA Deletion of base pair Adding a base pair