Exam IV Review Chapter 18

Chapter 18 Mutations and DNA repair Importance of fidelity of DNA replication ? If DNA is not replicated corrected, it has effects on transcription, mRNA processing, the mRNA stability, effects on translation, and it has effects on post-translational protein processing. Definitions Somatic mutations ? These types of mutations cannot be transmitted to the next generation, but can be used through vegetative propagation. Gametic mutations ? These types of mutations are passed on to 50% of the offspring. All cell of the offspring will carry the mutation. Transition ? Is the most common type of base substitutions. A purine is replaced by another purine and a pyrimidine is replaced by another Pyrimidine. Transversion ? During this type of base substitution a purine is replaced by a pyrimidine and a pyrimidine is replaced by a purine. Insertion ? an extra nucleotide is inserted into the sequence altering the reading frame and may change many codons. Deletion ? a nucleotide is deleted from the sequence also altering the reading frame and it also may change many codons. Expanding tri-nucleotide repeats ? This is the cause of several human genetic diseases. In this mutation copies of a trinucleotide are increased greatly causing a fragile chromosome. Nonsense mutations ? changes a sense codon into a nonsense codon (one that terminated translation). If this type of mutation occurs early on, the created protein is much shorter and generally useless. Missense mutations ? changes a codon in the mRNA, resulting in a different amino acid in the protein. Silent mutations ? alters a codon but, thanks to the redundancy of the genetic code, the codon still specifies the same amino acid. Mutation rate ? this is the frequency of changes in one biological unit (may be per cell division, per gamete, or per round of replication). (For example 4 mutations in 100,000 gametes = 4x10-5). Mutation frequency ? the incidence of a specific type of mutation within a group of individual organisms. (1 out 20,000 individuals = 2x10-4) Causes of mutations Biological: mistakes during replication ? they are also called spontaneous mutations. Some biological agents can also cause mutations as their genome can be integrated into the host?s DNA. Strand slippage ? this occurs when one nucleotide strand forms a small loop. If the looped-out nucleotides are on the newly synthesized strand, and insertion results. If they are not then deletion is the result. Expanding trinucleotide repeats (special case of strand slippage) ? copies of a trinucleotide are increased by a mutation which causes a hairpin loop to form, which then results in instability of the chromosome. Unequal crossing over ? caused by misaligned pairing. This results in one DNA molecule with an insertion and the other with a deletion. Duplicated or repetitive sequences are more likely to misalign during pairing than stretches of repeats (trinucleotide repeats or homopolymeric repeats), which are more prone to strand slippage and expanding trinucleotide repeats. Mismatch because of non-standard basepairing ? this can occur as a result of the flexibility in DNA structure. Thymine and guanine can pair through Wobble between normal bases. This is possible because of the flexible nature of the DNA helical structure. Normal, protonated, and other forms of the bases are able to pair in the flexible helical structure of the DNA. Chemical: Chemicals that react with DNA (alkylating agents, etc.) Alkylating agents donate an alkyl group to nucleotides and form analogs. This affects the base pairing, which leads to transitions. Some examples include Mustard gas, Ethylmethane sulfonate (EMS), and Diethyl Sulfate (DES). For example EMS cause a transition from T-A C-G. This example is reversible by simply adding more EMS. Deamination the Cytosine is transformed to Uracil using nitrous acid, which also lead to T-A C-G. Nitrous acid also changes adenine into hypoxanthine, which pairs with Cytosine, leading to T-A C- G. Hydroxylamine is a very specific base-modifying mutagen. It adds a hydroxyl group to cytosine. This conversion increases the frequency of a rare tautomer that pairs with adenine instead of Guanine. Because it only reacts with Cytosine the reverse reaction cannot be generated. Chemicals that pose as nucleotides (base analogs) ? These mutagenic chemicals can substitute for purines or pyrimidines. 5-BU is a thymine analog and can pair with G. 2-AP is an adenine analog and can pair with C. They lead to tautomeric shifts and transitions. Intercalating agents ? are agents that cause frameshift mutations. They include Acridine orange, ethidium bromide, and proflavin. Physical X-rays (chromosome breakage) ? the high energy radiation ionizes water, this forms free radicals, which are highly reactive molecules that can cause single and double stranded breaks. Single stranded breaks are repairablem whereas double stranded breaks can lead to chromosome rearrangement and base modification. UV-radiation (thymidine dimmers) ? this is lethal!! ? it causes pyrimidine-dimers that block replication. It links adjacent pyrimidines on a DNA strand. These dimers do not base pair normally, which leads to stalled replication, and misincorporation during replication, which then leads to microbial death. Existence and mechanisms of different DNA repair mechanisms ? This avoids the accumulation of mutations. Proof reading as a way to minimize mistakes ? this is a function of certain DNA polymerases. They have 3? to 5? exonuclease activity and recognize specifically unpaired bases. They also recognize lack of or improper hydrogen bonding. It works prior to replication as this is the only way mutation can be prevented. Mismatch repair ? the errors not corrected by proofreading cause a shape change of the DNA, this activates the mismatch repair mechanism. It removes the distorted segment and re-synthesizes the corrected segment. Example from E.coli; methylated A in GATC sequence for template strand recognition, followed by replacement of section with mismatch ? The repair enzyme recognizes the unmethylated daughter strand which contains the error and preferentially binds to the unmethylated strand and repairs the DNA. Base excision ? classic mismatch repair. It recognizes and replaces modified or incorrect bases. They have minor effects and do not block DNA replication or transcription Local repair ? works on the base only Base is removed, than ribose and phosphate, then fill in ?the process is completed in 5 steps. Modified base is identified and excised (DNA glycosylase) endonuclease cuts the phosphodiester bond and removes the deoxyribose DNA polymerase fills the gap Complementary to the intact strand Prokaryotes: DNA polymerase I Eukaryotes: DNA polymerase beta DNA ligase seals the nick Nucleotide excision ? Mechanism to repair lesions that distorts the shape of the double helix caused by dimers, damaged DNA, and such lesions that impede DNA replication and transcription. It is found in all mechanisms. Regional repair Detection of disturbed double helix and repair of section of DNA around the mistake ? Direct repair ? Its goal is to return the damaged bases to their ?original? state. It involves a series of enzymatic reactions. Reverting chemical changes without replacement (photolyase in E.coli) ? first the methyl groups are removed by methyltransferase. Then the pyrimidine dimers are undone via ?Photoreactivation repair?. This reaction is blue light dependend. Photolyase is a photoreactivation enzyme. This enzyme associates with the dimer in the dark. After absorbing a photon of light, the thymine dimer bond is broken and normal base paring is restored.