Chapter 11

11.5: Proteins Control Translesion DNA Synthesis and the Repair of Double-Strand Breaks • By what types of DNA damage is translesion DNA synthesis triggered? • What enzyme is responsible for this repair? What is the consequence of translesion DNA synthesis? • If double stranded DNA breaks (DSBs) are not repaired by the cell, what are the possible consequences? • Compare and contrast NHEJ and SDSA, including when each happen and the possibility of errors introduced by each. • Describe how SDSA uses strand invasion, the formation of a D loop, and DNA polymerase to produce a sister chromatid based on the other. • What does a D loop look like for DNA polymerase?

???

• If mutations are not directed by the environment, how is it that environmental factors can cause mutations?

???

11.6: DNA Double-Strand Breaks Initiate Homologous Recombination • When does homologous recombination take place in eukaryotes? Prokaryotes? • Describe the process by which homologous recombination occurs in eukaryotes, including the formation of double Holliday junctions, resulting in heteroduplex DNA. • By what mechanisms are Holliday junctions resolved? What is similar and different about these mechanisms? Which results in recombinant chromosomes? • Translesion DNA synthesis • SOS response • DNA double-stranded break (DSB) • Nonhomologous end joining (NHEJ) • Synthesis-dependent strand annealing (SDSA) • Strand invasion • D loop • Holliday junction • Heteroduplex region

????

• Why are some missense mutations also called neutral mutations?

Because they are missense mutations but do not change the function of the protein

• Recognize and explain the effects of the various types of mutations and their effects: o point mutations (insertion, deletion, base-pair substitution) o silent (synonymous), missense (nonsynonymous), nonsense, neutral and frameshift mutations

Point mutations: Occur at a specific, identifiable position in a gene or a specific location anywhere else in the genome --Base-pair substitution mutations: replacement of 1 nt base pair with another. Transition and transversion --Insertion: inserting one or more base pairs, causes frameshift mutation --Deletion: removal of one or more base pairs, causes frameshift mutation --Frameshift mutation: insertion or deletion that causes a change in the reading frame --Silent (synonymous): base-pair change that does not alter the resulting amino acid --Missense (nonsynonymous): base-pair change that results in an amino acid change in the protein --Nonsense: base-pair change that introduces a stop codon instead of an amino acid --Neutral: base-pair change that causes a missense mutation, but does not alter the protein's function. Ex: Lysine to Argenine because both basic so no change in function.

• How does p53 regulate DNA repair, cell cycle, and cell death?

Role of the p53 Repair Pathway • The p53 repair pathway controls cell responses to mutation by deciding to: 1. Pause the cell cycle at the G1-to-S transition to allow time for repair; or 2. Direct the cell to undergo programmed cell death • p53 levels are low in healthy cells • ATM increases p53 levels in response to DNA damage --• initiates G1 arrest; this allows for time to repair damaged DNA • Completed repair depletes p53 and allows the cell cycle to proceed • If the p53-induced pause of the cell cycle persists too long, apoptosis is induced (via another pathway)

• What is the Ames test and how is it used to study the effect of chemical mutagens? o Describe the experimental set up, use of S9 liver enzyme extract, the results observed for his- to his+ reversions, and the conclusion made about a chemical's mutagenic properties based on these results. o What do you expect a graph to look like that compares the mutagenic properties of 2 chemicals after the Ames Test with one that is highly mutagenic and one that is less mutagenic? How would the mutagenic property observed change with increased dosage?

The Ames Test • Mimics what happens when animals are exposed to compounds, and determines if the compound or any of its breakdown products is mutagenic • Bacteria are exposed to experimental compounds in the presence of mammalian liver enzymes • In animals, ingested chemicals are transported to the liver, where they are broken down by enzymes • Most commonly, strains of Salmonella typhimurium are used that carry point mutations interfering with their ability to synthesize histidine • The bacteria are exposed to the chemical to be tested, plus an extract of purified liver enzymes, and plated on medium lacking histidine • S9 extract provides liver enzymes to metabolize chemicals • Assay number of reversion mutations (his- à his+) compared to controls Ames Test Results (look at slide 14) • The number of reversion mutations from his- to his+ are assayed --• Not 100% accurate --• Can develop a dose response curve • If a compound is mutagenic, observe a significant increase in the reversion rate in treated strains relative to controls • Also compare strains with one type of mutation (e.g., base substitution) compared to those with another (e.g., frameshift), and untreated strains Set up: 1. S9 added to mutant strains of his- S typhimurium 2. his-1 is a base-substitution mutant, his-2 is a frameshift mutant 3. S9 bacterial mixture from each strain is spread on one experimental plate and one control plate 4. A paper disk is put into each plate. The test compound is added to the experimental plate disk but not to control-plate disks. 5. The presence of a significant number of revertant colonies indicates the test compound induces base-substitution mutations 6. The control plates determine the rate of spontaneous his- --> his+ reversion 7. An insignificant number of revertant colonies indicates the test compound does not induce frameshift mutations.

• How does non-Watson-and-Crick base-pairing occur and what is the consequence after 1 round of replication? 2 rounds of replication? o How do tautomeric shifts contribute to this? What nucleotides are more typically affected by these tautomeric shifts? o How do modification of nucleotides contribute to this? What nucleotides are more typically affected by these modifications?

Through DNA base mispairing, similar to third-base wobble, sometimes occurs during DNA replication. Tautomeric shifts lead to this. After 1 round: incorporated error - mispairing of a nucleotide in a newly synthesized DNA After 2 rounds: replicated error (mutation) - incorporated error converts it into a mutation, G-T pairing or C-A pairing Tautomeric shifts are changes that occur at hydrogen atoms, that alter the molecule. Tautomeric shifts change pairing between pyrimidines and purines. For normal pyrimidines or purines and that do this it causes enol to form (with 3 bonds instead of 2) or imino to form (with 2 bonds instead of 3) improper base pairing to occur. All of these do not change structure of double helix but will be incorporated. Depurination: loss of one of the purines (adenine or guanine) caused by breakage of covalent bond between base and sugar. This forms a DNA lesion known as an apurinic (AP) site that lacks a purine nucleotide base. Most AP sites are repaired before replication; if left unrepaired, DNA polymerase will usually compensate by putting an adenine into the site during replication. The missing nucleotide base can be replaced by DNA repair processes, but if it is not repaired the base will be missing during the next round of DNA replication. Deamination: The loss of an amino group (NH2). Cytosine is the most often seen. When deaminated, the NH2 is replaced with an O, forming Uracil. DNA mismatch repair readily recognizes uracil as an RNA nucleotide base and removes it from DNA. The excised uracil is replaced by cytosine, and wild-type sequence is restored. When methylated cytosine is deaminated, a thymine base is produced, which is now base- paired with guanine. DNA mismatch repair (MMR) can repair this

• What is the difference between a transversion and a transition base-pair mutation? o What is the effect of each on the double-helix structure?

Transition: which one purine replaces the other (A → G; G → A) or one pyrimidine replaces the other (C → T; T → C). Would not have an effect on the geometry. Transversion: a purine is replaced by a pyrimidine (A → T, A → C, G → T, and G → C), or a pyrimidine is replaced by a purine (T → A, T → G, C → A, or C → G). This would cause swelling or pinching in the shape of the helix.

• How do different organisms respond to UV-induced damage?

UV radiation is the most common mutagen that most organisms encounter on a daily basis. UV exposure induces the formation of photoproducts that can inhibit DNA replication as well as lead to mutation. One common way organisms identify and repair UV-induced DNA damage is through photoreactive repair. This direct DNA repair mechanism is found in bacteria, single-celled eukaryotes, plants, and some animals (e.g., Drosophila) but not in humans. Photoreactive repair utilizes the enzyme photolyase to bind to a UV-induced photo product. Once bound, photolyase uses visible light to direct energy into breaking the bonds that produce the photoproduct. In E. coli, photolyase is the product of the phr (photoreactive repair) gene. Mutations of this gene result in a substantial increase in UV-induced mutations in bacteria. Photolyase mutations in other organisms similarly result in increases in the mutation rate.

• How do amber suppressors affect protein translation?

mutation in tRNA so it recognizes stop codon, but carries an amino acid

11.3: Mutations May Be Caused by Chemicals or Ionizing Radiation • How do mutagens contribute to DNA damage? Include examples.

• Agents that cause DNA damage leading to mutations • Interact with DNA in specific ways to cause particular types of sequence changes • Induced mutations are produced by mutagens in an experimental setting to study types of damage caused, the mutation process itself, or repair responses to damage • Radiation: • Non-ionizing (UV) radiation • Ionizing radiation (X-rays) --• May cause breakage of covalent bonds in sugar-phosphate backbone --• Low levels may cause point mutations • Chemical mutagens: classified by their modes of action on DNA as follows Chemical mutagens: classified by their modes of action on DNA as follows: 1. Nucleotide base analogs 2. Deaminating agents 3. Alkylating agents 4. Oxidizing agents 5. Hydoxylating agents 6. Intercalating agents • Base analogs and intercalating agents (which are used anticancer drugs) depend on replication to cause mutations

• Describe (briefly) how mutations/DNA damage can be repaired, including methyl-directed mismatch repair, base excision repair, and nucleotide excision repair o Consider what type(s) of damage trigger each. o Compare and contrast the repair mechanisms of each. o What type of organisms undergo each?

• Base excision repair (BER) --• Repairs mutations caused by depurination and deamination --• Repairs damage to a base or replaces an incorrect base 1. Base pair mismatch recognized. 2. Removes incorrect base, creates AP site. 3. AP endonuclease generates single-stranded nick on 5' side of AP site. 4. DNA polymerase removes and replaces several nucleotides of the nicked strand (nick translation) 5. DNA ligase seals the gap. • Nucleotide excision repair (NER) --• Corrects damaged-induced distortions in the DNA helix (i.e. thymine dimers), so bulkier repairs -- Used to repair UV induced damage 1. Enzymes recognize and bind to the damaged region 2. Corrects damaged-induced distortions in the DNA helix (e.g., thymine dimers) 3. A segment of nucleotides is removed from the damaged strand 4. DNA polymerase fills in the gap and DNA ligase seals the sugar-phosphate backbone • Methyl-directed mismatch repair (MMR) --• Corrects mismatches left after DNA replication --• Mismatched nucleotides that escape DNA polymerase proofreading may be detected and repaired by MMR ----• Found in prokaryotes ----• Eukaryotes do not methylate their DNA in same way, so it is not known how the cell distinguishes parental vs. new strand, but mismatch repair does take place --• Repair enzymes distinguish between the template nucleotide and the new, mismatched nucleotide by methylation on the template strand ----• In E. coli, methylation is common on the adenine of 5¢- GATC-3¢ sequences --• Mutator genes: involved in DNA repair; loss of their function results in increased accumulation of mutations in the genome • Translesion DNA synthesis (TLS) and the SOS response --• Last resort response that allows replication to proceed past lesions that block DNA replication

• Why can't eukaryotes undergo methyl-directed mismatch repair?

• Eukaryotes do not methylate their DNA in same way, so it is not known how the cell distinguishes parental vs. new strand, but mismatch repair does take place

• What is the relationship between mutations in DNA repair genes (mutator genes) and cancer?

• Mutator genes: involved in DNA repair; loss of their function results in increased accumulation of mutations in the genome DNA Damage Signaling Systems • Biochemical mechanisms to recognize the presence of DNA damage and initiate a repair response • ATM & ATR communicate DNA damage through signal transduction to activate transcription of p53, thereby activating the p53 repair pathway

• What is strand slippage? o What enzyme is responsible and what kinds sequences does this more often occur?

• Strand slippage: occurs because of alterations in number of DNA repeats • DNA polymerase temporarily dissociates from the template and a portion of the newly replicated DNA forms a hairpin • Resumption of replication leads to re- replication of some of the repeats and an overall increase in the number of repeats on the daughter strand

• How do the additions of hydroxyl groups or alkyl groups affect nitrogenous bases? Removal of an amino group?

• interferes with DNA base pairing by distorting the DNA double helix

• When and how are DNA mutations introduced?

--primarily through errors in DNA replication or spontaneous changes in the chemical structure of a nucleotide base --• Usually occurs in repetitive sequences --Due to polymerase slippage

• Compare and contrast nucleotide base analogs, deaminating agents, alkylating agents, hydroxylating agents, DNA intercalating agents, ionizing radiation, and UV radiation. o What are the effects of each on DNA? How might each affect the structure of the double-helix?

1. nucleotide base analogs • A chemical compound with a structure similar to a DNA nucleotide base • Can pair with normal nucleotides • DNA polymerases cannot distinguish the analogs from normal nucleotides Example: • 5-bromodeoxyuridine (5-BU) acts as an analog of thymine (normal state; keto form) • 5-BU can also act as an analog of cytosine in a rare state (enol form) 2. deaminating agents • Removes amino groups from nucleotide bases • Deamination of adenine by nitrous acid produces hypoxanthine, which can mispair and lead to A-T to G-C base-pair substitutions 3. alkylating agents • Add bulky side groups to bases: methyl (CH3) and ethyl (CH3-CH2) groups --• interferes with DNA base pairing by distorting the DNA double helix • Ethyl methanesulfonate (EMS): alkylating agent used for mutagenesis --• induces transition mutations through its action on guanine 4. hydroxylating agents • Add hydroxyl groups to a recipient compound • Hydroxylamine adds a hydroxyl group to cytosine, creating hydroxylaminocytosine --• This can mispair with adenine, creating a C-G to T-A transition mutation 5. DNA intercalating agents • Molecules that fit between base pairs • Distort the DNA duplex • Leads to DNA nicking that is not efficiently repaired, resulting in added or lost nucleotides -- The consequence of lost or added nucleotides can effect how DNA is replicated and can also effect the function of a protein because this will cause different mRNAs to be made, which will lead to different amino acids being made and create a different protein. 6. ionizing radiation (X-rays) --• May cause breakage of covalent bonds in sugar-phosphate backbone --• Low levels may cause point mutations 7. UV radiation (UV is only non-ionizing radiation that induces mutations) --UV irradiation alters DNA nucleotides by inciting the formation of additional bonds that create aberrant structures called photoproducts • Photoproducts: aberrant structures with additional bonds involving nucleotides --• Thymine dimer: formed by covalent bonds between the 5 and 6 carbons of adjacent thymines --• 6-4 photoproduct: formed by a covalent bond between carbon 6 on one thymine and carbon 4 on the other • Must be repaired for replication to occur! • Bacteria, single-celled eukaryotes, plants, and some animals (not humans) can repair this • Resulting mutations are the primary cause of skin cancer due to UV exposure

11.4: Repair Systems Correct Some DNA Damage • What is the first "defense" against mutagenesis during DNA replication?

DNA polymerase

4. Most DNA mutations are caused by ___.

DNA polymerase errors

• What is the difference between an intergenic mutation/reversion and an intragenic mutation/reversion?

Forward mutation: converts wild-type to mutant. Reverse Mutation (Reversion): converts mutation to wild-type or near wild-type. True Reversion: DNA sequence reverts to encoding its original message due to a second mutation at the same site or within the same codon (think of AA and codons) Intragenic reversion: reversion occuring in a second mutation elsewhere in the gene (think of deletion then insertion) Intergenic Reversion: second-site mutation, suppressor mutation. Occurs by mutation in a DIFFERENT gene that compensates for the original mutation, restoring the organism to wild-type. Suppressor mutation: 2nd mutation "suppresses" the mutant phenotype caused by the 1st mutation

• Which types of point mutations can cause frameshift mutations? Why do these mutations change the "reading frame"?

Insertions and deletions, reading frame is read in 3's adding one or deleting one would move the reading frame.

11.1: Mutations Are Rare and Random and Alter DNA Sequence • What does it mean to say that most mutations are random?

It means that mutations do not happen on purpose, they are spontaneous Mutation: change in the DNA sequence. Mutations can cause changes in proteins that affect their functions and play a role in how organisms adapt to their environment.

• Could mutations outside of protein-coding regions affect the amino acid sequences of proteins?

Mutations outside of protein-coding regions could have an effect on recognition of where to start processes such as transcription and translation. Mutations outside of the protein-coding regions could affect amino acid sequence for the reasons stated above and if the mutation causes a frameshift in the coding region.

• Are all mutations that affect gene expression found in coding regions? If not, where else can they be found?

No, regulatory mutations (promoters, introns, and regions coding 5′-UTR and3′-UTR segments of mRNA)

• Are all mutations heritable?

Only those that are in germ line cells are heritable. The ones in somatic cells are not

• What are examples of photoproducts produced by UV/non-ionizing radiation? o What is their effect on DNA replication? o How do bacteria correct photoproducts? o What is the relationship between UV damage and skin cancer?

Photoreactive Repair • UV radiation is the most common mutagen of most organisms • Photoproducts of UV exposure can inhibit DNA replication and also lead to mutation • UV-induced damage can be addressed by photoreactive repair --• takes place in bacteria, single-celled eukaryotes, plants, and some animals (not humans) • Photolyase: uses energy from visible light to break the bonds producing the photoproduct --• E. coli phr (photoreactive repair) gene encodes photolyase

• What effects could mutations outside of protein-coding regions have? Consider specific regions and the effects on transcription.

Promoter Mutations: interfere with transcription initiation by reducing or eliminating transcription. . Splicing Mutations: result in splicing error and the production of mutant proteins due to the retention of intron in the mRNA Cryptic Splice Sites: produce new splice sites that replace or compete with authentic splice sites during mRNA processing Polyadenylation Mutations: processing of the 3' end of the mRNA has the polyadenylation signal. If a mutation happens in this sequence, it can block recognition of this sequence, which will generate abnormal mRNA and lead to severe reduction of functional protein

• Compare and contrast somatic cells and germline cells (gametes). o How might a mutation be inherited in organisms undergoing sexual vs. asexual reproduction? o How may heritable mutations contribute to evolutionary change in a population of organisms

Somatic: Not in germline, divide by mitosis, only asexual will carry the mutation, only direct descendants of the original mutated cell will carry the mutation Germline cells: give rise to sperm and egg, heritable from one generation to the next, sexual reproduction Not all mutations are heritable. Somatic cell mutations are not passed down while germline mutations are.

• What does it mean for mutations to be random and not directed?

They are random because they happen spontaneously. They are not directed because they do not create mutations to adapt to their environment, rather mutations spontaneously happen, and if they are beneficial to an organism in helping it survive, it will be passed to the next generation.

11.2: Gene Mutations May Arise from Spontaneous Events • What does it mean to say that most mutations are spontaneous?

arise in cells without exposure to agents capable of inducing mutation

3. Mutations may be found in the ___, as well as ___ of DNA.

coding region, regulatory regions

8. Homologous recombination occurs in ___, involves the formation and repair of ___, and can result in genetically different ___.

meiosis, double-stranded DNA breaks, gametes

6. The Ames test assesses the ___.

mutagenic properties of a chemical in a dose-dependent manner.

5. Mutations can be caused by external agents, called ___, such as ___.

mutagens, chemicals and radiation

Key concepts: 1. Mutations are very rare for any given nucleotide and occur ___ without regard to the needs of an organism.

randomly

2. Small-scale mutations include ___

single base-pair changes (substitutions), insertions and deletions.

7. DNA repair systems are used to repair mutations that arise ___ and by mutagens.

spontaneously