MCB 150: The Molecular and Cellular Basis of Life

Transcription

1 MCB 150 The Molecular and Cellular Basis of Life Mutations, Part II Today s Learning Catalytics Session ID is: Exam II information: 343 students (59%) stayed the same or increased from Exam I to Exam II; 172 of them increased by 10 points or more! increased by percentage points - 47 increased by percentage points - 8 increased by percentage points - 1 student increased by 41 percentage points! - 1 student increased by 50 percentage points!! 23 students have attended 4 or more office hours; 22 of them were above the mean on Exam II 173 students scored below 50% on the test; 5 attended office hours more than once 2

2 DNA sequences can be changed by many factors: Uncorrected mistakes in replication Chemical mutagens High-intensity radiation (x-rays, UV, etc.) Among these, uncorrected mistakes in replication account for the most mutations This is when millions to billions of new bonds are formed 3 How often do errors occur in DNA replication? Frequency of mutations that end up in E. coli is about 0.04 mutations/cell division - Can also be represented as 4 mutations/100 cell divisions Average in all organisms is 1 mistake per billion nucleotides But in vitro, E. coli DNA polymerases make ~400 mistakes/ cell division -- 10,000X the observed number! So how does E. coli (or any other organism) reduce the frequency of the errors it makes by 10 4? Molecular backspace keys and spell checkers 4

3 Proofreading (a DNA polymerase s backspace key ): an example of 3 5 exonuclease activity If this exonuclease activity is gone, error frequency is 100 times higher accounts for half of 10 4 reduction in error frequency 5 If proofreading doesn t catch the mistake: Mismatch repair system in E. coli scans recently synthesized DNA, looking for mismatches and hemimethylated DNA New DNA gets methylated at adenine residues within the sequence 5 GATC 3 shortly after replication 6

4 If proofreading doesn t catch the mistake: Mistake is in new strand Mismatch repair enzymes fix problem in unmethylated (new) strand Once DNA gets methylated, no way of distinguishing parent from daughter strand 7 Methyl-directed mismatch repair (MMR) in E. coli: MutH cuts nonmethylated strand. An exonuclease removes bases just beyond mismatch. DNA polymerase III fills in the gap. Ligase seals the nick. 8

5 If mismatch repair enzymes are missing, error frequency is 100 times higher One type of colon cancer can be traced to a mutation in the human mismatch repair system Proofreading and Mismatch Repair each decrease error frequency by 100X 100 x 100 = 10,000, which is the observed difference What about mutations that arise from sources other than errors in DNA replication? Other specialized systems of repair enzymes recognize damage and do their best to fix it 9 What if a mutation slips through uncorrected? 2 broad categories, many types of mutations: Point Chromosomal-level Base Substitution Frameshift Insertion Deletion Inversion Duplication Same sense Missense Nonsense Translocation 10

6 Base substitution mutations Replace one base pair with another, e.g. GC AT 11 How a base substitution becomes permanent: Note: only one daughter helix is shown from the first round of replication; the other is assumed to be accurate through both rounds of replication 12

7 The three categories of base substitutions: Template DNA after mutation -ATC- -AAC- Template DNA mrna = -UUG- = Leu mrna = -GUG- Val (missense) -CAC- mrna = -UAG- STOP (nonsense) mrna = -UUA- Leu (same sense) -AAT- 13 How do the different base substitution mutations affect the final protein function? If a base substitution mutation occurs in a protein-coding region of a gene, 3 effects are possible: promoter terminator DNA AUG UAA RNA Samesense Missense Nonsense 14

8 Same sense mutations: Nucleotide (usually in third position) changed to a different codon that specifies the same amino acid No effect on protein product or function What feature of the genetic code makes this possible? Nonsense mutations: Codon specifying an amino acid is changed to a stop codon, causing premature termination Protein is truncated and virtually always inactive (null phenotype) 15 Missense mutations: Codon specifying an amino acid is changed to a codon for a different amino acid; the effect on protein varies: IF the amino acid that has been altered is not critical for the folding or function of that protein OR if the amino acid that replaces it assumes those duties in roughly the same way THEN the protein is likely to retain at least some function, may even be fully functional. BUT if the altered amino acid is critical, and the duties aren t taken up by the amino acid that replaces it THEN the protein will be inactive. 16