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Exam 3
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What processes depend on nucleotide base pairing?
DNA Replication
Transcription
Translation
none are perfect
What type of DNA base pairing in the most reliable? Why?
DNA replication
It needs to be the most reliable mistakes in DNA alter the blueprint, which can affect all subsequent processes and are passed to daughter cells. You can always make a new transcript or protein, but you can’t make a new blueprint.
Define Mutations
Her
How do mutations differ in single-celled organisms vs. multi-cell organisms?
single-cell: all daughter cells have the mutation
multi-cell: mutations can be somatic (threat to individual, not species) or germ line/somatic (threat to individual and species)
What factors can cause changes in DNA sequences?
uncorrected mistakes in replication
chemical mutagens
high-intensity radiation (ex. x-rays, UV, etc.)biological agents (ex. viruses, transposons)
What factor accounts for the most mutations? Why?
DNA replication
Happens so frequently that there’s more possibility for error
How often do errors occur in DNA replication of E. coli? (mutations/cell division), (mutations/nucleotide)
0.04 mutations per cell division
1 mistake per billion nucleotides
How does E. coli reduce DNA replication errors by 104
Proofreading and Methyl-directed Mismatch Repair (MMR)
Steps for proofreading
DNA polymerase inserts incorrect base
Base moves to exonuclease site of DNA polymerase and is cleaved off (3’—5’ exonuclease activity)
DNA polymerase inserts correct base
molecular backspace key
reduces errors 100-fold
Steps for mismatch repair system (MMR)
The MMR corrects mistakes in the newly synthesize (unmethylated) strand missed during proofreading
once DNA gets method, no way to distinguish parent from daughter strand
MutS scans DNA for error and binds to it
MutH finds the hemimethylated cite (GATC sequence) closest to error and makes a nick n in the unmethylated strand (endonuclease activity)
MutL linkes MutS and MutH and loops out a region of DNA
exonuclease recruited to strip DNA from nick until slightly past error
DNA polymerase III fills in the gap
Ligase seals the nick
molecular spell check
reduces errors 100-fold
What happens to error frequency without proofreading or MMR?
error frequency increases 100 × 100 = 10,000 times without these mechanisms
One type of colon cancer is traced to mutation in MMR
What is done to address mutations that come from sources other than DNA replication (ex. chemical mutagens and high-intensity radiation)?
Nucleotide Excision Repair
Steps for nucleotide excision repair
a complex of proteins detects an irregularity (ex. kink) in DNA
an enzyme nicks DNA on both sides of damage
DNA helicase unwinds and removes region of damaged bases
DNA polymerase fills gap in 5’—3’
Ligase seals gap
used to address mutations induced by chemical mutagens and UV radiation
2 Broad Categories of Mutation Types
point mutations - small # BPs changed
chromosomal-level mutations - large # BPs changes
2 Categories of Point Mutations
Base substations
Frameshift
Types of Base Substitutions
Affect protein coding genes:
same sense - different codon, same amino acid
missense - different codon, different amino acid
nonsense - stop codon
transition - change one BP to another in same family
transversion - change one BP to another in a different family
Types of Chromosomal-level Mutations
Insertion
Deletion
Translocation - swap sections between chromosomes
Duplication - copy and paste
Inversion - flip segment (no BPs lost)
How do base substitutions become permanent?
2 rounds of replication
first round - BP mismatch happens
second round - uncorrected mismatch is replicated (“cemened”)
How do different base substitution mutations affect final protein function?
same sense - no effect
missense - depends on replacement (can it do similar function? yes - some function, no -innactive)
nonsense - early truncation of protein, typically leads to loss of function
Sickle-cell anemia
Missense
Defective beta subunit in hemoglobin. Changes shape or red bloods cells and lessens its ability to carry oxygen. Helps against malaria because cells constantly remade in attempt to correct error.
Phenylketonuria (PKU)
Missense
Newborns screened to prevent mental impairment. Effect can be prevented by special diet. Can’t process phenylalanine which leads to toxic buildup (toxic to nerves) and low levels of tyrosine (inhibits normal growth and skin pigment)
Predicting Amino Acid Changes for Single Base Substitutions
change first base - same relative position vertically
change second base - same row
change third base - same box
Define Frameshift Mutations and Explain Their Result and Effect on Protein Product
The addition or removal of a small number of bases not divisible by 3
They disrupt the normal reading frame, changing all subsequent codons
Typically leads to appearance of stop codon resulting in early truncation
Cystic Fibrosis
Removal of exactly 3 bases (one amino acid)
Results in faulty CFTR protein which removes chlorine from cells. Accumulation of Cl- in cells, do water doesn’t leave cell, dehydrating mucus layer
Lymphomas and Leukemias
Translocations
Oncogens
duplications
Cause them to become cancers
Fragile X syndrome
duplications
of short, repeated sequences
Huntington’s Disease
duplications
of short repeated sequences
Infertility
inversions
Haemophilia A
insertions
cause loss of clotting factor VIII
Duchenne’s Muscular Dystrophy
Deletion
Ames Test for Mutagenicity
Used to identify chemical mutagens
Dish 1: cells + growth medium + liver extract (conrtol - random mutation)
Dish 2: cells + growth medium + liver extract + chemical mutagen
Compare colonies that result. If Dish 2 has significantly more, it’s suspected the chemical mutagen causes mutation