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Ensuring DNA IntegrityEnsuring DNA Integrity
• Redundancy inherent in structureRedundancy inherent in structure
• DNA repair enzymologyDNA repair enzymology
• High precision in ReplicationHigh precision in Replication
DNA Polymerase III
• 10 protein subunits
• Restrictions on Nucleotide Addition
– Copies only single stranded DNA
– Can add only to pre-existing chains
– Adds only in the 5’ to 3’ direction
• Proofreading– 3’ to 5’ Exonuclease
Spontaneous MutationsSpontaneous Mutations
• Forward vs. Reverse MutationsForward vs. Reverse Mutations
• Range: 10Range: 10-4-4 to 10 to 10-8-8/Gene/Generation/Gene/Generation
• General TrendsGeneral Trends
Mutations Affecting Phenotype RareMutations Affecting Phenotype Rare
Genes Mutate at Different RatesGenes Mutate at Different Rates
Forward Rate > Reverse RateForward Rate > Reverse Rate
Bacterial Resistance to Bacteriophage
Observations:
1. Most Bacteria are sensitive to Bacteriophage
2. If a culture of sensitive bacteria are spread on plate containing
bacteriophage, no colonies grow.
3. Exception: a few colonies do grow, therefore they are resistant to
bacteriophage.
Assumption:
A mutation occurred that makes those bacteria resistant.
Two Hypotheses:
1. The mutation arises in response to the bacteriophage.
or
2. A few bacteria already have the mutation prior to being subjected
to the bacteriophage.
Expectation: Similar numbers of resistant colonies
Expectation: Fluctuation in numbers of resistant colonies
Ad
d S
elec
tive
Age
nt
Ad
d S
elec
tive
Age
nt
Fig. 6.4
THE CAT SAW THE DOG
Base SubstitutionTHE BAT SAW THE DOGTHE CAT SAW THE HOGTHE CAT SAT THE DOG
InsertionTHE CMA TSA WTH EDO G
DeletionTHE ATS AWT HED OG
Base Analogs
Alkylating Agents
Key Point: Chemical mutagens change the nature of the
complementary base pairing
Fig. 6.11
Benzer’s Fine Structure MappingBenzer’s Fine Structure MappingWhy T4 Bacteriophage?Why T4 Bacteriophage?
• Produce millions of progeny in a dayProduce millions of progeny in a day• rIIrII-- mutation mutation
– 1000s of mutant alleles available1000s of mutant alleles available– Unique phenotypeUnique phenotype
• rIIrII-- plaques plaques • rIIrII-- cannot lyse a specific bacterial strain cannot lyse a specific bacterial strain
– Can detect 1 recombinant/10Can detect 1 recombinant/1099 progeny progeny
Gene Structure Conclusions•Mutations can be order linearly•Genes can be divided internally
Fig. 6.16
a b c d
Enz.1 Enz.2 Enz.3
Mutant Cannot Grow Can Grow
Enzyme 1 a b, c, d
Enzyme 2 a, b c, d
Enzyme 3 a, b, c d
Arginine
Arg-H
enzyme
Argino-succinate
Arg-G
enzyme
Citrulline
Arg-F
enzyme
Ornithine
Arg-E
enzyme
Fig. 6.18
NHNH22---CHR---COOH---CHR---COOH
HH
NNHH
RR
CC
HH
CC OHOH
OO
Amino GroupAmino Group Carboxylic AcidCarboxylic Acid
Side ChainSide Chain