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

Proofreading:1. Mismatch Detected2. 3’-5’ Exonuclease3. 5’-3’ Polymerase

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

Results:Culture Number # resistant colonies

1 12 03 34 05 06 57 08 1079 0

10 64

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

Fig. 6.6

Depurination

Deamination

Fig. 6.6

Excision Repair

Fig. 6.7

Base Analogs

Alkylating Agents

Key Point: Chemical mutagens change the nature of the

complementary base pairing

Fig. 6.11

Perform a Complementation Test!

a b

“Fail to Complement”

Complementation Table

Fig. 6.13

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

a1 +

+ a2

X

+

a2a1

+

Gene Structure Conclusions•Mutations can be order linearly•Genes can be divided internally

Fig. 6.16

Fig. 6.17

X-Ray

Fig. 6.18

Fig. 6.18

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

Fig. 6.19

PrimaryStructure

SecondaryStructure

TertiaryStructure

Fig. 6.21

Fig. 6.22