11/30/2015

Gene Mutation

Fundamental Genetics
Lecture 13

Gene Mutation, DNA Repair,

and Transposable Elements

Any change in the nucleotide sequence of

a given DNA
Substitution, deletion or insertion of one
or more nucleotides
Could affect or not a given phenotype
The major basis of diversity among
organisms
The raw material of evolution

John Donnie A. Ramos, Ph.D.

Dept. of Biological Sciences
College of Science
University of Santo Tomas

Classification of Mutations
Based on nature of occurrence
Spontaneous mutation
Induced mutation

Based on cell type where it occurs

Autosomal mutation
Sex-linked mutation

Based on effect on the organism

Mutation affecting morphological trait
Mutation causing nutritional or biochemical variation
Mutation affecting behavior

Based on how it affects the regulation of other genes

Regulatory mutation

Others
Lethal mutation
Conditional Mutation

Caused (mostly) by mutagenic agents

Can be repaired by the body (in normal
conditions)

Detection of Mutation
Detection in bacteria and fungi
use of minimal culture medium
Prototrophs nutritional wild types
Auxotrophs needs specific supplements

Detection in Drosophila
Attached X-procedure

Detection in plants
Visual observation
Biochemical composition Analysis
Tissue culture

Detection in humans
Pedigree analysis
DNA sequencing
Microarray

Temperature-sensitive mutation

Pedigree Analysis

X-linked recessive Mutation

Molecular Basis of Mutation

Base substitutions or point mutation change

in the sense of information (missense)
Transition mutation purine-purine or
pyrimidine-pyrimidine mutation
Transversion purine to pyrimidine change or
vice versa
Frameshift mutation insertion or deletion of
one base.

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Tautomeric Shifts

Tautomeric Shift Causes Transition Mutation

Alternative base pairing (mutations) different from the AT and G-C pairs
First published by Watson and Crick
Formation of hydrogen between non-complementary
bases
Pairing still between purine and pyrimidine
Involves keto-enol pairs for T and G amino-imino pairs for
C and A

Base Analogs
Mutagenic chemicals
capable of susbtituting
purines or pyrimidines
during nucleic acid
biosynthesis
Analogs causes tautomeric
shift
Causes reverse mutation
reversion to the wild type
nucleotide sequence

Acridine Dyes
Aromatic molecules that mutations
Causes frameshift mutations by adding or
removing one or more bases in a given
sequence
Intercalates or wedge between purines and
pyrimidines

Alkylating Agents
Mutagenic chemicals capable of donating an alkyl group
such as CH3- or CH3-CH2- to amino or keto groups in
nucleotides
Examples:
Mustard gases (used as chemical warfare)
Ethylmethane sulfonate (EMS)

Apuric Sites
Spontaneous loss of one of the nitrogenous
bases in an intact double helix DNA
Occurs mostly on guanine or adenine
Involves the breaking of glycosidic bond linking
the 1-C of d-ribose and the 9 position of purine
ring

Induces contortions in a DNA helix causing

deletions or inserrtions
Examples:
Proflavin
Acrydin orange

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Nitrous Acid
Mutagenic agent causing deamination of
nitrogenous bases
In deamination, an amino group is converted to
keto group in cytosine and adenine thus
becomes uracil and hypoxanthine, respectively.
Examples:

UV and High Energy Radiation

Longer wavelengths than visible light has no effect on most
molecules
Shorter wavelengths than visible light interacts with most
molecules including DNA
Purines and pyrimidines absorbs UV at 260 nm
Example of effects: formation of Thymidine dimers

G-C pair converted to A-U pair then to A-T pair

after succeeding replications
A-T pair is converted to G-C (hypoxanthine
pairs with cytosine)

Ionizing Radiation
Causes ionization of molecules (transformation of stable
structures into free radicals and reactive ions)
x-rays, gamma rays, cosmic rays
Penetrate tissues and cells
Results in point mutations and disruption of phosphodiester
bonds.
Linear relationship between dose of radiation and percentage of
mutation it causes

Mutations in Humans
Result to both positive (variation, evolution, speciation) or
negative (diseases) effects.
Examples:
ABO Blood types (mutation in glycosyltransferase enzyme
converts H substance to A or O)
Muscular dystrophy (mutation in dystrophin muscle protein)
Duchenne muscular dystrophy (DMD) more common and severe
mostly frameshift mutation

Becker muscular dystriphy (BMD)

mostly substitutions

Trinucleotide repeats

Ames Test
Assay to detect mutations
(mutagenicity test)
Delvised by Bruce Ames
Uses different strains of
Salmonella typhimurium with
mutations on enzymes
involved in histidine
biosynthesis (auxotrophs) and
DNA repair
Assay measures the frequency
of reverse mutation

DNA Damage Repair Systems

Photoreactivation Repair
repairs damage caused by
UV (Thymine dimers)
found in prokaryotes only
activity of photoreactivation
enzyme (PRE)
needs blue light (visible light)

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DNA Damage Repair Systems

Nucleotide Excision Repair

Discovered by Paul Flanders I

E. coli

Excision Repair
Found in both prokaryotes and
eukaryotes
Light independent repair
Involved enzymes used in DNA
replication
Types:

Coded by genes called uvr

gene (ultrviolet repair)
Mutation of the gene causes
xeroderma pigmentosum
(severe skin lesions caused by
sunlight leading to skin cancer)

Base Excision Repair (BER)

Nucleotide Excision Repair
(NEP)

Proofreading and Mismatch Repair

DNA polymerases (I, II, III) exhibits ability to proofread
synthesized DNA bases
Mismatch repair functions when DNA polymerases
failed to correct mutations

Post-Replication Repair
Also called homologous recombination repair
Catalyzed by RecA protein
Indentified Miroslav Radman in E. coli

First proposed by Robin Hilliday

Recognizes mismatched bases after replication
Problem: how to recognize in a given DNA base pair which
is the template and which is the mutant base?
In prokaryotes, recognition is based on the DNA sequence
recognized by adenine methylase to perform DNA
methylation (addition of methyl group on template DNA)

5.GATC.3
3.CTAG.5

SOS Repair

Double-Strand Break Repair

Proposed by Phil Hanawalt and Pauyl Flanders (in E. coli)

Also called homologous recombinational repair

Also occurs post-replication

Occurs when mutation occurs in both strands of a

double helix DNA

DNA polymerase continue replication across a given lesion

No gap is produced
Unspecific DNA bases might be added (compromised DNA
base fidelity)
Involves the proteins coded by lexA, recA and uvr genes

Involves the separation of a segment of a gene

During replication, no template will used for the
synthesis of the excised DNA fragment
Replaced by homologous undamaged DNA from
the homologous chromosome
Involves ligases to bind DNA fragments
Implicated in Xray-hypersensitivity,
immunodeficiency, breast cancer and ovarian
cancer

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Site-Directed Mutagenesis
Experimental technique
used to produce a mutant
gene to create a mutant
protein
Based on DNA
hybridization principle

Gene Knockouts
Excision of an entire gene to observe its effect on an
entire organism
An experimental tool to study the function of a protein
encoded the knockout gene
Resulting organisms are called knockout organisms
Often results to loss of function

Insertion of a mutant
codon (resulting to
different amino acid to
produce a mutant protein)

Nude mouse (immune system knockout) used to

regenerate human ear

Transposable Genetic Elements

Jumping Genes in Corn

Also called transposons or jumping genes

Ac activator gene

Genetic elements (insertion sequences, <2000 bp) that

moves from chromosome to another

Ds dissociation gene
W theoretical gene

Results to posible disruption of a given gene if

movement occurred in the middle of a gene
First studied by Barbarra McClintock (1983 Nobel Prize
Winner)
Implicated in antibiotic resistance (in certain strains of
pathogenic bacteria)

Ac and Dc Elements

Transposable Elements in Drosophila

Copia gene organization

DTR direct terminal repeat (276 bp each) contains

ITR (inverted terminal repeat)