Gene-Lecture 13- Gene Mutation and Repair

7/23/2019 Gene-Lecture 13- Gene Mutation and Repair

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11/30/201

Gene Mutation, DNA Repair,

and Transposable Elements

Fundamental GeneticsLecture 13

John Donnie A. Ramos, Ph.D.Dept. of Biological Sciences

College of ScienceUniversity of Santo Tomas

Any change in the nucleotide sequence ofa given DNA

Substitution, deletion or insertion of one

or more nucleotides

Could affect or not a given phenotype

The major basis of diversity amongorganisms

The raw material of evolution

Caused (mostly) by mutagenic agents

Can be repaired by the body (in normalconditions)

Gene Mutation

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

Temperature-sensitive mutation

Classification of Mutations 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

Detection of Mutation

Pedigree Analysis

X-linked recessive Mutation

Molecular Basis of Mutation

Base substitutions or point mutation – changein the sense of information (missense)

Transition mutation – purine-purine orpyrimidine-pyrimidine mutation

Transversion – purine to pyrimidine change orvice versa

Frameshift mutation – insertion or deletion ofone base.



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Tautomeric Shifts

Alternative base pairing (mutations) different from the A-T 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 forC and A

Tautomeric Shift Causes Transition Mutation

Base Analogs

Mutagenic chemicalscapable of susbtitutingpurines or pyrimidinesduring nucleic acidbiosynthesis

Analogs causes tautomericshift

Causes reverse mutation – reversion to the wild typenucleotide sequence

Alkylating Agents

Mutagenic chemicals capable of donating an alkyl groupsuch as CH3- or CH3-CH2- to amino or keto groups in

nucleotides

Examples:

Mustard gases (used as chemical warfare)

Ethylmethane sulfonate (EMS)

Acridine Dyes

Aromatic molecules that mutations

Causes frameshift mutations by adding orremoving one or more bases in a givensequence

Intercalates or wedge between purines and

pyrimidines

Induces contortions in a DNA helix causingdeletions or inserrtions

Examples:

Proflavin

Acrydin orange

Apuric Sites

Spontaneous loss of one of the nitrogenousbases in an intact double helix DNA

Occurs mostly on guanine or adenine

Involves the breaking of glycosidic bond linkingthe 1’ -C of d-ribose and the 9 position of purinering



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Nitrous Acid

Mutagenic agent causing deamination ofnitrogenous bases

In deamination, an amino group is converted toketo group in cytosine and adenine thusbecomes uracil and hypoxanthine, respectively.

Examples:

G-C pair converted to A-U pair then to A-T pairafter succeeding replications

A-T pair is converted to G-C (hypoxanthinepairs with cytosine)

UV and High Energy Radiation

Longer wavelengths than visible light has no effect on most

molecules

Shorter wavelengths than visible light interacts with mostmolecules including DNA

Purines and pyrimidines absorbs UV at 260 nm

Example of effects: formation of Thymidine dimers

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 phosphodiesterbonds.

Linear relationship between dose of radiation and percentage ofmutation it causes

Mutations in Humans

Result to both positive (variation, evolution, speciation) ornegative (diseases) effects.

Examples:

ABO Blood types (mutation in glycosyltransferase enzymeconverts 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 withmutations on enzymesinvolved in histidinebiosynthesis (auxotrophs) and

DNA repair

Assay measures the frequencyof reverse mutation

DNA Damage Repair Systems

Photoreactivation Repair

repairs damage caused byUV (Thymine dimers)

found in prokaryotes only

activity of photoreactivationenzyme (PRE)

needs blue light (visible light)



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DNA Damage Repair Systems

Excision Repair

Found in both prokaryotes and

eukaryotes Light independent repair

Involved enzymes used in DNA

replication

Types:

Base Excision Repair (BER)

Nucleotide Excision Repair(NEP)

Nucleotide Excision Repair

Discovered by Paul Flanders IE. coli

Coded by genes called uvrgene (ultrviolet repair)

Mutation of the gene causesxeroderma pigmentosum (severe skin lesions caused bysunlight leading to skin cancer)

Proofreading and Mismatch Repair

DNA polymerases (I, II, III) exhibits ability to proofreadsynthesized DNA bases

Mismatch repair – functions when DNA polymerasesfailed to correct mutations

First proposed by Robin Hilliday

Recognizes mismatched bases after replication

Problem: how to recognize in a given DNA base pair whichis the template and which is the mutant base?

In prokaryotes, recognition is based on the DNA sequencerecognized by adenine methylase to perform DNAmethylation (addition of methyl group on template DNA)

5’….GATC….3’

3’….CTAG….5’

Post-Replication Repair Also called homologous recombination repair

Catalyzed by RecA protein

Indentified Miroslav Radman in E. coli

SOS Repair

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

Also occurs post-replication DNA polymerase continue replication across a given lesion

No gap is produced

Unspecific DNA bases might be added (compromised DNAbase fidelity)

Involves the proteins coded by lexA, recA and uvr genes

Double-Strand Break Repair

Also called homologous recombinational repair

Occurs when mutation occurs in both strands of adouble helix DNA

Involves the separation of a segment of a gene

During replication, no template will used for thesynthesis 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 ovariancancer



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Site-Directed Mutagenesis

Experimental techniqueused to produce a mutantgene to create a mutant

protein

Based on DNAhybridization principle

Insertion of a mutantcodon (resulting todifferent amino acid toproduce a mutant protein)

Gene Knockouts

Excision of an entire gene to observe its effect on anentire 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”

Nude mouse (immune system knockout) used to

regenerate human ear

Transposable Genetic Elements

Also called transposons or “jumping genes”

Genetic elements (insertion sequences, <2000 bp) thatmoves from chromosome to another

Results to posible disruption of a given gene ifmovement occurred in the middle of a gene

First studied by Barbarra McClintock (1983 Nobel PrizeWinner)

Implicated in antibiotic resistance (in certain strains ofpathogenic bacteria)

Jumping Genes in Corn

Ac –activator gene

Ds – dissociation gene

W – theoretical gene

Ac and Dc Elements Transposable Elements in Drosophila

Copia gene organization

DTR – direct terminal repeat (276 bp each) containsITR (inverted terminal repeat)

Documents

Gene-Lecture 13- Gene Mutation and Repair