chapter 13 mutation, dna repair,… © john wiley & sons, inc

Chapter 13Mutation, DNA Repair,…

© John Wiley & Sons, Inc.

Amino acids in polypeptides are joined by what type of bond?

a) Hydrogenb) no-Peptidec) Covalentd) Ionice) Weak

Which of the following is a secondary structure often observed in proteins?

a) α helixb) β sheetc) σ subunitd) α helix and β sheete) All of these

Which of the following is not a true statement about ribosomes?

a) They are structured into large and small subunitsb) They are comprised of RNA and proteinc) The ribosomes in prokaryotes are typically larger than those found in eukaryotesd) The ribosome subunits associate during the initiation of translatione) All of these are false statements

Chapter Outline Mutation: Source of the Genetic Variability Required for

Evolution The Molecular Basis of Mutation Mutation: Basic Features of the Process Mutation: Phenotypic Effects Assigning Mutations to Genes by the Complementation Test Screening Chemicals for Mutagenicity: The Ames Test

DNA Repair Mechanisms Inherited Human Diseases with Defects in DNA Repair DNA Recombination Mechanisms


Mutation: Source of the Genetic Variability Required for Evolution


Mutation--A change in the genetic material (molecular level)

Mutant --an organism that exhibits a novel phenotype

Types of Mutations– Changes in chromosome number and

structure– Point mutation--changes at specific

nucleotide in a gene (A,T,C,G)– Insertion mutations--insert fragment of

DNA– Deletion mutations--delete fragment of

DNA


“Virus”

Disruption of FGF -Receptor Function

Fibroblast Growth Factor Signaling is Essential for Mesoderm Production in Frog Embryos

Dominant mutations in human FGFR-3 (TM domain)

Achondroplasia

Mutation and Evolution

Mutation is the source of all genetic variation (e.g.,chromatin remodeling).

Natural selection preserves the combinations best adapted (or NOT) to the existing environment.


The Molecular Basis of Mutation

Mutations alter the nucleotide sequences of genes in several ways,

--the substitution of one base pair for another.

(A for T)

--the deletion (or addition) of one or a few base pairs. ( AT…….GC)


Tautomeric Shifts: --chemical fluctuations, --conformation states (stable==========unstable)

A:T

C:G.

Py

Pu

Tautomeric Shifts Affect Base-Pairing


C:T

T:G.

Mutation Caused by Tautomeric Shifts


Base Substitutions A transition replaces a pyrimidine with another

pyrimidine or a purine for another purine. A transversion replaces a pyrimidine with a

purine or a purine with a pyrimidine.


Frameshift Mutations: alteration of the open reading frame (ORF)


Mutation Frequency

Frameshift, transition, transversion mutations are infrequent– Bacteria and phage: 10–8 to 10–10 per nucleotide

pair per generation– Eukaryotes: 10–7 to 10–9 per nucleotide pair per

generation


1/107 to 1/109

Silent mutation: UCU=Ser; UCA, UCC, UCG = Ser

Induced Mutations Induced mutations occur upon exposure to

physical (energy) or chemical (reaction) mutagens.

Muller demonstrated that exposing Drosophila sperm to X-rays increased the mutation frequency.

Hermann J. Muller and Edgar Alternburg measured the frequency (>150 fold increase) of X-linked recessive lethal mutations in Drosophila.



C: crossover suppressorl: recessive lethal mutationB: bar-eye mutation

The Electromagnetic Spectrum

X-rays induce mutations through ionization.

(DNA ionization--radical anions and cations-- G.+)

Ultraviolet light induces mutations through excitation.


Ionizing Radiation Causes Changes in Chromosome Structure

Ionizing radiation breaks chromosomes and can cause deletions, duplications, inversions, and translocations


Stability of carbon-containing molecules

Mutagenesis by Ultraviolet Irradiation

Hydrolysis of cytosine to a hydrate may cause mis-pairing during replication

Cross-linking of adjacent thymine forms thymidine dimers, which block DNA replication and activate DNA repair mechanisms.


UV-A 320 to 400 nmUV-B/C <300 nm

Types of Chemical Mutagens

Chemicals that are mutagenic to both replicating and non-replicating DNA (e.g., alkylating agents and nitrous acid)

Chemicals that are mutagenic only to replicating DNA (e.g., base analogs and acridine dyes)


Chemical Mutagens


Alkylating Agents chemicals that donate alkyl groups to other molecules.

induce transitions, transversions, frameshifts, and chromosome aberrations (anomaly).

Alkylating agents of bases can change base-pairing properties. (GC to AT)

can also activate errors during repair processes.


A Base Analog: 5-Bromouracil


--similar structures--incorporated into DNA--increase frequency of mis-pairing

Mutagenic Effects of 5-Bromouracil


Nitrous Acid Causes Oxidative Deamination of Bases


Intercalation of an Acridine Dye Causes Frameshift Mutations


--(+) charges molecules

--Incorporated into DNA

--DNA is more rigid

--Change conformation

(non-bending)

Hydroxylamine(NH2OH)

Hydroxylamine is a hydroxylating (OH) agent.

Hydroxylamine hydroxylates the amino group of cytosine and leads to G:C A:T transitions.


.

Mutations Induced by Transposons(Repeats)


Fragmets/segments of DNA that are capable to shift / translocate/move from one location to another

Expansion of Trinucleotide Repeats

Simple tandem repeats are repeated sequence of one to six nucleotide pairs (CGG, CAG and CTG).

Trinucleotide repeats can increase in copy number and cause inherited diseases (Fragile X Syndrome, Huntington disease, Spinocerebellar ataxia)


• Mutations are induced by • chemicals, • ionizing irradiation, • ultraviolet light, and • endo(exo)genous transposable genetic elements.

• Point mutations are of three types:(1) Transitions—purine for purine and pyrimidine for pyrimidine substitutions,(2) Transversions—purine for pyrimidine and pyrimidine for purine substitutions,

and(3) Frameshift mutations—additions or deletions of one or two nucleotide pairs,

which alter the reading frame of the gene distal to the site of the mutation.

Chromatin remodeling==Epigenetics


Mutation: Basic Features of the Process

Mutations occur in all organisms from viruses to humans.

They can occur spontaneously or be induced by mutagenic agents.

Mutation is usually a random, non-adaptive process.


Mutation: Somatic or GerminalGerminal mutations occur in germ-line

cells and will be transmitted through the gametes to the progeny.

Somatic mutations occur in somatic cells; the mutant phenotype will occur only in the descendants of that cell and will not be transmitted to the progeny.


puberty (10-14 years)

Mutation: Spontaneous or Induced

Spontaneous mutations occur without a known cause due to unknown agents in the environment.

Induced mutations result from exposure or organisms to mutagens, physical and chemical agents that cause changes in DNA, such as ionizing irradiation, ultraviolet light, or certain chemicals.


Factors Influencing the Rate of Spontaneous Mutations

Accuracy of the DNA replication machinery

Efficiency of the mechanisms for the repair of damaged DNA

Degree of exposure to mutagenic agents in the environment


Mutation: Usually a Random, Non-adaptive Process

Is mutation random (intrinsic) or directed by the environment?

Replica plating was used to identify the presence of antibiotic (chemical) resistant bacteria prior to treatment with an antibiotic (chemicals).

Environmental stress does not cause mutations but selects for mutants that are best adapted to the environmental stress.


Mutation: A Reversible Process

Forward mutation—mutation of a wild-type allele to a mutant allele.

Reverse mutation (reversion)—a second mutation that restores the original phenotype.– Back mutation—a second mutation at the

same site.– Suppressor mutation—a second mutation

at a different location in the genome.



Phenotype is not suppressed

Phenotype is restored

Mutation: Phenotypic Effects

The effects of mutations on phenotype range from no

observable change to lethality.


DNA/RNA/ polypeptide

Types of Mutations Isoalleles have no effect on phenotype or small

effects that can be recognized only by special techniques.

Null alleles result in no gene product or totally non-functional gene products.

Recessive (Dominant) lethal mutations affect genes required for growth of the organisms and are lethal in the homozygous state.


X-linked Recessive Lethal Mutations Alter the Sex Ratio


Monoploid: (Recessive or Dominant) positive mutational effect (phenotype)Diploid: (Recessive)--positive mutational effect (phenotype)--homozygousX-linked .....hemizygous

Recessive Mutations Often Block Metabolic Pathways

(Recessive) lethal mutations

Neutral mutations ( no effect on phenotype)

Expression of Wild-type and Mutant Alleles


Mutations in Human Globin Genes Adult hemoglobin (Hemoglobin A) contains two α

chains and two β chains.

Hemoglobin in patients with sickle-cell anemia (Hemoglobin S) differs from Hemoglobin A at only one position.

The sixth amino acid in the β chain is glutamic acid in Hemoglobin A (HBBA) and is valine in Hemoglobin S (HBBS). This substitution is caused by mutation of a single base pair (T:A substitution).


Tay-Sachs Disease

Tay-Sachs disease is an autosomal recessive disease.

The mutation causing Tay-Sachs disease is in the gene encoding hexosaminidase A.

© John Wiley & Sons, Inc.not linked to sex chromosome

Conditional Lethal Mutations(Experimental)

Conditional lethal mutations are– Lethal in the restrictive condition but– Viable in the permissive condition.

Mutants with conditional lethal alleles can be propagated under the permissive condition, and the phenotype can be studied under restrictive condition.


Conditional Lethal Mutants

Auxotrophs are unable to synthesize an essential metabolite that is synthesized by prototrophs. Auxotrophs can grow only when the essential metabolite is supplied in the medium.

Temperature-sensitive mutants will grow at one temperature but not at another.

Suppressor-sensitive mutants are viable only when a second genetic factor, a suppressor, is present.


Morphogenesis in Bacteriophage T4

This pathway was identified using mutants, electron microscopy, and biochemistry.

Temperature-sensitive and suppressor-sensitive


# =gene

http://www.endocytosis.org/Dynamin/Shibire.html

http://www.endocytosis.org/Dynamin/Shibire.html

Assigning Mutations to genes by the Complementation test

The complementation or trans test can be used to determine

whether two mutations are located in the same chromosome or in two

different chromosomes.


Genetic tool to demonstrate: One gene....One polypeptide


Assigning Mutations

Double heterozygote:Two mutations (m1 and m2)Wild-type (m1+ and m2+)Co-exists in one or twochromosomes

coupling

repulsionArrangement (organized)


Apr and W arerecessive mutations

X-linked mutations


(head) (tail)


?

Screening Chemicals for mutagenicity: The Ames test

The Ames test provides a simple and inexpensive method for detecting the mutagenicity of chemicals (Carcinogens)

-intracellular

-extracellular

-enviromental

© John Wiley & Sons, Inc.Auxotrophic prototrophic

DNA Repair Mechanisms

Living organisms contain many enzymes that scan their DNA for

damage and initiate repair processes when damage is

detected.


DNA Repair Mechanisms in E. coli

Light-dependent repair (photo-reactivation).Excision repair.Mismatch repair.Post-replication repair.Error-prone repair system (SOS response).


UV Light-Dependent Repair:

Photolyase Cleaves Thymine

Dimers.


--No endonuclease--No Poly--No ligase

Excision Repair (steps) A DNA repair endonuclease or endonuclease-

containing complex recognizes, binds to, and excised the damaged base or bases.

A DNA Polymerase fills in the gap, using the undamaged complementary strand of DNA as a template.

DNA ligase seals the break left by DNA polymerase.


Types of Excision Repair

Base excision repair pathways remove abnormal or chemically modified bases.

Nucleotide excision repair pathways remove larger defects, such as thymine diners.



( )

Nucleotide Excision Repair


Mismatch Repair in E. coli Mismatching or mispairing of G and T (DNA polymerase/exonuclease proofreading activity)

The A in GATC sequences is methylated subsequent to DNA replication.

In newly replicated DNA, the parental strand is methylated, but the new strand is not. This difference allows the mismatch repair system to distinguish the new strand from the old strand.

The mismatched nucleotide is excised from the new strand and replaced with the correct nucleotide, using the methylated parental strand as a template.


Exonuclease

Endonuclease

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Mismatch Repair in E. coli MutS recognizes mismatches and binds to

them to initiate the repair process. MutH and MutL join the complex. MutH cleaves the unmethylated strand at

hemimethylated GATC sequences on either side of the mismatch.

Excision requires MutS, MutL, MutU (DNA helicase II), and an exonuclease.

DNA polymerase III fills in the gap, and DNA ligase seals the nick.


Me

MeMe

Me

Post-replication Repair in E. coli A thymine dimer in the template strand blocks replication

(DNA Polymerase III does not recognize thymidine dimer)

DNA Polymerase III restarts DNA synthesis past the dimer, leaving a gap in the nascent strand.

RecA binds to the single strand of DNA at the gap and mediates base pairing with the homologous segment of the sister double helix to fill the gap.

DNA polymerase fills the gap in the sister double helix, and DNA ligase seals the nick.


The SOS Response in E. coli If DNA is heavily damaged by mutagenic agents, the

SOS response, which involves many DNA recombination, DNA repair, and DNA replication proteins, is activated.

DNA dependent DNA Polymerase V replicates DNA in damaged regions, but sequences in damaged regions cannot be replicated accurately.

This error-prone system eliminates gaps but increases the frequency of replication errors (Pol II, IV and V are low-fidelity polymerases)


Induction of the SOS Response In the absence of DNA damage, LexA binds to DNA

regions that regulate transcription of SOS response genes and keeps their expression levels low.

When extensive DNA damage occurs, RecA binds to single-stranded regions of DNA in damaged regions.

This activates RecA, which stimulates LexA to inactivate itself. When LexA is inactivated, the SOS response genes are expressed.


Inherited Human Diseases with Defects in DNA Repair

Several inherited human disorders result from defects in DNA repair

pathways.


Xeroderma Pigmentosum (XP) Individuals with XP are

sensitive to sunlight (UV light).

The cells of individuals with XP are deficient in the repair of UV-induced damage to DNA.

Individuals with XP may develop skin cancer or neurological abnormalities.


DNA Recombination Mechanisms

Recombination between homologous DNA molecules involves the activity of

numerous enzymes that

1-cleave,

2-unwind,

3-stimulate single-strand invasions of double helices (RecA proteins),

4-repair, and

5-join strands of DNA.© John Wiley & Sons, Inc.

Recombination

In eukaryotes, crossing over is associated with the formation of the synaptonemal complex during prophase of meiosis I (Chiasmata).

Crossing over involves the breakage of parental chromosomes and rejoining of the parts in new combinations.

The Holliday model and the double-strand break model are two explanations of the molecular basis of recombination.


The Holliday Model: single strand break model

The Holliday Model: single strand break model

--DNA-dependent ATPase.

--can hold a single strand and double strand together.

--DNA synapsis reaction between a DNA double helix and a homologous region of single stranded DNA.

--catalyzes branch migration begins.

The Recombination Intermediate is a Chi Structure


http://web.mit.edu/engelward-lab/animations/DSBR.html

5) Which of the following is considered a point mutation?1. Substitution of the base A for the base C2. Deletion of the base T3. Insertion of the base G

a) 1b) 2c) 3d) 1 and 2e) All of these

Which base pair combinations can form when nitrogenous bases are present in their rare imino or enol states?

a) A:Tb) C:Gc) A:Cd) A:T and C:Ge) C:G and A:C

Which of the following enzymes performs light dependent repair?

a) DNA gyraseb) DNA polymerasec) DNA photolyased) RNA polymerasee) DNA helicase

chapter 13 mutation, dna repair,… © john wiley & sons, inc

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