Genetics Chapter 19 - Gene Mutation, DNA Repair and Recombination

Mismatch Repair Systems Recognize and Correct a Base Pair Mismatch

- A base mismatch is another type of abnormality in DNA - The structure of the DNA double helix obeys the AT/GC rule of base pairing: however, during DNA replication an incorrect base may be added to the growing strand by mistake - DNA polymerases have 3'-5' proofreading ability that can detect base mismatches and fix them - If proofreading fails, the mismatch repair system comes to the rescue - Mismatch repair systems found in all species - Important aspect of these systems is that they are specific to the newly made strand - Molecular mechanism of mismatch repair in E. coli: MutL, MutH, MutS detect the mismatch and direct its removal from the newly made strand - The proteins are named Mut because their absence leads to a much higher mutation rate than normal - Key characteristic of MutH: can distinguish between parental strand and daughter strand. Prior to replication, both parental strands are methylated, immediately after replication, the parental strand is methylated whereas the newly made daughter strand is not. - MutS protein slides along the DNA and finds a mismatch. The MutS/MutL complex binds to MutH, which is already bound to a hemimethylated sequence

Changes in Chromosome Structure Can Affect Gene Expression

- A chromosomal rearrangement may affect a gene because the chromosomal breakpoint (site of breaking and rejoining) occurs within the gene. - Alternatively, a gene may be left intact, but its expression may be altered because of its new location: position effect - Two common reasons for position effects: 1. movement to a position next to regulatory sequences 2. Movement to a heterochromatic region

Tautomeric Shift

- A temporary change in base structure - The common, stable form of thymine and guanine is the keto form: rarely, T and G convert to an enol form - The common, stable form of adenine and cytosine is the amino form: rarely, A and C can convert to an imino form - Causes mistakes in base pairing - To cause mutation, a tautomeric shift must occur immediately prior to replication

Oxidative Stress Leading to DNA Damage and Mutation

- Aerobic organisms produce Reactive Oxygen Species (ROS) including: hydrogen peroxide, superoxide, hydroxyl radical - Body tried to block buildup of ROS: enzymes such as superoxide dismutase and catalase, antioxidants - Oxidative stress: an imbalance between the production of ROS and an organism's ability to break them down - ROS overaccumulation can lead to oxidative DNA damage - Ex. guanine can be converted to 7,8-dihydro-8-oxoguanine (8-oxoG), pairs with adenine during replication. Causes GC base pair to go to TA base pair

Induced Mutations

- Agents that alter structure of DNA and thereby cause mutations - called mutagens - Concerned about mutagens for 2 main reasons: 1. mutagens often involved in development of human cancers 2. mutagens can cause gene mutations that may have harmful effects in future generations - Mutagenic agents usually classified as chemical or physical mutagens

Base Excision Repair Removes a Damaged Base

- Base excision repair (BER) involves a category of enzymes known as DNA N-glycosylases - These enzymes can recognize an abnormal base and cleave the bond between it and the sugar in the DNA - Depending on the species, this repair system can eliminate abnormal bases such as Uracil; 3-methyladenine; 7-methylguanine

DNA Repair

- Because most mutations are deleterious, DNA repair systems are vital to the survival of all organisms - Living cells contain several DNA repair systems that can fix different type of DNA alterations - In most cases, DNA repair is multi-step process 1. An irregularity in DNA structure is detected 2. The abnormal DNA is removed 3. Normal DNA is synthesized

Base Analogues

- Become incorporated into into daughter strands during DNA replication - Ex. 5-bromouracil is a thymine analogue - It can be incorporated into DNA instead of thymine - It can be incorporated into DNA instead of thymine - A tautomeric shift can result in pairing with guanine

Non-Homologous End Joining

- Broken ends are recognized by end-binding proteins: formation of crossbridge - Processing may result in deletion of small region: not error free

Point Mutation

- Change in single base pair - Can involve a base substitution - Transition: change of pyrimidine (C, T) to another pyrimidine or a purine (A, G) to another purine - Transversion: change of a pyrimidine to a purine or vice versa - Transitions more common than transversions

Mutations Can Be

- Changes in chromosome structure - Changes in chromosome number - Changes in DNA of a single gene: can affect molecular and phenotypic expression of genes

Intercalating Agents

- Contain flat planar structures that intercalate themselves into the double helix - Distorts the helical structure - When DNA containing these mutagens is replicated, the daughter strands may contain single-nucleotide additions and/or deletions resulting in frame-shifts - Ex. acridine dye, proflavin

Base Modifiers

- Covalently modify the structure of a nucleotide - Ex. nitrous acid replaces amino groups with keto groups (-NH2 to =O) - This can change cytosine to uracil and adenine to hypoxanthine - These modified bases do not pair with the appropriate nucleotides in the daughter strand during DNA replication - Some chemical mutagens disrupt the appropriate pairing between nucleotides by alkylating bases within the DNA - Ex. nitrogen mustards and ethyl methanesulfonate (EMS)

Double-Strand Breaks in DNA Can be Repaired by Recombination

- DNA double-strand breaks are very dangerous - Breakage of chromosomes into pieces - Caused by ionizing radiation and chemical mutagens: also caused by reactive oxygen species which are the by-products of cellular metabolism, 10-100 breaks occur each day in a typical human cell - Breaks can cause chromosomal rearrangements and deficiencies - May be repaired by two systems known as homologous recombination repair (HRR), and nonhomologous end joining (NHEJ) - Double-strand break is processed by the short digestion of the DNA strands - Sister chromatids are only available during S and G2 of cell cycle: used for strand exchange, rarely, HRR can occur between non-identical chromosomes - The unbroken strands are used as templates to synthesize DNA - Strands are then broken and then rejoined in a way that produces separate chromatids - Because sister chromatids are genetically identical, homologous recombination can be an error-free repair mechanism

Gene Mutations Outside of Coding Sequence

- Gene mutations outside outside of coding sequences can affect gene expression and phenotype - Mutations in core promoter can change levels of gene transcription - Up promoter mutations: increase transcription - Down promoter mutations: decrease transcription

Mutations in Germ-Line or Somatic Cells

- Germ-line Cells: cells that give rise to gametes such as eggs and sperm - Somatic Cells: all other cells - Germ-line Mutations: those that occur directly in a sperm or egg cell, or in one of their precursor cells. Occur in gametes - passed to half of the gametes in the next generation; mutation found in the whole body - Somatic Mutations: those that occur directly in a body cell that is not part of the germ-line. results in patches of affected area - size of patch will depend on timing of the mutation. The earlier the mutation, the larger the patch. An individual with somatic regions that are genotypically different from the rest of the body is called a genetic mosaic, mutations not present in gametes

Causes of Gene Conversion

- Homologous recombination can cause two different alleles to become identical alleles: this proces,s whereby one of the alleles is converted to the other, has been termed gene conversion, the converted allele is close to the crossover site - Gene conversion can occur in one of two ways: 1. DNA mismatch repair 2. DNA gap repair synthesis - Gap Repair Synthesis: gene conversion by gap repair synthesis according to the double-strand break model. The top chromosome carries the recessive b allele, and the bottom chromosome carries the dominant B allele

Nucleotide Excision Repair Removes Damaged DNA Segments

- Important general process for DNA repair - This type of system can repair many types of DNA damage, including: thymine dimers and chemically modified bases, missing bases, some types of crosslinks - NER is found in all eukaryotes and prokaryotes - However, its molecular mechanism is better understood in prokaryotes - In E. coli the NER system requires 4 key proteins: UvrA, UvrB, UvrC, UvrD - Named because they are involved in Ultraviolet light repair of pyrimidine dimers: they are also important in repairing chemically damaged DNA - UvrA, B, C and D recognize and remove a short segment of damaged DNA, DNA polymerase and ligase finish the repair job - Several human disease have been shown to involve inherited defects in genes involved in NER - These include xeroderma pigmentosum (XP), cockayne syndrome (CS), and PIBIDS - A common characteristic of all three syndromes is an increased sensitivity to sunlight - XP can be caused by defects in seven different NER genes

Damaged Bases Can be Directly Repaired

- In a few cases, the covalent modifications of nucleotides can be reversed by specific enzymes - Photolyase: can repair thymine dimers It splits the dimers restoring the DNA to its original condition Uses energy of visible light for photoreactivation - Alkyltransferase: repairs alkylated bases It transfers the methyl or ethyl group from the base go a cysteine side chain within the alkyltransferase protein Surprisingly, this permanently inactivates alkyltransferase

Gene Mutations Given Names that Describe How They Affect Genotype and Phenotype

- In natural population, the wild-type is the relatively prevalent genotype. Genes with multiple alleles may have two or more wild-types. - Forward mutation: changes the wild-type genotype into some new variation - Reverse mutation: changes a mutant allele back to the wild-type (reversion)

Homologous Recombination

- Involves crossing over between identical or homologous regions of chromosomes - Occcurs in meiosis I and occasionally during mitosis, involves the alignment or a pair of homologous chromosomes, followed by breakage at analogous locations and exchange of corresponding segments - Crossing over occurs between sister chromatids: sister chromatid exchange (SCE) - Sister chromatids are genetically identical to each other - SCE does not produce a new combination of alleles - Crossing over that occurs between homologous chromosomes may produce new combinations of alleles

Damaged DNA May be Replicated by Translesion DNA Polymerases

- It is inevitable that some lesions may escape all repair systems: such lesions may be present when DNA is replicated - Replicative DNA polymerases, such as DNA pol III in E. coli, are sensitive to geometric distortions in DNA: they are unable to replicated through DNA lesions, this type of replication requires specialized DNA polymerases - Specialized enzymes assist in replicative DNA polymerase in the translesion synthesis (TLS) procession - The translesion-replicating polymerases contain an active site with a loose, flexible pocket - They can accommodate aberrant structures in the template strand - Negative consequence of translesion-replicating polymerases is their low fidelity - The mutation rate is typically in the range of 10^-2 - 10^-3, much higher than 10^-8 of replicative polymerase - Specialized enzymes assist in DNA polymerase in translesion synthesis (TLS) process - The translesion-replicating polymerases contain an active site with a loose, flexible pocket - They can accommodate aberrant structures in the template strand - A negative consequence of translesion-replicating polymerases is their low fidelity: the mutation rate is typically in the range of 10^-2-10^-3, much higher than 10^-8 of replicative polymerase - When a replicative DNA polymerase encounters a damaged region, it is swapped for a TLS polymerase, region is duplicated with error-prone replication

Testing Methods to Determine if an Agent is a Mutagen

- Many different tests have been used to evaluate mutagenicity - One commonly used test is the Ames test - Developed by Bruce Ames: The test uses a strain of Salmonella typhimurium that cannot synthesize the amino acid histidine: it has a point mutation in a gene involved in histidine biosynthesis - A second mutation (that is, a reversion) may occur, thereby restoring the ability to synthesize histidine - The Ames test monitors the rate at which this second mutation occurs - Different strains with transition, transversion or frameshift mutations can be used - The control plate indicates that there is a low level of spontaneous mutation - Mix together the suspected mutagen, a rat liver extract, and a salmonella strain that cannot synthesize histidine. The suspected mutagen is omitted from the control sample.

Various Proteins Facilitate Homologous Recombination

- Molecular studies in two different yeast species suggest that double-strand breaks initiate the homologous recombination that occurs in meiosis - In other words, double-strand breaks create sites where a crossover will occur - In saccharomyces cerevisiase, formation of DNA double-strand breaks requires at least 10 different proteins: one of them, Spo11, is instrumental in actually breaking the DNA - Homologous recombination is found in all species, the cells of any given species may have more than one molecular mechanism for homologous recombination - The enzymology of homologous recombination is best understood in E. coli

More Recent Models Have Refined the Steps of Recombination

- More detailed studies of genetic recombination have led to a refinement of the Holliday model - In particular, more recent models have modified the initiation phase of recombination - Two nicks in the same location on two strands is unlikely - Rather, it is more likely for a DNA helix to incur a break in both strands of one chromatid

Mutations Based on Their Effects on Wild-Type Phenotype

- Mutations can also be described based on their effects on the wild-type phenotype - Often characterized by differential ability to survive - Deleterious mutations: decrease chances of survival, most extreme are lethal mutations -Beneficial mutations: enhance the survival or reproductive success of an organism, the environment can affect whether a given mutation is deleterious or beneficial - Conditional: affect phenotype only under defined set of conditions, ex. temperature-sensitive mutation

Depurination

- Removal of a purine (guanine or adenine) from the DNA forms an apurinic site - Covalent bond between deoxyribose and a purine base is somewhat unstable - It occasionally undergoes a spontaneous reaction with water that releases the base from the sugar, mammalian cells lose approximately 10,000 purines per 24 hours at 37 degrees - Apurinic sites can be repaired: however, if the repair system fails, a mutation may result during subsequent rounds of DNA replication. polymerase will add a random base - During replication, 3 out of 4 bases (A, T, and G) are the incorrect nucleotide - 75% chance of mutation

Deamination of Cytosine

- Removal of amino group from the cytosine base: other bases not readily deaminated - DNA repair enzymes can recognize uracil as an inappropriate base in DNA and remove it - However, if the repair system fails, a C-G to A-T mutation will result during subsequent rounds of DNA replication - 5-methylcytosine can be deaminated into thymine, a normal constituent of DNA - Repair enzymes cannot determine which of the two bases on the 2 DNA strands is the incorrect base - For this reason, methylated cytosine bases tend to create hot spots for mutation

Mutations Due to Trinucleotide Repeats

- Several human genetic diseases caused by unusual form of mutations: trinucleotide repeats expansion (TNRE) - Certain regions of chromosome contain trinucleotide sequences repeated in tandem - In individuals without disease symptoms, these sequences are transmitted from parent to offspring without mutation - However, in persons with TNRE disorders, the length of a trinucleotide repeat has increased above a certain critical size (disease symptoms occur) - In some cases, the expansion is within the coding sequence of the gene - Typically the trinucleotide expansion is CAG (glutamine) - Therefore, the encoded protein will contain long racks of glutamine: this causes the proteins to aggregate with each other, this aggregation is correlated with the progression of the disease, but may not cause disease symptoms - In other cases, the expansions are located in noncoding regions of genes: some of these expansions are hypothesized to cause abnormal changes in RNA structure, some produce methylated CpG islands which may silence the gene - Some TNRE disorders progressively worsen in future generations - may depend on which parent the mutant allele comes from: In Huntington disease, the TNRE is more likely to occur if inherited from the father, in myotonic muscular dystrophy, the TNRE s more likely to occur if inherited from mother. This suggests that TNRE can occur more frequently during oogenesis or spermatogenesis, depending on gene involved: anticipation

Spontaneous Mutations

- Spontaneous Mutations: result from abnormalities in cellular/biological processes - ex. errors in DNA replication. Underlying cause originates within the cell - Induced mutations: caused by environmental agents, agents that are known to alter DNA structure are termed mutagens - can be chemical or physical agents - Spontaneous mutations can arise by 3 types of chemical changes: 1. Depurination 2. Deamination 3. Tautomeric shift

Experiment Testing Random Mutation Theory

- Studied resistance of E. coli to infection by bacteriophage T1 - tonr (T one resistance) - Wondered if tonr is due to spontaneous mutations that occur at a low rate or to a physiological adaption - physiological adaption hypothesis predicts that the number of tonr bacteria is very low unless there is selection for T1 resistance. - Random mutation hypothesis: predicts that mutations will happen randomly and will occur without selection - Mutations had occurred randomly in the absence of selection by T1 - Became observable with selection but new colonies did not appear due to the presence of T1 - supports random mutation hypothesis - random mutation theory

Suppressor Mutations

- Suppressor mutations reverse phenotypic effects of another mutation - Intragenic suppressors: second mutation is within the same gene as the first mutation, typically, the first mutation causes an abnormality in protein structure and second mutation restores normal protein structure. - Intergenic suppressors: second mutation is in a different gene from the first mutation. Examples: redundant function, common pathway, multimeric proteins, transcription factors

Mechanism of Trinucleotide Repeat Expansion

- TNREs contain at least one C and one G: this allows formation of a hairpin - During DNA replication, a hairpin can lead to an increase or decrease in the length of DNA: polymerase can slip off DNA, hairpin forms and pulls strand back, DNA polymerase hops back on - begins synthesis from new location - These changes can occur during gamete formation: offspring will have very different numbers of repeats - Can also increase repeats in somatic cells: this can increase severity of the disease with age

Holliday Model for Homologous Recombination

- The Holliday model can account for the general properties of homologous recombination during meiosis deduced from genetic crosses in fungi - A particularly convincing piece of evidence came from electron micrographs of recombination structures: the structure has been called a chi (X) form

Mutation Rates and Frequencies

- The term mutation rate is the likelihood that a gene will be altered by a new mutation - Commonly expressed as the number of new mutations in a given gene per cell generation - Range of 10^-5 - 10^-9 per generation: humans add 100-200 new mutations/generation - The mutation rate for a given gene is not constant: it can be increased by the presence of mutagens - Mutation rates vary substantially between species and even within different strains o the same species - Mutation frequency for a gene is the number of mutant genes divided by the total number of genes in a population - If 1 million bacteria were plated and 10 were mutant: mutation frequency would be 1 in 100,000 or 10^-5 - The mutation frequency is important in areas of genetics such as population genetics - Mutation frequencies may become greater than mutation rates, due to natural selection and genetic drift

Mutagens Alter DNA Structure

1. Base Modifiers: some covalently modify base structure, others disrupt pairing by alkylating bases 2. Intercalating Agents: directly interfere with replication process 3. Base Analogues: incorporate into DNA and disrupt structure, some tautomerize at a high rate - Physical mutagens include radiation: x-rays, gamma rays, ionizing radiation, UV light

Nonsense Mutations

Base substitutions that change a normal codon to a stop codon

Mutation

Heritable change in genetic material, provide allelic variation - On the positive side, mutations are the foundation for evolutionary change needed for a species to adapt to changes in the environment - On the negative side, new mutations are much more likely to be harmful then beneficial to the individual and often are the cause of diseases - Since mutations can be harmful, organisms have developed ways to repair damaged DNA

Frameshift Mutations

Involve the addition or deletion of a number of nucleotides that is not divisible by three - This shifts the reading frame so that translation of the mRNA results in a completely different amino acid sequence downstream of the mutation

Homologous Recombination

Involves exchange of identical or similar DNA segments between homologous chromosomes; enhances genetic diversity; involved in DNA repair

Types of Physical Mutagens

Ionizing radiation: includes X-rays and gamma rays - Has short wavelength and high energy - Can penetrate deeply into biological molecules - Creates chemically reactive molecules termed free radicals - Can cause: base deletions, oxidized bases, single nicks in DNA strands, cross-linking, chromosomal breaks. - Nonionizing radiation: includes UV light - has less energy, cannot penetrate deeply into biological molecules, causes the formation of cross-linked thymine dimers, thymine dimers may cause mutations when that DNA strand is replicated

Missense Mutation

base substitutions in which an amino acid change does occur. Ex. Sick Cell, unlike sickle cell disease, a missense mutation may have no detectable effect on protein function, and the mutation is said to be neutral. This is more likely to occur if the new amino acid has similar chemistry to the amino acid it replaced. Glutamic acid replaced with valine

Silent Mutations

base substitutions that do not alter the amino acid sequence of the polypeptide, due to the degeneracy of the genetic code