Genetics Ch. 19

Why are we concerned with mutagens?

- involved in the development of human cancers - new mutations may be harmful and may have detrimental effects on their future offspring.

Steps in DNA repair via N-glyosylase

1. N-glycosylase recognizes an abnormal base and cleaves the bond between the base and the sugar forming an apyrimidinic site. 2. AP endonuclease recognizes a missing base and cleaves the DNA backbone at the 5' side of the missing base. 3 ways it could go: 3a. In E. coli DNA polymerase I uses in its 5' to 3' exonuclease activity to remove the damaged region and then fills in the region with normal DNA. DNA ligase seals the region. 3b. In eukaryotes, such as humans, DNA polymerase beta can remove the apyrimidinic site and replace it with the correct nucleotide. DNA ligase seals the region. 3c. In eukaryotes such as humans, DNA polymerase delta or epsilon synthesizes a short segment of DNA, which generates a flap Flap is removed by flap endonuclease. DNA ligase seals the region

replica plating experiment procedure

1. place individual bacterial cells onto growth media 2. Incubate overnight to allow the formation of bacterial colonies (the master plate) 3. press a velvet cloth (wrapped over a cylinder) onto the master plate, and then lift gently to obtain a replica of each bacterial colony. Press the replica onto 2 secondary plates that contain T1 phage. Incubate overnight to allow growth of mutant cells.

What happens at an apurinic site during DNA replication?

Because a complementary base is not present to specify the incoming base for the new strand, any of the four bases are added to the new strand in the region that is opposite the apurinic site which may produce a new mutation.

How do DNA repair enzymes repair deamination? What happens if the repair enzymes do not do their job?

DNA repair enzymes can recognize uracil as an inappropriate base within DNA and subsequently remove it. If repair does not take place, a mutation may result because uracil hydrogen bonds with adenine during DNA replication. Therefore if a DNA template strand has uracil instead of cytosine, a newly made strand will incorporate adenine instead of guanine.

What can happen at a low rate to G or T and A or C?

G or T can convert to an enol form, and A or C can change to an imino form

How do we judge if an agent is a mutagen?

Researchers compare the mutation rate in the presence and absence of the suspected mutagen

AP endonuclease

a DNA repair enzyme that recognizes a DNA region that is missing a base and makes a cut in the DNA backbone near that site.

Homologous recombination

a DNA repair system that occurs at double-stranded breaks or when DNA repair damage causes a gap in synthesis during DNA replication. The strands of the normal sister chromatids are used to repair a damaged sister chromatid.

somatic mutation

a mutation in a somatic cell

overaccumulation of ROS is implicated in what medical conditions

cardiovascular disease, Alzheimers disease, chronic fatigue syndrome, and aging.

amino form

the common form of adenine and cytosine

Uvr stands for

ultraviolet light repair - for thymine dimers and repairing chemically damaged DNA

Steps of E. coli NER system

1. The UvrA/UvrB complex tracks along the DNA in search of damaged DNA (distorted double helix). 2. After damage is detected, UvrA is released and UvrC binds. 3. UvrC makes cuts on both sides of the thymine dimer. 4. UvrD, which is a helicase, removes the damaged region. UvrB and UvrC are also released. 5. DNA polymerase fills in the gap and DNA ligase seals the region. (diagram in 9.5)

How can spontaneous mutations arise (3)?

Depurination Deamination Tautomeric shifts

non-ionizing radiation

Ex. UV light has less energy and so it penetrates only the surface of an organism such as the skin can cause mutations

nucleotide excision repair (NER)

a DNA repair system in which several nucleotides in the damaged strand are removed from the DNA and the undamaged strand is used as a template to resynthesize a normal strand. used to repair bulky, helix-distorting lesions can repair thymine dimers, chemically modified bases, missing bases, and certain types of crosslinks in all eukaryotes and prokaryotes

Nonhomologous ends joining

a DNA repair system that occurs at double-stranded breaks. The broken ends are recognized by proteins that keep the ends together; the broken ends are eventually rejoined.

Base excision repair and nucleotide excision repair

a DNA repair system where an abnormal base or nucleotide is first recognized and removed from the DNA, and a segment of DNA in this region is excised. Then the complementary DNA strand is used as a template to synthesize a normal DNA strand

direct repair

a DNA repair system where an enzyme recognizes an incorrect alteration in DNA structure and directly converts the structure back to the correct form.

2-aminopurine

a base analog that acts as a chemical mutagen incorporated during DNA replication

Missense mutation + Ex.

a base substitution that leads to a change in the amino acid sequence of the encoded polypeptide. Ex. sickle cell disease - mutation in the beta-globin gene changes the sixth amino acid from glutamic to valine

induced mutation

a change in DNA structure caused by an environmental agent

spontaneous mutations

a change in DNA structure that results from natural biological or chemical processes

point mutation

a change in a single base pair within DNA

position effect

a change in phenotype that occurs when the location of a gene changes from one chromosomal site to a different one.

conditional mutant

a mutant whose phenotype depends on the environmental conditions, such as temperature-sensitive mutant.

suppressor or suppressor mutation

a mutation at a second site that suppresses the phenotypic effects of another mutation

germ-line mutation

a mutation in a cell of the germ line. if the gamete participates in fertilization, all cells of the offspring will have the mutation

down promoter mutation

a mutation in a promoter that decreases the rate of transcription

up promoter mutation

a mutation in a promoter that increases the rate of transcription

reversion

a mutation that changes a mutant back to a wild-type allele

silent mutation

a mutation that does not alter the amino acid sequence of the encoded polypeptide even though the base sequence has changed

beneficial mutation + Ex.

a mutation that enhances the survival or reproductive success of an organism Ex. Heterozygous for sickle cell

neutral mutation

a mutation that has no detectable effect on protein function or no detectable effect on survival of the organism

deleterious mutation

a mutation that is detrimental with regard to its effects on phenotype - decreases the chance of survival and reproductive success

lethal mutation

a mutation that produces an allele that results in the death of a cell or an organism

transversion

a point mutation in which a purine is interchanged with a pyrimidine or vice versa

base substitution

a point mutation in which one base is substituted for another

transition

a point mutation involving a change of pyrimidine to another pyrimidine (like C to T) or purine to another purine (like A to G)

breakpoint

a region where two chromosome pieces break apart and rejoin with other chromosome pieces when it occurs in the middle of a gene = inhibit gene function

wild type

a relatively prevalent genotype in a natural population

apurinic site How is it created?

a site in the DNA that is missing a purine base The covalent bond between deoxyribose and a purine base is somewhat unstable and occasionally undergoes a spontaneous reaction with water that releases the base from the sugar, thereby creating an apurinic site.

intragenic suppressor

a suppressor mutation that is within the same gene as the first mutation that it suppresses. - often produces a change in protein structure that compensates for an abnormality in protein structure caused by the first mutation.

intergenic suppressor

a suppressor mutation that occurs in a different gene than the gene that contains the first mutation the change in expression of one gene compensates for a loss-of-function mutation affecting another gene.

replica plating

a technique in which replicas of bacterial colonies are transferred to new growth plates

tautomeric shift

a temporary change in chemical structure, such as an alteration between the keto and enol forms of the base that are found in DNA.

Ames test

a test using strains of a bacterium, Salmonella typhimurium, to determine in a substance is a mutagen

5-bromouracil What happens at a high rate?

a thymine base analog that acts as a chemical mutagen incorporated during DNA replication base pairs with adenine At a high rate, 5BU undergoes a tautomeric shift and base-pairs with guanine - transition mutation because the adenine has been changed to a guanine

base excision repair (BER)

a type of DNA repair in which a modified base is removed from a DNA strand. Following base removal, a short region of the DNA strand is removed and then resynthesized using the complementary strand as a template.

photoreactivation

a type of DNA repair mechanism of thymine dimers that involves photolyase and requires light directly restores the structure of DNA

ethyl methanesulfonate (EMS)

a type of chemical mutagen that alkylates bases (i.e attaches methyl or ethyl groups)

nitrous acid

a type of chemical mutagen that deaminates bases, replacing amino groups with keto groups.

acridine dye

a type of chemical mutagen that intercalates between adjacent base pairs in DNA and causes frameshift mutations.

trinucleotide repeat expansion (TNRE) + Ex.

a type of mutation that involves an increase in the number of tandemly repeated trinucleotide sequences typically involves CAG repeats which encode for glutamine long glutamine tracts cause the proteins to aggregate

random mutation theory

according to this theory, mutations are random events- they can occur in any gene and do not require exposure of an organism to an environmental condition that causes specific types of mutations.

proflavin

an acridine dye, which is a chemical mutagen that causes frameshift mutations

mutagens

an agent that can alter the structure of DNA, causing a mutation

nitrogen mustard

an alkylating agent that can cause mutations in DNA a type of mustard gas used during WWI to damage skin, eyes, mucous membranes, lungs and blood-forming organs

mutant allele

an allele that has been created by altering a wild-type allele by mutation

photolyase What does it contain?

an enzyme found in bacteria, fungi, most plants, and some animals that can recognize and split thymine dimers, which returns the DNA to its original condition contains two light-sensitive cofactors

DNA N-glycosylase

an enzyme that can recognize an abnormal base and cleave the bond between it and the sugar in the DNA backbone involved in (BER)

alkyltransferase

an enzyme that can remove methyl or ethyl groups from guanine bases that have been mutagenized by alkylating agents such as nitrogen mustard and EMS. transfers the methyl or ethyl group from the base to a cysteine side chain within itself

oxidative stress

an imbalance between the production of reactive oxygen species (ROS) and an organism's ability to break them down.

genetic mosaic

an individual that has somatic regions that differ genotypically from each other

somatic cells

any cell of the body except for gametes and germ-line cells that give rise to gametes.

can apurinic sites be fixed?

apurinic sites are recognized by DNA repair enzymes which can fix them but if the repair system fails, a mutation may result during subsequent rounds of DNA replication

How does a suppressor differ from a reversion?

because a suppressor occurs at a different site in the DNA from the first mutation.

What can the presence of enol and imino forms of the bases cause?

can cause a mutation because these rare forms do not conform to the AT/GC rule of base pairing. Instead, if one of the bases is in enol or imino form, hydrogen bonding will promote T-G and C-A base pairs.

oxidative DNA damage Where does it happen?

changes in DNA structure that are caused by ROS DNA bases are very susceptible to oxidation especially Guanine bases oxidative DNA damage to guanine causes it to base-pair with adenine during DNA replication cause mutations where a G-C base pair becomes a T-A base pair = transversion mutation.

tautomers

chemically similar forms of certain small molecules, such as bases, which can spontaneously interconvert. bases which exist in keto and enol forms or amino and imino forms

Deamination changes... cytosine to_____ adenine to _____

cytosine to uracil adenine to hypoxanthine

What prevents damage by ROS?

enzymes such as superoxide dismutase ad catalase prevent the build up of ROS small molecules such as vitamin C may act as antioxidants as well as blueberries etc.

How does TNRE occur?

formation of a hairpin during DNA replication can lead to an increase in the length of a DNA region if it occurs in the newly made daughter strand After hairpin forms, DNA polymerase slips off the template strand and goes back on to resume replication from the end of the hairpin When this occurs, DNA polymerase is synthesizing most of the hairpin region twice.

nonsense mutations

involve a change from a codon that specifies an amino acid to a stop codon. This change terminates the translation of the polypeptide earlier than normal, producing a truncated polypeptide

Frameshift mutation

involve the addition or deletion of a number of nucleotides that is not divisible by 3. Because the codons are read in multiples of 3, this type of mutation shifts the reading frame.

Effect of mutation on the splice recognition sequence

may alter the ability of pre-mRNA to be properly spliced

Effect of mutation on the 5'-UTR/ 3'-UTR

may alter the ability of the mRNA to be translated, may alter mRNA stability

Effect of mutation on the regulatory element/operator site

may disrupt the ability of the gene to be properly regulated

Effect of mutation in the promoter

may increase or decrease the rate of transcription

alkylation

methyl or ethyl groups are covalently attached to the bases

results of replica plating experiment

on the secondary plates, only those mutant cells that are resistant to lysis by T1 phage (called ton^r mutants) could grow. A few colonies were observed and shockingly they occupied the same location on each plate these results indicate that ton^r mutations occurred randomly while the cells were growing on the nonselective master plate. The presence of the T1 phage in the secondary plates simply selected for the growth of previously occurring ton^r mutants support random mutation hypothesis

How do position effects alter gene expression?

one possibility is that a gene may be moved next to regulatory sequences for a different gene, such as silencers or enhancers, and those then influence the expression of the relocated gene. another possibility is that a chromosomal rearrangement may reposition a gene from a less condensed or euchromatic region of a chromosome, where it is active, to a very highly condensed, heterochromatic region. - may produce a variegated phenotype = expression of gene is variable

How can ROS be good?

produced by the immune system as a means of killing pathogens and ROS are used in cell signaling

reactive oxygen species (ROS)

products of oxygen metabolism that are produced in all aerobic organisms and that can, if they accumulate, damage cellular molecules, including DNA, proteins, and lipids Ex. hydrogen peroxide, superoxide, and hydroxyl radical unwanted by-products of energy production in mitochondria

ionizing radiation

radiation of short wavelength and high energy can alter DNA structure Ex. X-rays and gamma rays produces free radicals Exposure to high doses of ionizing radiation results in base deletions, single-stranded and double stranded-breaks in the DNA backbone, crosslinking and oxidized bases.

germ line

refers to cells that give rise to gametes (sperm and egg)

hot spots

regions within a gene that are more likely to mutate than others.

What happens if a 5-methylcytosine is deaminated? What does this prove about cytosines?

results in thymine but this causes the DNA repair system not to be able to distinguish which is the incorrect base. = why cytosines are hot spots for mutation

Mismatch repair

similar to excision repair except that the DNA defect is a base pair mismatch in the DNA, not an abnormal nucleotide. The mismatch is recognized, and a segment of DNA in this region is removed. The parental strand is used to synthesize a normal daughter strand of DNA.

trinucleotide repeat expansion disease examples

spinal and bulbar muscular atrophy (SBMA), Huntington disease (HD), spinocerebellar ataxia (SCA1), fragile X sydromes (FRAXA and FRAXE) and myotonic muscular dystrophy often expansions are located in non-coding regions

keto form

the common stable form of guanine and thymine

mutation rate

the likelihood that a gene will be altered by a new mutation

mutation frequency

the number of mutant genes divided by the total number of copies of a gene within a population

anticipation + Ex.

the phenomenon in which severity of an inherited disease tends to get worse in subsequent generations. Ex. increased copies of the tandem repeats

depurination

the removal of a purine base from DNA. purine = adenine or guanine The covalent bond between deoxyribose and a purine base is somewhat unstable and occasionally undergoes a spontaneous reaction with water that releases the base from the sugar

deamination + Ex.

the removal of an amino group from a molecule. For example, the removal of an amino group from cytosine produces uracil. Other bases are not readily deaminated

How and when does a tautomeric shift cause a mutation?

the shift must occur immediately prior to DNA replication. After the strands unwind, a tautomeric shift may occur. a base mismatch is created which can be repaired by DNA polymerase proofreading function or via a mismatch repair system but if this does not occur there will be incorrect bases paired causing a mutation.

How do tautomers interconvert?

through a chemical reaction that involves the migration of a hydrogen atom and a switch of a single bond and an adjacent double bond

thymine dimers What causes them?

two adjacent thymine bases in a DNA strand that have become covalently linked - UV light can cause the formation of these dimers