BIS 101 Mutations and Mutagenesis (Ch12) final

You have screened 250,000 pollen grains for texture variations and identified 4 mutant grains. What is the mutation rate?

1.60 x 10^(-5) ∙mutation frequency is the number of times mutation alters a specific gene or 4/250,000

How many rounds of DNA replications are needed to fix a mutation in the chromosome?

2 rounds

If a nonsense mutation occurs in the following sequence which of theses amy be the result? Wild type 5' TAC AGA CGG ACT 3'

5' TAG AGA CGG ACT 3' ∙a nonsense mutation introduces a premature stop codon into the sequence (UAA, UAG, UGA)

5' TAT GCA TGT TAC GAT 3' Given the above sequence, how might you introduce a mutation to have this sequence generate the amino acid sequence Tyr-Ala-Trp-Leu-Arg

5' TAT GCA TGG TTA CGA 3'. ∙a frameshift mutation adds or deletes nucleotides in such a way that it alters the sequence of the protein beginning at the site of the point mutation

The following gene has been mutated, as indicated below wild type 5'TCA GCC CAT TCA GAT 3' mutant 5' TCA GCC CAG TCA GAT 3' You decide to take the mutant, induce additional mutations and identify secondary mutations that represent a true reversion. Which sequence exhibits both a true reversion mutation and a silent mutation

5' TCC GC CAC TCA GAT 3' A true reversion must both code for the same amino acid and a reversion should restore the original amino acid sequence

What is the correlation between mutagens, teratogens and carcinogens

90%

Gross mutations

Bc of the high energy of Xrays, most mutations involved chromosome breakage or gross mutations. ∙They are not helpful for fine genetic analysis bc they can involve thousands/millions of base pairs

Which specialized enzyme plays a role in SOS repair?

Bypass polymerase (pol V) ∙SOS repair is described as a last-ditch effort on the part of a heavily damaged bacterial cell to replicate its DNA and divide before succumbing to DNA damage.

Which enzymes removes chemically modified nucleotides from DNA during nucleotide base excision repair?

DNA glycolyase and AP endonuclease ∙DNA glycolyase recognizes and removes the modififed purine to yield an AP site and then the AP endonuclease removes the remainder of the apurinic nucleotide

Specific excision repair

DNA glycosylases

Cytosines of CpG dinucleotides are frequent targets for methylation in mammalian promoters, in which methylation helps regulate transcription. These CpG dinucleotides are hotspots of mutation as a result of which type of spontaneous DNA nucleotide lesion?

Deamination of methylated cytosine. Adenine and guanine are purines, so cytosine cannot undergo depurination

Who found the first mutagen in the form of X rays

Herman Muller

Hydroxylamine (NH2OH)

Hydroxylating agent - adds hydroxyl groups

which proteins is/are involved in the first step of nonhomologous end joining (NHEJ) in which double strand breaks are recognized

Ku70-Ku80

BrdU is a chemical mutagen commonly used to label proliferating cells. it is derived from uracil and most often base pairs with adenine but can base pair with guanine. What type of mutagen is it?

Nucleotide base analog.

Following UV irradiation, which type of DNA damage might you expect to find?

Pyrimidine dimers

what type of mutation is seen here? Wild type" 5' TTC AGG CAG TGG 3' Mutant 5' TTC AGA CAG TGG 3'

Silent mutation. ∙both AGG and AGA encode the a.a. Arg so the mutation has no effect on protein structure or function

Which type of mutation may result in expression of intronic sequence or loss of part of an exon?

Splice site mutation ∙splice site mutation may cause a gene to retain intron sequence or exclude exon sequence from mature mRNA

Which error free DNA repair mechanism can repair double stranded breaks occuring after the completeion of DNA replication?

Synthesis dependent strand annealing (SDSA) ∙double stranded breaks still present a different type of challenge to postreplication cells but teh presence of identical sister chromatids can be exploited in an error free repair process (as opposed to NHE) which is error prone and repairs DNA breaks prior to replication)

What is the purpose of the Ames test and what type of extract is used in this test?

The ames test examines the mutagenic potential of a chemical using s9 liver extract.

Whats the best test for fragile X

a PCR based test using primers flanking the CGG repeats

Silent mutation

a base substitution mutation that changes one codon to a synonymous codon and does not alter the amino acid sequence of a polypeptide

missense mutation

a point mutation in which a single nucleotide change results in a codon that codes for a different amino acid.

Post replication or mismatch repair

adenine methylase

Point mutations

affect only one or 2 bases in a DNA sequence. a single base change, addition or deletion are examples of point mutations.

Common mutagen and carcinogen on peanuts

aflotoxin

Ethyl methane sulfanate (EMS)

alkylating agent - adds ethyl groups

Ames test

bacterial assays that allows us to quickly screen thousands of chemicals for their potential genotoxicity and carcinogenicity. the basis for this test is the reversion rate for a series of well characterized mutations in salmonella bacteria

Dimerization

can occur between adjacent C & C, U and U, C & U but T & T (thymine dimers (are the most common photoproduct in UV irradiated DNA

Strong UV irradiation

can produce chromosomal breaks, however lower doses can produce permenant photoproducts called pyrimidine dimers.

mutations and cancer

cancer is caused by multiple DNA mutations that allow cells to proliferate uncontrollably

Deaminating agents

capable of removing amino groups from any nucleotide. Deamination of 5-meC can lead to G-C to A-T base pair substitution.

teratogens

cause of birth defects

genetic variation

changes to the DNA with the processes of independent assortment, recombination and gene conversion creates a pool of genetic variation for natural selection to choose the most fit organism for a particular niche

Genotoxic

compounds that are capable of damaging the DNA in our chromosomes

Protomutagen

doesn't become a mutagen until synthesized through the liver and processed by the body

RecA protein

in the DNA-RecA-SSB complex is an active form that also activates transcription of several genes, including pol V

Forward mutations

induced with well characterized mutagens at a frequency characteristic for that mutagen

Frameshift mutation

is a genetic mutation caused by indels (insertions or deletions) of a number of nucleotides in a DNA sequence that is not divisible by three.

what's P53 encoded by

its a eukaryotic DNA binding protein. In humans its encoded by the TP53 gene

lacUV5

lacUV5 promoter mutation was created with UV irradiation

Spontaneous mutations

mutations are rare and occur as a result of errors in DNA replication, tautomeric shifts in certain bases, permanent chemical changes in nucleotides or by slippage in DNA replication

Induced mutations

mutations can be caused or induced by chemicals or physical agents such as ionizing radiation (Xrays & UV lights). These mutagens can be used to increase the rate of mutations by several order of magnitude

suppressor mutations

mutations can be reversed and gene function by a back or reverse mutation or by a supporessors. A suppressor is a second mutation within the gene (intragenic) or in another gene (extragenic)

Transversion

mutations that change purines to pyrimidines and vice versa(A→C; G→T)

Transition

mutations that change purines to pyrimidines to their alternate forms (A→G; C→T)

Li Fraumeni syndrom is a human familial cancer syndrome caused by a defect in which gene involved in DNA damage repair

p53. ∙acts as a transcription factor which initiates G1 arrest of the cell cycle by inducing syntheses of the protein p21 that inhibits formation of Cdk-Cyclin complexes. The p53 induced pause in the cell cycle allows time for the repair of DNA damage. The completion of DNA repair depletes p53 and the cell cycle transitions to the S phase

If E. Coli are UV irradiated, which DNA repair system will be inhibited if the cells are kept in the dark?

photoactive repair. ∙dimers produced by radiation are bound by the enzyme photolyase, which uses light energy to break bonds forming the dimer. This allows for excision and repair of the mutated base.

nonsense mutation transition

point mutation results in a nonfunctional codon

Xeroderma Pigmentosum

rare heritable form of skin cancer in humans. ∙autosomal recessive ∙defect in 1 of 8 complementation groups involved in excision repair of UV induced DNA damage. ∙Complementation group XPC is the most common and XPG is associated with neuro disorders

Recombination repair

recA

Premutation

repeats in the 6-200 range appears normal. Males w/in this range are called "normal transmitting males". Repeats of >200 show the disease phenotype. . ∙CG in the CGG repeat is a target for DNA methylation (5' methyl cytosine) which seems to reduce the expression of the fmr-1 genes. One strand of the repeated region has been show to form a hairpin structure that blocks DNA synthesis. ∙methylation means "the gene should be turned off"

extragenic suppression

second mutation can occur outside the gene or on another gene. ∙Most common type is a mutant tRNA in which a base substitution in the anticodon loop enables the tRNA to recognize one of the stop codons (UAA, UGA, UAG)

Molecular basis of mutation

single base pair substitutions, insertions or deletions can change the way the triplet code of the correct open reading frame (zero reading frame) is read. These forward mutations can lead to loss of function or a gene or create a new function

HERP%

the risk of a real or potential mutagen can be assessed by calculating the HERP%: Human Exposure/Rodent Potency percentage ((150mg/70kg)/1.5mg/kg) x 100 = 10,000&

reversion analysis

the study of the frequency, cause and nature of back mutations

Excision-repair pathways

uvrA, uvrB, uvrC

Intragenic suppression

when the second mutation can occur in the mutated gene

auxotrophs

wild type bacteria (prototrophs) are mutagenized with well characterized mutagens and mutant strains are isolated that are incapable of growth on minimal medium. ∙mutants have lost the ability to perform biochem functions such as ability to use other sugars

what happens when you have a nonfunctional P53 gene

you get colon cancer along with aneuploidy and rapid cell proliferation

Nitrous acid mutagen (HNO2)

∙Deaminates (removes amino group) adenine to produce Hypoxanthin (Hx) to bind to C. ∙Nitrous acid produces an AT to GC transitions. ∙2 rounds of DNA replication are required to fix the mutation into the gene

Strand slippage

∙During DNA replication, the nascent/template strand may slip and add a base on one strand (nascent) or delete a base on the template strand ∙happens most frequently when there are repeats ∙Causes a hairpin look that will go back through the DNA pol to get fixed

How can mutations arise

∙Errors in DNA replication ∙Temp alteration in H bonding between bases (tautomeric shifts) ∙physical and chemical agents called mutagens that alter DNA structure

What features are shared by genes identified as "hotspots" for mutations

∙Genes are often large

Ames test protocol

∙Spread each of 6 histidine minimal agar plates (lacking histidine) with 10^9 almonella cells of his- mutants ABC respectively ∙make a sterile solution of unknown compound X ∙Soak 3 small filters disks in X and place them in the center of 3 histidine minimal agar plates ∙soak 3 dishes in sterile water and place them in the center of the other 3 plates as negative controls ∙incubate plates at 37 C for 20 hours and count the number of his+ colonies around each disk

How did Dr. Ames discover the test

∙Used a series of defined his-mutants (grows on mediums with histidine only) of Salmonella typhimurium. ∙Test uses a common reversion analysis ∙Chemicals could be quickly test for positive mutagenicity for potential carcinogenicity

When do pyrimidine dimers become mutagenic

∙When they go uncorrected. DNA pol doesn't recognize pyrimidine dimers as good templates and will either stall ar the dimer or delet or add a nucleotide. ∙UV irradation is useful in producing point mutations of the single nucleotide insertion or deletion

How does P53 act as a tumor suppressor?

∙bc of it's role in monitoring DNA damage, apoptosis and cell cycle control to prevent cancer

what is P53 is called ?

∙called the guardian of the genome for its role in preserving genome integrity by preventing mutations from propagating from one cell to the next

Fragile X syndrome

∙characterized and found to contain a tandemly repeated trinucleotide sequence (CGG) near it's 5' end. The mutation responsible for fragile X syndrome involves expansion of this repeat segment by DNA slippage. The number of CCG repeats in the FMR1 genes of the general population varies from 6-50

back mutations

∙intragenic suppression, reversion involves a back mutation that restores the original base pair in the wild type strain or cell ∙can occur spontaneously or they may be induced by a mutagen

GO system

∙mutM ∙mutY ∙mutT

Direct reversal of damage of DNA

∙photlase ∙alkyltransferase

What process causes mutations responsible for altering the number of DNA repeats as seen in many trinucleotide repeat disorders?

∙strand slippage ∙strand slippage occurs when the DNA pol of the replisome temporarily dissociates from the template strand as it moves across a region of repeating DNA sequence, creating a temporary double stranded hairpin. When replication resumes, a protion of the repeat region is re-replicated, and this is why these regions are hotspots for mutation

Tautomeric shift (tautomerization)

∙the spontaneous, rare, raid and reversible isomerization of the N bases to alternative H bonding forms (imino/enol forms of pyrimidines and amino/keto forms of purines) due to proton migration around purine rings) ∙tautomerization also creates the possible non-canonical base pairing in DNA or RNA producing wobble in the anticodon loop of tRNA

E Coli: Excision repair

∙uvrA detects the distortion in the DNA and guides uvrB to the damaged site ∙uvrA dissociates and uvrC binds uvrB at the damaged site ∙uvrC makes cuts about 12 bases apart in the damaged DNA strand ∙Helicase unwinds the 12-mer from the helix ∙DNA Pol 1 synthesizes new DNA in the gap ∙sometimes when base modifications are sometimes too subtle to be recognized by the uvrA protein. So DNA glycosylases are used to remove damage bases