Mutation and DNA repair

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Why is the potential mutagen mixed with liver enzymes?

In the liver we tend to process lots of stuff. The potential mutagen is mixed with liver enzymes because when molecules get modified within our liver they can either become mutagenic or could be mutagenic for bacteria and less harmful for us this way we can gather information on how the chemical would behave in a mammal.

Ethyl methanesulfonate (EMS)

Is a commonly used mutagen to generate point mutations in genetic screens

keto-enol forms

T and G

How does the DNA repair after Excision?

The presence of a DNA strand complementary to the damaged strand provides a template for resynthesis of the defective sequences—DNA polymerase and DNA ligase fill in and seal up the resulting gap.

Tautomeric forms

alternate forms that differ in only a single proton shift (affects purines and pyrimidines

deamination

an amino group in cytosine or adenine is converted to a keto group.

Mutagens

are natural or artificial agents that induce mutations

deamination of adenine

is converted to hypoxanthine.

What are the biologically important tautomers?

keto-enol forms of T and G and the amino-imino forms of C and A

Types of point mutations

silent mutations, missense mutations, non-sense, frame-shift,

What information about a mutagen is gained by using bacterial strains with known mutations in genes of the histidine operon?

...

Why are his-auxotrophic strains used in the Ames test?

...

Why did some colonies grow in the absence of a mutagen?

2 induced or spontaneous. Colonies on the left are spontaneous, mutations that arose in the histidine genes spontaneously without any chemicals. In this experiment if you plate a chemical with a known mutagen, you expect to see more colonies on it. if the chemical is not a mutagen you expect to see the same number of colonies.

To minimize the number of errors, our cells have three DNA repair mechanisms:

A) Proofreading B) Mismatch repair C) Excision repair

Types of mutagens

A) alkylating agents, (B) nucleotide analogs, (C ) acridine dyes, and (D) radiation.

Deletions mutations

Are a loss of a chromosomal segment.

Duplications

Are a repeat of a segment.

Nonsense mutations

Are base substitutions that substitute a stop codon. Change in base translates to an early stop codon. The shortened proteins are usually not functional.

Point mutations

Are mutations of single genes. Result from: -addition or subtraction of a base -the substitution of one base for another Caused by: -mistakes during DNA replication -environmental mutagens

Germ-line mutations

Are mutations that occur in cells that give rise to gametes. Germ line mutations provide genetic diversity for evolution, but usually produce an organism that does poorly in its environment.

Somatic mutations

Are passed on during mitosis, but not to subsequent generations. Somatic mutations do not affect offspring, but can cause cancer.

Induced mutations

Are permanent changes caused by some outside agent (mutagen). A result of external factors, such as chemical or radiation.

Proofreading

As a DNA polymerase III adds a new base to a growing strand, the enzyme makes a proofreading check.

amino-imino forms

C and A

The hydrolysis of an -NH2 group from a base is called ________, while intercalating agents such as acridine orange function as mutagens by causing ________:

C) deamination; deletions or insertions

Acridine dyes

Cause frameshift mutations by intercalating (inserting) between DNA bases which could result in production of daughter cells with inserted or deleted regions. Chemicals such as ethydium bromide, benzopyrene from smoke and yellow and red acridine dyes can cause frameshift mutations in DNA. DNA polymerase copies the altered strand, adding or deleting base pairs around the bulge formed by the bound mutagen.

Chromosomal mutations

Changes in the arrangements of chromosomal DNA segments.

frame-shift mutation

Consists of the insertion or deletion of a single base in a gene. This type of mutation shifts the code, changing many of the following codons to different codons. These shifts almost always lead to the production of nonfunctional proteins.

Which of the following mutations would be most likely to have a harmful effect on an organism? A) a base-pair substitution. B) a single base deletion near the middle of an intron. C) a single base deletion close to the end of the coding sequence. D) a single base insertion near the start of the coding sequence. E) a silent mutation.

D) a single base insertion near the start of the coding sequence.

Types of Chromosomal mutations

Deletions, Duplications, Inversion, Translocating

Alkylating agents

Donate an alkyl group (C2H5) to amino or keto groups in nucleotides to alter base-pairing affinity.

Is the following statement about missense mutations True or False? "Because they only affect a single amino acid, missense mutations do not have a significant effect on protein function."

False

Excision repair proteins

Operate over the life of a cell. They "inspect" the cell's DNA for damage, then cut the damaged strand and remove it.

2 types of mutations (alteration of DNA nucleotide sequence)

Point mutations and Chromosomal mutations

Inversions mutations

Result from breaking and rejoining when segments get reattached in the opposite orientation.

Translocations mutations

Result when a portion of one chromosome attaches to another

what are the 2 types of mutations in multicellular organisms

Somatic mutations and Germ-line mutations

Radiation (2 types of damage)

UV radiation and ionizing radiation

Ames Test

Used to Assess the Mutagenicity of Compounds using any of a dozen strains of Salmonella typhimurium

How does DNA polymerase III proofread a growing DNA strand

When DNA polymerase III recognizes an error, the epsilon (ε) subunit (which can act as an exonuclease) removes the wrong nucleotide and tries again.

what type of mutation causes sickle cell anemia?

a missense mutation. a defect in the β-globin subunits of hemoglobin. The β-globin in sickle-cell differs from the normal by only one amino acid.

Spontaneous mutations

a result of natural biological and chemical processes - Are permanent changes, caused by any of several mechanisms: -errors in replication -nondisjunction and translocation in meiosis -tautomeric shift (change in structure)

epsilon (ε) subunit

act as an exonuclease removing the wrong nucleotide during proofreading

base-pairing of cytosine after deamination

base-pairs with Adenine

base-pairing of hypoxanthine after deamination

base-pairs with Cytosine

missense mutations

cause an amino acid substitution. may reduce the functioning of a protein or disable it completely. a base change resulted in translation of a different amino acid.

Tautomeric shifts

change the bonding structure of the molecule allowing hydrogen bonding between non complimentary bases.

Nucleotide analogs

compete with the normal nucleotide during DNA replication and cause point mutations. Nucleotide analogs have a similar chemical structure to a normal nucleotide. - Undergo tautomeric shifts - is not particularly stable - less stable than normal bases

Mutations

comprise any change in the base-pair sequence of DNA. - are heritable changes in DNA—changes that are passed on to daughter cells. - is a source of genetic variation and provides the raw material for natural selection. - also the source of genetic damage that contributes to cell death or genetic diseases including cancer. -Can occur spontaneously or be induced

deamination of cytosine

converted to uracil

UV radiation

creates pyrimidine dimers that distort the DNA conformation in such a way that errors tend to be introduced during DNA replication

Why does the Ames Test use Salmonella typhimurium?

increased sensitivity to mutagens and their ability to reveal the presence of specific types of mutations.

How does a tautomeric shift affect the ability of a cytosine to perform base pairing?

makes 2 hydrogen bonds with adenine instead of 3 hydrogen bonds with guanine.

How does a tautomeric shift affect the ability of a thymine to perform base pairing?

makes 3 hydrogen bonds with guanine instead of 2 hydrogen bonds with adenine.

What is the effect of a tautomeric shift of a cytosine on one strand of DNA upon the 2 daughter molecules formed by one round of DNA replication of this sequence? 3. What are the sequences of the 4 DNA molecules formed by two rounds of DNA replication of the sequence above?

one daughter cell will be unaffected. The cytosine of the second daughter cell will have the innate ability to base-pair with adenine. the unaffected strand will continue to replicate normally producing 2 normal daughter cells. The erronenous insertion of an Adenine during replication using the imino Cytosine as a template results in an A=T mutation on one daughter cell. The imino Cytosine will revert to the amino form on the 2nd daughter cell.

Silent mutations

result in no change in the amino acids in the protein due to redundancy in the genetic code. A change in one base still codes for the same amino acid.

mismatch repair mechanism

scans new DNA for mismatched base pairs before the new DNA strand is methylated. can distinguish between the methylated template strand and the unmethylated new strand. Thus it can determine which base is correct (the base on the template strand) and which base needs to be replaced on the new strand.

Mutagens

some outside agent that induces mutation altering DNA in several ways: 1. Altering covalent bonds in nucleotides 2. Adding groups to the bases 3. Radiation damages DNA: - Ionizing radiation (X rays) produces free radicals. - Ultraviolet radiation is absorbed by thymine and causes interbase covalent bonds to form.

Ionizing Radiation

such as X-ray and gamma radiation energize electrons, causing them to leave atoms and molecules, forming highly reactive ions. This can cause chromosomal damage.

error rate of DNA poly III in proofreading

~1 in 10,000 or one base in a billion


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