7.2: Molecular Mechanisms that Alter DNA sequence

1. internal factors (metabolic biproducts or replication errors)

2. external factors (UV light and chemicals)

How is DNA constantly being damaged? [2]

DNA polymerase reads the damaged strand and permanently copies it into the new strand

Once replication is complete, the change is permenently locked into the genetic code of the daughter cells

If DNA replication occurs before the repair machinery can fix the alteration, what happens?

1000 times/hour

How often does purine base hydrolysis occur in every human cell?

Apurinic site that does not specify a complementary base.

purine base hydrolysis is called depurination, leading to:

Since the site does not specify a complementary base pair, a random baes is introduced and the probability of mutation is 75%

What is probability of mutation at an apurinic site?

Deamination

Removal of an amino from a nucleobase cytosine

Changes it to Uracil. Following DNA replication, this leads to a transition from C to T

What is the result of deamination of cytosine?

Breaks the sugar-phosphate backbone leading to DNA strand breaks and potential deletions

How can X rays cause mutations?

Causes adjacent Thymines to form dimers, which leads to substitutions when DNA is replicated

How can UV rays cause mutations?

By generating oxidized Guanine.

How can oxidative stress damage DNA?

It incorrectly mispairs with Adenine, which ultimately generates a G:C T:A transversion mutation

How can oxidized guanine (8-oxodG) change DNA?

1 mistake every 10^6 base pairs due to proofreading activity of DNA polymerase

how often do DNA polymerase make mistakes?

DNA polymerase proofreading function is in form of 3’-5’ exonuclease activity that is active when polymerase makes a mistake.

Exonuclease removes the wrongly incorporated base so that DNA polymerase adds the correct base.

How does DNA polymerase proofreading work?

Tautomers

Structural isomers of a molecule that spontaneously interconvert with each other. It exists in a chemical equilibrium.

Tautomerization

Mechanism that triggers DNA repliation errors by polymerase. If the base is in this form when DNA polymerase is active, the wrong base will be incorporated.

CAG

PolyQ diseases are caused by this triplet repeat:

Trinucleotide repeat diseases

this type of repeat disease exemplifies the impactful consequences of mutaitons in humans

Huntingtons

Example of polyQ disease

Fragile X syndrome

Example of Non polyQ disease:

Mutagen

Any physical or chemical agent that raises the frequency of mutations above the spontaneous rate

Base analog

Mutagens that are similar in chemical structure to the normal nitrogenous bases in DNA that the replication mechanism can incorporate them into DNA in place of normal bases

5-bromo uracil. ONe isomer pairs with A and one pairs with G

Example of base analog

Cause base substitutions on the complementary strand synthesized in the next round of DNA replicatoin

Incorporation of base analogs can cause what?

1. hydroxylation

2. alkylation

3. deamination

Agents can alter base structure through: [3]

ethyl or methyl group that causes the wrong base to be paired (substitution)

Alkylating agents add what to the base?

Deamination

Removal of an amine group

Nitrous acid

example of deamination agent

MOdifies cytosine to uracil, which pairs with A instead of G, modifies ademine to hypoxyanthine, a base that pairs with C instead of T

How does deamination affect cytosine?

Intercalator

Class of chemical mutagens composed of flat, planat molecules that can sandwhich themselves between successive base pairs, disrupts the machinery of DNA replication, and cause mutation

Proflavin: cause single base insertion and deletions

Example of intercalator

Cancer

Somatic mutations can lead to:

screen for chemicals that cause mutations in bacterial cells

The ames test is used to screen:

histidine oxotroph to histidine prototroph

The ames test is to see if bacteria convert from what to what?

Prototrophy

ability to grow in medium lacking histidine, able to provide nutrient/synthesis to grow

Generates mutation

If bacteria convert from his- to his+, this could mean that

TO make sure a compound is not carcinogenic

Principle of the ames test