DNA Repair & Mutagenesis

Question 1

With the great number of mutations that occur in the genome, why is the effect largely mitigated?

Answer 1

Most of DNA is non-coding; less than 2% of the human genome contains “informative” information. Mutations that do cause a cellular defect would most likely kill the cell, so it will typically not cause diseases such as cancer. Overall, most mutations are inconsequential.

Question 2

Name some examples of DNA lesions that require repair.

Answer 2

Missing base, altered base, incorrect base defects, bulge, linked pyrimidines, or strand breaks.

Question 3

What effect might acid, heat, or glycosylases have on DNA?

Answer 3

It may cause a missing base lesion.

Question 4

What effect might radiation or alkylating agents have on DNA?

Answer 4

It may cause an altered base lesion.

Question 5

What effect might tautomers or proofreading errors have on DNA?

Answer 5

It may cause incorrect base defects.

Question 6

What effect might intercalating agents have on DNA?

Answer 6

It may cause a bulge in the DNA.

Question 7

What effect might UV irradiation have on DNA?

Answer 7

It may cause linked pyrimidines/a pyrimidine (thymidine) dimer.

Question 8

What effect might ionizing radiation or chemical agents have on DNA?

Answer 8

It may cause strand breaks.

Question 9

How are tautomeric forms of the DNA bases made?

Answer 9

Tautomeric forms of the DNA bases occur by the rearrangement of hydrogen bonds and by the repositioning of hydrogens in the purine and pyrimidine bases. Tautomers may lead to spontaneous mutations.

Question 10

What are the normal and tautomeric forms of adenine and cytosine called?

Answer 10

The normal form is the amino form and the tautomeric the imino form.

Question 11

What are the normal and tautomeric forms of guanine and thymine called?

Answer 11

The normal form is the keto form and the tautomeric the enol form.

Question 12

Is the normal form or tautomeric form of DNA bases more common?

Answer 12

The amino and keto forms (the normal forms) are much more common than the rare imino and enol forms.

Question 13

What causes a transition mutation?

Answer 13

When a mutation occurs as a result of the tautomer of cytosine, cytosine changes its structure so that it can bond with adenine (this is the imino form), resulting in a transition mutation.

Question 14

What are the three general DNA repair pathways?

Answer 14

1. The DNA polymerases proofread the DNA during DNA replication.
2. Mismatch repair (also known as post-replication repair or two-types excision repair).
3. Excision repair.

Question 15

Which structures have 3’-5’ proofreading exonuclease activity?

Answer 15

Prokaryotic polymerase III and eukaryotic polymerases gamma, delta, and epsilon.

Question 16

Describe the process of base excision repair.

Answer 16

An individual damaged base is recognized by an enzyme called uracil DNA glycosylase. The glycosylase will cleave the uracil and allow a repair nuclease to create a substrate for DNA polymerase, which will fill in that gap. DNA ligase fully closes the gap.

Question 17

Describe the process of nucleotide excision repair.

Answer 17

A special repair nuclease removes about 30 nucleotides. The DNA is resynthesized by DNA polymerase, creating a substrate for DNA ligase.

Question 18

What is the difference between base excision repair and nucleotide excision repair?

Answer 18

Nucleotide excision repair repairs larger mutations than base excision (which are generally only a defect in one base).

Question 19

What are the two damage recognition pathways of nucleotide excision repair?

Answer 19

1. Transcription coupled via Cockayne syndrome B protein (CSB). This is a rapid pathway that allows prioritization of genes that are actively expressed.
2. Global. This is much slower. It recognizes lesions (such as bulging thymidine dimers) with the XPC protein.

Question 20

In bacteria, what do the hypermutable genes (“mut” genes) do?

Answer 20

They recognize and repair lesions.

Question 21

Describe the steps of MMR repair.

Answer 21

1. Two enzymes, mutS and mutL, detect the mismatch in DNA.
2. mutS and mutL complex loops DNA to join with mutH near hemimethyl site.
3. MutH nicks the daughter strand and travels 3’-5’ toward mismatch with UvrD helicase to separate the helix and new strand.
4. A trailing exonuclease digests ssDNA ending just past mutS and mutL complex. The mismatch is removed.
5. The gap is filled by DNA polymerase III with hemi-CH3 strand as a template.
6. The DNA ligase completes the repair.

Question 22

How does the transcription-coupled pathway of DNA repair recognize the lesions that need to be repaired first (ie on the genes that are actively transcribed)?

Answer 22

Repair of a template strand occurs as the DNA is being transcribed, and the presence of the lesion may be signaled by a stalled RNA polymerase. This preferential repair pathway ensures that those genes of greatest importance to the cell, which are the genes the cell is actively transcribing, receive the highest priority on the “repair list.”

Question 23

List the steps of nucleotide excision repair.

Answer 23

1. The lesion is recognized.
2. Enzymes that possess helicase activity separate the two strands of the duplex.
3. The damaged strand is cut on both sides of the lesion by a pair of endonucleases.
4. The segment of DNA between the incisions is released.
5. The gap is filled by a DNA polymerase.
6. The strand is sealed by DNA ligase.

Question 24

How is base excision repair initiated?

Answer 24

BER is initiated by a DNA glycosylase that recognizes the alteration in the DNA sequence and removes the altered base by cleavage of the glycosidic bond holding the base to the deoxyribose sugar.

Question 25

In E. coli, during the process of mismatch repair, how are the parental strand and the daughter strand differentiated?

Answer 25

They are distinguished by the presence of methylated adenosine residues on the parental strand.