Mutations and repair

Question 1

Mutation rate

Answer 1

the probability that a given bp or DNA region (e.g. gene, genome)
changes over time and it is characteristic for each organism. Proofreading activity
of DNA polymerase and DNA repair systems decrease the mutation rate (1 in 10 nt
to 1 in 108 and 1 in 1011), while mutagens increase the mutation rate

Question 2

Types of spontaneous mutations

Answer 2

Point mutations, Insertions and deletions,
Replication Slippage and Tautomerization

Question 3

Point mutations

Answer 3

An exchange only one base pair. It doesn’t affect the length of the DNA sequence. There are two types:
Transitions & Transversions

Question 4

Transitions:

Answer 4

interchanges of purines (A, G) or pyrimidines (T, C).
It is the most common, it doesn’t affect DNA structure

Question 5

Transversions

Answer 5

substitutions of purines for pyrimidines or vice versa. Less common, modify DNA structure

Question 6

Insertions and deletions

Answer 6

Insertions or deletions can cause frameshift mutations which leads to aberrant proteins. Frameshift mutations are caused by indels that are NOT a multiple of three base pairs. They change they frame in which triplets are translated into polypeptides

Question 7

Replication slippage

Answer 7

Microsatellites (<10 bp) and minisatellites (10 – 100 bp)
consist of tandem repeats of short sequences. Unstable regions of the genome with multiple insertions, deletions and genome rearrangements.
A replication slippage event occurs when the daughter DNA strand slips back one repeating unit during replication.

Question 8

Tautomerization

Answer 8

Tautomers are structural isomers of chemical compounds (e.g. nucleotides).
During a tautomer shift or tautomerization, protons are
transferred from one tautomer to another.
It is a transient change to an alternative form of the
molecule (nucleotide).

Question 9

Common types of Chemical mutagens:

Answer 9

• Base analogs
• Deaminating agents
• Alkylating agents
• Intercalating agent

Question 10

Base analogs

Answer 10

5- Bromouracil is an analog of thymine. It contains a bromine atom in place of thymine’s methyl group.
It can be incorporated in DNA instead of thymine.
It causes a tautomeric shift from a keto form to an enol form

Question 11

Deaminating agents

Answer 11

The mutagen Nitrous acid performs an oxidative
deamination that converts Cytosine into Uracil.
It causes a point mutation (transition)
Removal of amino group

Question 12

Alkylating agents

Answer 12

Alkylation is the transfer of an alkyl group from one
molecule to another.
Methylation is a type of alkylation, where methyl groups are added to DNA.
Methylation changes DNA activity without changing the
sequence. It is a very important biological process
involved in epigenetics.

Question 13

Intercalating agents

Answer 13

Ethidium bromide used in agarose gel electrophoresis to visualize DNA.
It intercalates between base pairs, it distorts the double helix and the DNA polymerase can add extra bases during DNA replication.
Leads to insertions and deletions

Question 14

Physical mutagens

Answer 14

Ionizing radiation, Ultraviolet radiation, Heat

Question 15

Types of DNA Repair System:

Answer 15

I. Direct Repair
II. Excision Repair
a. Base excision repair (BER)
b. Nucleotide excision repair (NER)
c. Mismatch excision repair (MMR)
III. Homologous recombination
IV. Nonhomologous end-joining (NHEJ)

Question 16

Direct repair system

Answer 16

Rare. Direct repair systems reverse the damage back to normal of single bases that affect the structure of DNA. without modifying the DNA sequence.
Repairs alkylation -> MGMT removes the methyl group

Question 17

Base excision Repair

Answer 17

Base excision repair removes and replaces a single damaged base in the DNA.
Two main pathways (Both start with Glycosylase)
Glycosylases cleave the glycosidic bond between the damaged base and the deoxyribose, leaving an AP site/baseless site.

Question 18

Long-patch pathway (Base excision Repair)

Answer 18

• Endonuclease APE1 cleaves on the 5’ side of the AP site
• Replication complex with DNA polymerase synthesizes 2-10 nt in the 5’-3’ direction creating a 5’ flap
• FEN1 (flap endonuclease) removes the displaced DNA
• Ligase seals the nick

Question 19

Long-patch pathway (Base excision Repair)

Answer 19

• Endonuclease APE1 cleaves on the 5’ side of the AP site
• Replication complex with DNA polymerase synthesizes 2-10 nt in the 5’-3’ direction creating a 5’ flap
• FEN1 (flap endonuclease) removes the displaced DNA
• Ligase seals the nick

Question 20

Short-patch pathway (Base excision Repair)

Answer 20

-Lyase breaks the sugar ring creating a nick on the 3’ side of the AP site
- APE1 and DNA polymerase replace a single nucleotide
- Ligase seals the nick

Question 21

Nucleotide excision repair (NER)

Answer 21

Nucleotide excision repair removes longer DNA sequences around the damaged base and synthesizes a new stretch of
DNA.

Question 22

Steps in Nucleotide excision repair

Answer 22

1. Recognition step, damaged is identified by surveillance proteins.
2. Incision step, an endonuclease cleaves the DNA strand on both sides of the damage
3. Excision step, an exonuclease and/or a helicase removes the damaged strand that has been previously cleaved
4. Gap-filling step, a DNA polymerase synthesizes the replacement DNA and DNA ligase seals the nick

Question 23

Mismatch repair (MMR)

Answer 23

Mismatch excision repair system repairs errors that occur during DNA replication
It ONLY repairs the daughter strand

Question 24

How does the MMR system distinguish between the
daughter and the parent strand?

Answer 24

In E. coli, Dam methyltransferase methylates the adenines
on both strands in the sequence GATC.
After replication only the parental strand is methylated
(hemimethylated state).
So the daughter strand is recognized because it is not yet methylated

Question 25

Double strand breaks (DBS) will lead to?

Answer 25

DBS is a severe type of DNA damage. If it is not repaired
properly it will lead to:
1 chromosomal instability
2 Increase the rate of mutation
3 Increase the probability of cancer and serious diseases
It can be repaired by:
- Homologous recombination (HR)
- Non-homologous end joining (NHEJ)

Question 26

Homologous recombination

Answer 26

Recombination repair systems function after DNA replication. Fix single (SSB) and double strand breaks (DSB).
HR uses homologous sequences to repair the damage. If after replication, the daughter duplexes are different, the homologous single strand from the normal duplex is used to
fill the gap of the damaged strand.
NO genetic information is lost during the repair

Question 27

Non-homologous end joining

Answer 27

Repairs Double Strand Breaks (DSB) by joining and
ligating the blunt ends together
It is error-prone

Question 28

Ku protein complex in Eukaryotes

Answer 28

• Ku dimer recognizes the damage and binds to the DNA ends. It forms a scaffold that holds both DNA ends together.
• It recruits other enzymes to repair the damag

Question 29

The SOS system in E. coli

Answer 29

The SOS response is a global response to severe
DNA damage that has be bypassed during replication. It stops the cell cycle and DNA repair systems are induced. It is an error-prone system
activated by the RecA-protein.
1. LexA is a repressor of many genes involved in
DNA repair
2. When severe damage in the cell induces RecA,
RecA triggers the cleavage of LexA and several
genes involved in DNA repair systems are expressed and repair the damage

Question 30

Glycosylases

Answer 30

cleave the glycosidic bond between the damaged base and the deoxyribose, leaving an AP site/baseless site.
Uracil-DNA Glycosylase repairs uracil bases added by
deamination of cytosine