9.1 - DNA Mutations/Repair & SOS Response

Question 1

Why are there so many different repair mechanisms?

Answer 1

Because there is a constant need to repair the genome. In fact, DNA is constantly damaged as a result of UV light, as well as other chemicals termed mutagens.

Question 2

What is the Ames mutagenesis test?

Answer 2

A test that uses bacteria to test whether a given chemical can cause mutations in the DNA of the test organism, i.e. to detect whether a given chemical is a mutagen.

Question 3

What do we place the mutagen disk on in the Ames mutagenesis test?

Answer 3

A minimal media like M9

Question 4

What is an example of a kind of medium that can be used for the Ames mutagenesis test?

Answer 4

Histidine can be excluded from the medium so only bacteria that are capable of synthesizing their own histidine will be able to grow.

Question 5

What must be plated as well in the Ames mutagenesis test? (histidine example)

Answer 5

Bacterial cells with a mutation in the histidine biosynthetic gene are plated

Question 6

What does it mean if cells grow in the Ames mutagenesis test? (Histidine example)

Answer 6

It means that the cells have regained the ability to synthesize histidine, thereby indicating that their DNA was mutated such that the mutation in the histidine biosynthetic gene was reversed.

Question 7

What does the fact that cells became His+ confirm?

Answer 7

The mutagenic nature of the chemical plated.

Question 8

What is the SOS response?

Answer 8

Response to drastic DNA damage

Question 9

What happens when DNA is damaged?

Answer 9

E.coli stops dividing but continues to grow. The cells keep getting longer and longer. Cell division however will be blocked since DNA is damaged.

Question 10

When do bacteria stop cell division?

Answer 10

When they sense that there DNA is damaged

Question 11

Do all biosynthetic pathways stop when cell division is stoped?

Answer 11

No, all biosynthetic pathways will continue

Question 12

What is the target of the SOS regulation mechanism?

Answer 12

FtsZ which is involve in cell division

Question 13

How many genes in the SOS regulon are regulated by LexA?

Answer 13

20

Question 14

What is a regulon?

Answer 14

A regulon is a group of genes that are regulated as a unit.

Question 15

What is LexA?

Answer 15

LexA is a repressor that, under normal conditions, binds promoters of the SOS response genes and inhibits their expression.

Question 16

Is RecA still expressed when DNA is damaged?

Answer 16

Yes, but in low concentrations

Question 17

What is RecA?

Answer 17

RecA is a ubiquitous protein and is part of the AAA+ family of proteins and the SOS response proteins. RecA is a key protein in homologous recombination and DNA repair.

Question 18

What does RecA do?

Answer 18

It mediates “strand invasion” by coating ssDNA and help it invade the dsDNA helix where DNA damage has occurred.

Question 19

When is the expression of RecA induced?

Answer 19

When the cell senses DNA damage (e.g damage due to UV)

Question 20

What happens when DNA damage is sensed by RecA?

Answer 20

it will bind to LexA and cause it to cleave itself (auto-proteolysis). So RecA is a co-protease of LexA repressor.

Question 21

What happens when LexA is cleaved?

Answer 21

LexA is no longer functional and the SOS response genes are expressed

Question 22

What is SulA

Answer 22

One of the SOS response genes which bind FtsZ and inhibit its polymerization and the formation of the Z ring.

Question 23

Why would you want to inhibit FtsZ polymerzation during the SOS response?

Answer 23

Because if you do not form the Z ring then there is no cell division

Question 24

What are some other regulons involved in the SOS response?

Answer 24

RecBCD: this is a helicase and a nuclease (capable of cutting the DNA).
RecJ.

Question 25

What happens if a bacterium has been infected with lambda phage?

Answer 25

The lambda phage can establish lysogeny under good conditions. Lysogeny is maintained by the repressor C1. But under conditions of stress such as DNA damage, the phage repressor C1 is inhibited, resulting in entry into the lytic cycle so that the phage DNA can excise out.

Question 26

What is the lambda phage repressor called?

Answer 26

cI

Question 27

What does the lambda phage repressor inhibit?

Answer 27

Transcription of genes required for the lytic life cycle and therefore the phage maintains lysogeny.

Question 28

What is the translesion bypass system?

Answer 28

The translesion bypass system is part of the SOS system and is made up of umuCD (also known as DNA Pol V), which is an error prone DNA polymerase induced during the SOS response.

Question 29

How many genes make up the translesion bypass system?

Answer 29

2 genes adjacent to each other, umuD and umuC

Question 30

What happens to umuD and umuC under normal conditions?

Answer 30

The transcription of umuC and D is repressed by LexA

Question 31

What does functional umuCD require?

Answer 31

Cleavage by RecA whose expression is induced by DNA damage.

Question 32

What happens when RecA binds umuD?

Answer 32

umuD’s auto-proteolysis is induced

Question 33

What does the cleaved umuD do?

Answer 33

It associates with umuC, resulting in a functional umuCD.

Question 34

What does the functional umuCD do?

Answer 34

The functional umuCD will go to the damaged DNA and add nucleotides. However, there will be a lot of errors since umuCD is an error prone DNA pol.

Question 35

What can the substrates of umuCD be?

Answer 35

Damaged DNA with gaps and barely readable sequences. It will then fill the gaps with semi-random plausible DNA.

Question 36

When can mistakes be beneficial?

Answer 36

If they confer advantageous elements to the bacteria, such as antibiotic resistance.

Question 37

How do cells recover (i.e. restart cell division)?

Answer 37

A protease called Lon will selectively recognize and cleave SulA. Inactive SulA will allow FtsZ to form the Z ring so that cell division can occur

Question 38

What does Lon do?

Answer 38

Degrades SulA. When you delete Lon you do not have septation but cells keep growing (long cells)

Question 39

What happens if you delete SulA?

Answer 39

With deletion of SulA you go from long cells to short cells (repressor mutation) SulA is a suppressor of long cells