Bacterial DNA Replication/Repair - Khan

Question 1

Which direction does DNA synthesis occur?

Answer 1

5’ to 3’

Question 2

Explain how replication is semi-discontinuous.

Answer 2

There is a leading strand which replicates continuously, and a lagging strand which replicates discontinuously.

Question 3

What is an Okazaki fragment?

Answer 3

Small pieces of synthesized DNA on the lagging strand.

Question 4

What does it mean that DNA cannot be synthesized de novo?

Answer 4

Replication requires a free 3’ OH which is usually achieved by adding a short RNA primer by DNA primase.

Question 5

Which enzyme unzips DNA during replication?

Answer 5

Helicase

Question 6

What is meant by DNA replication is semi-conservative?

Answer 6

Once replicated, dsDNA will consist of one parental (template) strand and one newly synthesized strand.

Question 7

Does DNAP have exonuclease activity?

Answer 7

Yes.

Question 8

How is the clamp loader associated with DNA polymerase?

Answer 8

DNAP stalls when it reaches an RNA primer, causing it to be released from clamp. A new DNAP-clamp is assembled at the next RNA primer.

Question 9

Sources of DNA mutations (4)?

Answer 9

1. Polymerase errors
2. Chemical alteration of nucleotides
3. Radiation
4. Spontaneous alterations of nucleotides

Question 10

Describe depurination.

Answer 10

Glyosidic bond breakage. DNAP doesn’t know which nucleotide to add (no template).

Question 11

Describe deamination

Answer 11

The amino group on cytosine are replaced with oxygen. The new base is now a U which is structurally similar to T. DNAP will add an A instead of G.

Question 12

Describe Base Excision Repair

Answer 12

1. Glycosylase recognizes a deaminated base, and removes it.
2. AP endonuclease cuts out the sugar-phosphate with missing base.
3. DNAP replaces correct base and ligase seals nick.

Question 13

Describe Base Excision Repair

Answer 13

1. Bulky DNA lesion identified
2. A nuclease cuts either side of the lesion
3. A helicase removes the damaged section
4. DNAP + ligase replace and seal corrected sequence.

Question 14

What is translesion synthesis?

Answer 14

A separate DNAP is called in to continue replication across a lesion with relatively little error.

Question 15

Describe mismatch repair in E. coli

Answer 15

BacDNA has methylated As at GATC sites. Exonucleases will recognize the template strand by this methylation pattern and cut the newly synthesized strand.

Question 16

Describe the SOS response in E. coli

Answer 16

In response to DNA damage, activated proteins will induce genes that facilitate repair or damage tolerance at much higher rates.

Question 17

Describe homologous recombination

Answer 17

One newly synthesized strand can invade the broken strand (they are identical) allowing the break to be repaired.

Question 18

What are transposable elements?

Answer 18

Small pieces of DNA that can randomly insert at many sites in the genome. Includes insertion sequences and transposons.

Question 19

What are some consequences of transposable element?

Answer 19

They can insert into a promoter sequence and activate a gene. They can also disrupt genes.

Question 20

What are insertion sequences?

Answer 20

Small sequences that only have genes required for movement.

Question 21

What are transposons?

Answer 21

Contain genes required for movement as well as additional genes (antibiotic resistance etc.)

Question 22

Describe replicative transposition

Answer 22

The Tn gets replicated and now there are multiple copies of the element.

Question 23

Describe conservative transposition

Answer 23

Simple cut and past of the insertion at the target site. `

Question 24

What gene is required for movement of a transposon and what enzyme does it make.

Answer 24

Tn3 and transposase

Question 25

What is meant by transposons can be inter and intramolecular?

Answer 25

They can jump between genomes (via plasmids) or within the same genome

Question 26

Describe the CRISPR-Cas9 System in bacteria

Answer 26

CRISPR is a part of the bacterial immune system against viruses. Cas9 is an endonuclease associated with crRNA.

Question 27

Explain how CRISPR-Cas9 can be used in the laboratory

Answer 27

CRISPR-Cas9 can be guided by a specially designed RNA molecule (guide RNA) which brings Cas9 to a target site where it can cut out a piece of specific DNA.