Lecture 9

Question 1

How is helix first separated at Ori sequence?

Answer 1

Initiation factor DNAa recognizes and binds 9-mer repeats, then separates the DNA at 3AT-rich tandem repeats

Question 2

Which protein unwinds the helix as replication occur?

Answer 2

DNAb helicase complexes proceed bidirectionally

Question 3

How is re-annealing prevented in prokaryotes and eukaryotes?

Answer 3

Single Strand DNA-binding protein (SSB) in prokaryote

Replication Factor A in eukaryote

Question 4

What does DNA polymerase need for for replication (5)?

Answer 4

1. all four dNTPs
2. fragment of DNA that acts as template
3. Magnesium ions
4. primer providing a free 3’-OH group
5. A sliding clamp

Question 5

Why is RNA less stable than DNA?

Answer 5

RNA has 2’-OH

Question 6

Direction of DNA synthesis and template reading

Answer 6

5’-3’ for synthesis

3’-5’ for reading

Question 7

Why does dNTP need to be in the form of triphosphate?

Answer 7

it gives energy for formation of phosphodiester bond.

Question 8

Why is sliding clamp important during DNA replication?

Answer 8

DNA pol assembles with clamp to form a highly processive enzyme. Without it, DNA pol would dissociate after each nucleotide synthesis, and have to have primer again to reattach to the template.

Question 9

Which enzyme remove primer in Okazaki fragments?

Answer 9

Ribonuclease

Question 10

Why does ligase have to join okazaki fragments not DNA pol?

Answer 10

Because DNA polymerase can work only with triphosphates, but fragment is monophosphate.

Question 11

What is the difference between DNA polaymerase I and III? (2 same function, 1 different function)

Answer 11

Both of them synthesize nucleotide,
Both of them have 3’-5’ exonuclease activity (direct repair). When there is mistake, pol pauses, and 3’end of strand is transferred to exonuclease site, for removal of mispaired base.

but Type I has 5’-3’ exonuclease activiy which function as ribonuclease. So it can remove primers and replace it with nucleotides at the same time. so it removes RNA primers of Okazaki fragment and elongates the previous Okazaki fragment.

Question 12

What is role of DNA gyrase?

Answer 12

relieve positive supercoils ahead of the fork.

Both strand of dsDNA is cut, and another duplex DNA is then passed through the cut ends and the ds break resealed.

Question 13

What is the name of protein used for replication termination?

Answer 13

Tus protein

Question 14

What is the role of Tus protein

Answer 14

it binds tot he Ter sequence, then prevents DNA duplex from unwinding by blocking the replication fork and inhibiting DNAb (helicase), so it is called contrahelicase.

Question 15

After termination, resulting progeny chromosomes are interwined, so what is done to resolve this?

Answer 15

Gyrase makes cut in dsDNA to unwind them.

Question 16

Why azidothymidine (AZT) used in treatment of HIV?

Answer 16

AZT is analogue of thymidine, so it gets incorporated into reverse transcription process. Since it does not have 3’OH (it has 3’N), it terminates reverse transcription process. (Since it doesn’t have 3’OH synthesis of next nt is not possible by DNA pol)

Question 17

Why is didanosine used for treatment of viral infection?

Answer 17

DDI has hydrogen replcing 3’-OH on sugar. Thus terminating the reverse transcription process.

Question 18

What is the advantage of using didanosine?

Answer 18

it has much higher affinity to viral reverse transcriptase than human polymerase. So drug is much more specific.

Question 19

What is side effect of didanosine?

Answer 19

it has effect on gamma polymerase in mitochondria so side effect would lack of ATP

Question 20

Why is tenofovir used for viral infection?

Answer 20

it is deoxyadenosine 5’triphosphate analogue. Tenofovir is phosphrylated by host kinase and gets incorporated into reverse transcription process. It lacks 3’-OH on sugar.

Question 21

Why is acyclovir used for viral infection?

Answer 21

It also lacks 3’-OH, and is analogue of deoxyguanosine. , causing termination of viral DNA synthesis.

Question 22

Why is acyclovir advantageous?

Answer 22

It needs to be phosphorylated by kinase before incorporation into viral DNA, but viral thymidine kinase is much more effecive in catalysing 1st phosphorylation than host thymidine kinase, so acyclovir is only activated in infected cells, thus being more potent against viral DNA polymerase than host.

Question 23

What diseases is acyclovir highly specific? (2)

Answer 23

Herpes simplex and varicella zoster

Question 24

How does Gyrase inhibitor behave?

Answer 24

it prevents re-ligation of the cleaved DNA duplex by DNA Gyrase, which turn into permanent DNA strand breaks causing cell death of bacteria.

Question 25

Name gyrase inhibitor based antibiotics

Answer 25

ciprofloxacin

Question 26

What is the drug family of hyrase inhibitor

Answer 26

Fluoroquinoloes, quinolones

Question 27

What is the difference between topoisomerase I and II

Answer 27

I : make a cut in only one strand of ds DNA

II: make a cut in both strands of DNA

Question 28

Which enzyme has reverse-transcriptase activity during DNA replication in eukaryote?

Answer 28

telomerase

Question 29

What is the role of DNA polymerase alpha?

Answer 29

priming during replication initiation

Question 30

What is the role of DNA polymerase gamma? (2)

Answer 30

lagging strand synthesis (including gap filling)

also has 3’-5’ exonuclease

Question 31

what is the role of DNA polymerase ebsylon (2)

Answer 31

leading strand synthesis

has 3’-5’ exonuclease

Question 32

Which enzyme is responsible for removal of RNA primer during DNA replication in eukaryotes? (2)

Answer 32

RNase H or Flap endonuclease 1 (FEN1)

Question 33

What is the effect of cytosine arabinoside (araC)

Answer 33

Termination of elongation

Question 34

For what disease is araC used?

Answer 34

Leukemia, lymphoma

Question 35

What is the mechanism of araC

Answer 35

It has 3’OH that allows further chain elongation when incorporated, however it has another OH at position 2, which prevents polymerase adding the next dNTP due to stereo restraints, which leads to termination.

Question 36

What is the mechanism of araA

Answer 36

It has 3’OH, but planar configuration of the arabinoside prevents DNA elongation due to OH at position2.

Question 37

What is araA used for?

Answer 37

for treatment of relapsed childhood acute lymphoblastic leukaemia (anti-neoplastic agent)

Question 38

what are 2 drugs that interfere with eukaryotic topoisomerases?

Answer 38

etoposide, camptothecin

Question 39

What is the name of group of drugs that interfere eukaryotic topoisomerases?

Answer 39

topotecan

Question 40

For which disease is topotecan used?

Answer 40

ovarian cancer, lung cancer, acute myelocytic leukemia

Question 41

Difference between etoposide and camptothcin

Answer 41

etoposide target topoisomerase II, camptothecin target topoisomerase I

Question 42

What is the mechanism of topotecan?

Answer 42

traps the enzyme topoisomerase when it cleaves DNA causing double strand breaks, preventing DNA replication

Question 43

What is actinomycin D?

Answer 43

Inhibitors of replication

Question 44

What is the mechanism of actinomycin D and its effect (2)

Answer 44

It binds to G-C rich region of DNA. Then, prevents formation of regions of ssDNA.
Also, inhibit RNA transcription (cell needs synthesis of specific RNA’s and protein for cell cycle)

Question 45

What disease is actinomycinD used for?

Answer 45

Wilm tumor, Kaposi sarcoma, testicular cancer