Copying DNA and RNA

Question 1

DNA and RNA are copied by a set of ____ called ______.

Answer 1

Enzymes. Polymerases.

Question 2

The naming of DNA and RNA polymerases is based on what?

Answer 2

The product being made, not the template being used.

Question 3

What is the difference between E.coli replication and human replication (because we use it as an example).

Answer 3

E.coli has no nucleus and doesn’t wrap DNA into histones.

Question 4

E.coli has __ replication forks. They move in _____ directions. How does this aid in replication?

Answer 4

2. Opposite. Two strands open up in the origin of replication (OriC) and form a replication bubble.

Question 5

What is it called when polymerases form new strands?

Answer 5

Bi-directional replication.

Question 6

What does semi-conservative refer to?

Answer 6

Replication is semi-conservative meaning new strands are synthesised to have each daughter cell with one strand of DNA from the parent and one newly synthesised. (Physical card 6).

Question 7

The first enzyme capable of copying DNA found was ______. Why couldn’t it be named the main enzyme responsible?

Answer 7

It didn’t work fast enough to copy the whole genome.

Affinity for DNA not that strong - falls off really quickly and has low processivity.

Question 8

What is the role of DNA polymerase 1?

Answer 8

Important in repairing damage - UV light damage.

Has a 5’ to 3’ exonuclease activity which means it can digest and hydrolise nucleotides that were just created.

Question 9

What is the most important DNA polymerase for replication/which does most of the copying of DNA.

Answer 9

DNA polymerase 3.

Question 10

How many subunits does DNA polymerase 3 have? How do some of these subunits work to create a greater affinity for DNA?

Answer 10

10. Large circular donut-like pair of subunits clamp the enzyme to DNA (beta diamer). Can hold on for more than 40 minutes.

Question 11

What are the similarities between DNA and RNA polymerases?

Answer 11

1. Always copy in 5’ to 3’ direction.
2. Substrate is always nucleotide triphosphate
3. Require a template
4. Form a phosphodiester bond
5. Release pyrophosphate (PPi)

Question 12

What is distinct about DNA polymerase from RNA polymerase?

Answer 12

Needs a primer to be told where to start copying and often proofread the last nucleotide 3’ to 5’ exonuclease activity.
Uses deoxynucleoside triphosphate (dNTPs) as a substrate.

Question 13

Primer for DNA replication is made by _______?

Answer 13

RNA polymerase.

Question 14

Polymerase adds what to the template after the primer is layed?

Answer 14

The dNTP (deoxy-nucleotide triphosphate) that base pairs to the template.

Question 15

How is each nucleotide added in replication?

Answer 15

Sequentially, one after the other as directed by the template.

Question 16

How are the nucleotides bonded together once they are base-paired to the template?

Answer 16

From the 3’ hydroxyl (OH group) of the previous base pair that was formed to the 5’ carbon of newly added.

Question 17

What happens if an incorrect base/nucleotide is added?

Answer 17

It can be cleaved by the 3’ to 5’ exonuclease proofreading activity.

Question 18

Refer to physical card 7 for the run down of the replication process.

Answer 18

Card 7.

Question 19

How do antiviral drugs such as AZT work?

Answer 19

They modify the 3’ OH bond of the virus’ DNA or remove it so that the virus can not replicate its DNA. If there is no functioning 3’ OH there is no extension as this is where the phosphodiester bond forms between the sugars with the bases attached.

Question 20

Why don’t antiviral drugs affect the human cell?

Answer 20

Our DNA has proofreading abilities so that the nucleotide will not be incorporated into the human cell - viruses are not as complex.

Question 21

What are some of the challenges of DNA replication?

Answer 21

Can only be done once just before cell division. Only on or off - no basal activity. Must be coordinated and the whole genome must be copied at once. Both strands must be copied at the same time in the same place.

Question 22

What is reverse transcriptase? How does it work?

Answer 22

Uses an RNA template to produce complementary DNA or cDNA. Works in a 5; to 3’ direction and needs a primer.

Question 23

What can perform reverse transcriptase?

Answer 23

Retroviruses e.g HIV/

Question 24

What is a Klenow enzyme or fragment?

Answer 24

Digest polymerase 1 for a short amount of time with a protease (proteolysis) and you get 2 fragments:
66kD with polymerase 3’ to 5’ exonuclease and a 33kD fragment with 5’ to 3’ exonuclease activity.

Question 25

What is Taq polymerase useful for?

Answer 25

It doesn’t get denatured in increasing temperatures so good to use in reactions such as PCR when you want to melt DNA strands. Amplifies sections of the DNA by copying it over and over. Do need a primer for this and only works 5’ to 3’.

Question 26

When does replication occur?

Answer 26

Only once before cell division.

Question 27

What is the role of Helicase in DNA replication?

Answer 27

Unwinds the DNA

Question 28

What is the role of Single-Stranded Binding Protein (SSBP) in DNA replication?

Answer 28

Two strands once unwound want to hydrogen bond and so ssbp coats to stop the two ss DNA from joining back together.

Question 29

What is the role of Topoisomerase in DNA replication?

Answer 29

untangles and relaxes the DNA as it is unwound so we don’t get supercoiling.

Question 30

What is the role of Primase in DNA replication?

Answer 30

Lays down the short RNA primers.

Question 31

What is the role of DNA polymerase 1 in DNA replication?

Answer 31

Fills in the gaps in the lagging strand.

Question 32

What is the role of Ligase in DNA replication?

Answer 32

Seals the sugar-phosphate backbone.