DNA Replication

Question 1

In what Phase of the cell cycle does DNA replication happen

Answer 1

S phase

Question 2

How many replication forks do E.coli have and in what direction do they move?

Answer 2

Two that move in opposite (bidirectional) directions

Question 3

What does helicase do?

Answer 3

Unwinds and separate the 2 DNA strands by breaking the weak hydrogen bonds

Question 4

What do single-strand binding proteins do?

Answer 4

Attach to keep the two strands of DNA separate and untwisted or stabilize the single strand of DNA

Question 5

What does topoisomerase do?

Answer 5

Attaches at the 2 replication forks to relieve stress on the DNA molecule as it separates

Question 6

What does Primase do?

Answer 6

Synthesizes RNA primers to start the addition of new DNA

Question 7

Where can DNA pol add nucleotides to?

Answer 7

the 3’ end of the template DNA

Question 8

What does ligase do?

Answer 8

Joins the okazaki fragments to make one strand

Question 9

In prokaryotes which DNA pol carries out most synthesis and which completes lagging strand synthesis?

Answer 9

DNA pol III carries out most but pol I completes lagging strand synthesis

Question 10

What is the 3’ exonuclease activity of DNA pol I?

Answer 10

Serves as the proofreading function

Question 11

What is the 5’ exonuclease activity of DNA pol I?

Answer 11

Functions in both replication and DNA repair

Question 12

What is the purpose of Mg2+ in replication?

Answer 12

Help the Negative 3’ OH group and alpha-phosphate form the phosphodiester bond by shielding the negative charges and stabilizing the transition states

Question 13

Do RNA polymerases need a primer to begin synthesis?

Answer 13

No

Question 14

Which is the leading strand and the lagging strand?

Answer 14

Leading strand goes 5’ to 3’ in the direction of the the opening of the fork. Lagging strand 5’ to 3’ moving away from the opening of the fork.

Question 15

What does the lagging strand have to do so that it can synthesize the daughter strand in the right direction?

Answer 15

It needs to loop around so the 2 polymerases can go in the same direction.

Question 16

What is the holoenzyme made up of?

Answer 16

2 DNA Pol III’s, 2 Tau subunits and structural subunits.

Question 17

What is the role of the sliding clamp?

Answer 17

The sliding clamp is the component of the holoenzyme complex that encircles the DNA and keeps Pol associated with the template strand. It also increases the speed that DNA pol can add bases.

Question 18

How many types of sliding clamps are there?

Answer 18

There are 3. prokaryotes = beta w/ 2 subunits. Humans = PCNA 3 subunits. Bacteriophage have gp45 3 subunits

Question 19

Single stranded binding proteins recruit each other in what kind of manner

Answer 19

Cooperative manner

Question 20

What direction does helicase work in? What is its purpose?

Answer 20

5’ to 3’. Unwind DNA for replication

Question 21

What is the mechanism for Type 1 topoisomerase?

Answer 21

Topos unwind DNA for replication. Type 1 cuts one strand and unwinds by transferring the phosphodiester bond between the enzyme and the DNA molecule. They do not use energy in this process.

Question 22

What is the mechanism for Type 2 topoisomerase?

Answer 22

Type 2 cuts both strands of DNA and introduce supercoils. In prokaryotes Gyrase uses ATP energy to cut both strands to switching the positive supercoiling to negative supercoiling to better facilitate unwinding

Question 23

DNA twisted in the opposite direction of the double helix

Answer 23

Negative supercoiling. Opposite of the natural, right-handed turn of DNA.

Question 24

DNA twisted in the same direction of the double helix

Answer 24

Positive supercoiling. This is the natural way DNA turns.

Question 25

What enzyme mechanism is needed in eukaryotes to deal with supercoiling?

Answer 25

(Not gyrase, a different topo II) They only need to relieve positive supercoils downstream of the replication fork because eukaryotic DNA is net negatively supercoiled around histones. Negative supercoils will not be introduced.

Question 26

In prokaryotes, how many DNA Pol III molecules are replicating the DNA?

Answer 26

3 - 1 on the leading strand and 2 on the lagging strand

Question 27

How do you initiate replication in E.coli (prokaryote). Then what happens?

Answer 27

The initiator protein binds to the oriC sequence of the chromosome. The initiator binding recruits helicase, primase and pol III complex.

Question 28

Where do eukaryotes initiate replication?

Answer 28

Sites that have an AT-rich sequence, because it only has 2 H-bonds as opposed to GC region with 3 H-bonds

Question 29

What enzyme removes RNA primers and adding DNA bases?

Answer 29

DNA pol I

Question 30

What does DNA pol III need to be able to add a base?

Answer 30

3’ hydroxyl group

Question 31

What cells have active telomerase?

Answer 31

Germline cells, eggs and sperm. Somatic cells do not have active telomerase.

Question 32

What is telomerase?

Answer 32

It is an RNA dependent DNA polymerase. Uses RNA as a template but brings in DNA bases AKA reverse transcription

Question 33

After telomerase extends the 3’ overhang what happens next?

Answer 33

Primase lays an RNA primer comes in and binds to the region and then DNA pol III adds new bases. Then DNA pol I removes the RNA primer.

Question 34

What do Telomere binding proteins do?

Answer 34

They protect the DNA as well as regulate telomere length.

Question 35

What is the difference between dNTPs and ddNTPs?

Answer 35

dNTPs have a 3’ hydroxyl group and ddNTPs have a 3’ hydrogen group

Question 36

How does sanger sequencing work and how does high throughput sequencing work?

Answer 36

Sanger was slower and needed multiple tubes for reactions and was radioactive. High-throughput uses a single tube to carry out all reactions and use fluorescent dye