DNA Replication

Question 0

Requirements for synthesis

Answer 0

1. Deoxynucleoside triphosphates (dNTPs) - DNA - rNTPs - RNA

2. Primer - template junction

Question 1

Types of polymerase

Answer 1

1. DNA-directed polymerase
   -replication of DNA
   -requires a 3’ end
2. DNA-directed RNA polymerase
   -transcription of DNA
   -primer formation during DNA replication (does not require 3’
   end)

Question 2

Compare RNA polymerase and DNA polymerase in ability when starting replication

Answer 2

• RNA polymerase can start new RNA chains de novo

- DNA polymerase just needs a 3’ end ( can use one on RNA or DNA)

Question 3

In what direction does synthesis occur

Answer 3

Occurs by extension of 3’ end - replication is 5’-3’ but moves along the template 3’-5’

Question 4

Sn2 reaction

Answer 4

• dNTP pairs with its base pair (A-T, C-G) by hydrogen bonding
• 3’OH attacks αphosphate on the dNTP.
• phosphate β and γ stay together (pyrophosphate) but separates from dNTP
• the strong phosphodiester bond is not formed under this reaction (not enough energy)

Question 5

Creation of the phosphodiester bond

Answer 5

• Hydrolysis of pyrophosphates yields the required energy
• the phosphodiester bond forms
• extremely favorable form - hard to break down

Question 6

DNA polymerase ensures that the correct nucleotide is incorporated into the growing strand of DNA by __________.

A. detecting the identity of the incoming nucleotide
B. specifically binding only the correct nucleotide in the active site
C. selecting the correct nucleotide in a exonuclease active site in the DNA polymerase
D. monitoring the ability of the incoming nucleotide to form either an A:T or G:C base pair

Answer 6

D. monitoring the ability of the incoming nucleotide to form either an A:T or G:C base pair

Question 7

How does DNA polymerase know it has the correct base?

Answer 7

No base pairing if incorrect base

Question 8

Why are rNTPs not incorporated?

Answer 8

rNTP cannot bond because is pushed to far that it cannot bind it’s phosphate group to the 3’OH

Question 9

Hand model of DNA polymerase - palm function

Answer 9

Catalytic site

Question 10

Hand model of DNA polymerase - fingers

Answer 10

Bend template and interact with dNTPs

Question 11

Hand model of DNA polymerase - thumb

Answer 11

Holds polymerase and DNA together

Question 12

What are the important amino acids found on the “finger” of polymerase

Answer 12

• Tyr
• Arg
• Lys

Question 13

Function of Tyr amino acid on finger of polymerase

Answer 13

Interacts with base - pushes base so hydrogen bonding can occur

Question 14

Function of Arg/Lys amino acid on finger of polymerase

Answer 14

Interacts with phosphate group - puts in place with right orientation

Question 15

Catalysis in polymerase

Answer 15

• Happens in palm
• A preps primer
• B preps dNTP
• B stabilizes pyrophosphate

Question 16

Function of metal ions in catalysis that occurs in palm

Answer 16

Two metal ions present
A. Interacts with 3’-OH - reduces association between O-H
B. interacts with triphosphate group of dNTP - neutralizes negative charge

Question 17

Mispaired base pair

Answer 17

• mismatch disrupts palm-DNA interaction
• creates higher affinity between exonuclease and ss 3’ DNA compared to polymerase site.
• end of prom moves to exonuclease active site
• mismatch removed - affinity changes
• primer moves back to polymerase active site

Question 18

Compare replication between leading and lagging strands

Answer 18

1. Leading strand -> continuous replication

2. Lagging strand -> segmented (Okazaki fragments)

Question 19

Unwinding of DNA

Answer 19

1. Helicase unwinds DNA
2. Ring-shaped = high degree of processivity
3. Pulls DNA through center of ring
4. Requires ATP hydrolysis for energy
5. Usually bonds to lagging strand

Question 20

Problem of separating DNA strands

Answer 20

1. Supercoiling occurs ahead of replication fork

2. If not relieved replication stops

Question 21

Solution to the problem of unwinding DNA

Answer 21

1. Topoisomerase relieves the supercoiling of DNA
2. Cuts DNA ahead of fork, changes the flips the direction of the coil (positive to negative supercoil).
3. Reseals DNA

Question 22

How does the cell prevent annealing?

Answer 22

Single stranded DNA-binding proteins prevent annealing

Also protects against possible mutations (DNA is at its most vulnerable form).

Question 23

Basic needs of DNA polymerase

Answer 23

1. Template DNA
2. Primer
3. dNTPs
4. Metal ions

Question 24

DNA polymerase can initiate DNA replication

Answer 24

False - needs a primer (requires primase)

Question 25

Function of the sliding clamp

Answer 25

Quickens polymerase reattaching to DNA for replication increasing processivity 1. Polymerase detaches every 20-100base pairs 2. Sliding clamps keeps the polymerase from diffusing away

Question 26

Prokaryote polymerase

Answer 26

DNA pol I-V - DNA pol I - DNA pol III

Question 27

Eukaryote DNA polymerase

Answer 27

Have DNA pol (Greek letters) - DNA pol switching - DNA pol α - DNA pol δ and ε

Question 28

Prokaryotic DNA polymerase III holoenzyme

Answer 28

1. 3 pol III - primary enzyme involved in chromosome replication - polymerase activity - exonuclease activity 2. 2 sliding clamps 3. 1 clamp loader 4. Highly processive 5. Proof reading function

Question 29

Prokaryotic DNA pol I

Answer 29

1. Removes RNA-DNA linkage left behind by RNase H (5' exonuclease) 2. Fills in gaps left behind by RNase H 3. Not highly processive 4. Has no clamp

Question 30

Removing a primer

Answer 30

1. RNase removes almost all RNA strands on DNA 2. Cleaves only RNucleotide 3. The H of RNase H = hybrid

Question 31

Binding backbone

Answer 31

DNA polymerase cannot bind backbone | Ligase binds backbone

Question 32

Eukaryotic DNA polymerases switch

Answer 32

Have multiple subunits 1. DNA pol α - primase - involved in initiating new DNA strands - creates RNA primer and then start DNA copy 2. DNA pol ε - synthesizes leading strand 3. DNA pol δ - synthesizes lagging strand