DNA replication

Question 1

DNA structure

Answer 1

• Strands run in opposite polarity
• New nucleotides added in the 5’ –> 3’ direction (added onto the 3’ end)
• Complementary base pairing (held together by hydrogen bonds)

Question 2

Requirements for DNA replication

Answer 2

• template DNA to be copied
• raw materials (nucleotides)
• enzymes

Question 3

DNA replication must be . . .

Answer 3

• accurate
• fast
  (bacteria is 1000 bp/sec)
  (humans are 33 bp/sec)

Question 4

Who is credited for discovering DNA replication?

Answer 4

Watson and Crick publish DNA has a double helix in 1953

Question 5

DNA replication potential models

Answer 5

• Conservative
• Semi- conservative
• Dispersive

Question 6

Conservative potential model

Answer 6

Both strands serve as template (parental and new)

Question 7

Semi conservative potential model

Answer 7

One strand serves as template

Question 8

Dispersive

Answer 8

Parental DNA is “Dispersed” randomly

Question 9

When did Matt Meselson and Franklin Stahl meet?

Answer 9

Met in summer 1954

Question 10

Meselson & Stahl Experiment

Answer 10

• they grew E. coli in a medium containing 15N- incorporated into DNA bases, which made DNA “Heavy”
• grew long enough so both strands are labeled
• Parent DNA is Heavy:Heavy (H:H)
• Transferred the DNA to medium containing 14N and grew several generations
• “Light” nucleotides are incorporated into newly made DNA
• Pattern reveals model of replication
• Labeled DNA analyzed on CESIUM CHLORIDE gradients

Question 11

Conservative

Answer 11

L:L and H:H

Question 12

Semi-conservative

Answer 12

H:L and H:L

Question 13

Dispersive

Answer 13

H:L and H:L

Question 14

What does DNA migrate to in the experiment?

Answer 14

its buoyant density: heavy labeled DNA runs further
- H:H DNA runs near bottom of tube
- H:L DNA (15 N & 14N) runs in between
- L:L DNA runs near top of tube

Question 15

When were the different models ruled out and which model won?

Answer 15

Conservative was ruled out first because bands did not match up. Dispersive was ruled out after second round because two different bands. Therefore, Semi-conservative model won!

Question 16

DNA replication is bi-directional

Answer 16

DNA synthesis initiates at an origin of replication and proceeds outwards in opposite directions

Question 17

Prokaryotic DNA replication

Answer 17

• Millions of base pairs
• 1000 bp/sec
• ONE origin of replication

Question 18

Eukaryotic DNA replication

Answer 18

• 100s of millions of base pairs (billions
• 33 bp/sec
• MULTIPLE origins of replication (solves slow rate of elongation)

Question 19

Replicon model

Answer 19

Proposed in E. coli
- Contains:
~ Origin of replication (replicator)
~ Initiator (DnaA)
~ Replication forks

Question 20

Origin of replication (replicator)

Answer 20

sequence where DNA replication starts

Question 21

Initiator (DnaA)

Answer 21

protein(s) that bind to the origin and initiate replication

Question 22

Replication forks

Answer 22

move away from the origin in a bidirectional fashion

Question 23

DNA replication in eukaryotes

Answer 23

• multiple origins of replication
• opens multiple replication bubbles that fuse together to complete replication

Question 24

First step in DNA Replication

Answer 24

1.) Find the origins of replication
- Short stretches of DNA having a specific nucleotide sequence
- DnaA (the initiator) binds to OriC (the origin)
- Looping causes stress to form in nearby AT-rich sequence= short stretch unwinds
- Unwound section provides access for DNA replication enzymes

Question 25

Second step in DNA Replication

Answer 25

2.) Two DNA helicase attach and unwind the DNA molecule by breaking hydrogen bonds between nucleotides - Move away from each other and separate the two parental strands, making them available as template strands - Creates replication forks: Y-shaped region where DNA is unwound

Question 26

Third step in DNA Replication

Answer 26

3.) Untwisting of the double helix causes strain ahead of the replication fork - Topisomerase keeps the tension down

Question 27

Fourth step in DNA Replication

Answer 27

4.) Single-Stranded Binding Proteins (SSB) bind to unpaired DNA strands, keeping them from repairing - keeps unraveled DNA single-stranded/stabilizes DNA

Question 28

Fifth step in DNA Replication

Answer 28

5.) Unwound sections of parental DNA are now able to serve as templates for synthesis of new complementary DNA strands

Question 29

One problem with DNA replication (5th step)

Answer 29

Enzymes that synthesize DNA cannot initiate synthesis of a polynucleotide - Can only add nucleotides to the end of an existing chain that is base-paired with the template strand

Question 30

Solution to problem with DNA replication (5th step)

Answer 30

PRIMASE - can synthesize a short stretch of RNA called the RNA primer - new DNA strand can start from the 3' end of the RNA primer

Question 31

Sixth step of DNA Replication

Answer 31

6.) DNA polymerase III catalyzes the synthesis of new DNA - Adds complementary (to the parental DNA strand) nucleotides to the 3' -OH end of the RNA primer - Only adds in the 5' --> 3' direction! - each nucleotide added comes from triphosphate nucleotide --> when added, releases two phosphates as pyrophosphate (broken down by pyrophosphatases)

Question 32

Seventh step of DNA Replication

Answer 32

7.) DNA added to both strands in different ways depending on if leading strand (continuous) or lagging strand (not continuous) - helicases continue to unwind parental DNA - LEADING strand: synthesizes continuously - LAGGING strand: once enough of the parental DNA is unwound, primase lays down another RNA primer (DNA polymerase III extends unit it hits the previous primer)

Question 33

Leading strand

Answer 33

Template strand with 5' end at replication fork - Continuous synthesis in the 5' --> 3' direction - Grows towards the replication fork - Only ONE primer needed and DNA polymerase III remains attached to this strand

Question 34

Lagging strand

Answer 34

Template strand with 3' end at replication fork - Discontinuous synthesis in the 5' --> 3' direction - Grows away from the replication fork - MULTIPLE primers needed to be laid down by primase - DNA polymerase III extends until it hits the previous primer = creates Okazaki fragments

Question 35

Which strand creates Okazaki fragments?

Answer 35

Lagging strand

Question 36

Eighth step of DNA Replication

Answer 36

8.) RNA primers are removed and Okazaki fragments are ligated

Question 37

DNA polymerase 1

Answer 37

removes the RNA primer and fills gap with DNA nucleotides

Question 38

DNA ligase

Answer 38

covalently links the Okazaki fragments together

Question 39

When does termination occur?

Answer 39

When replication bubbles merge

Question 40

Issues with the 5' end of lagging strand

Answer 40

- RNA primer removed from lagging strand, but DNA polymerase III cannot add to it because can only add to 3' end - Therefore, next round of replication: shorter leading strand and shortened/staggered lagging strand

Question 41

Do prokaryotes have an issue with 5' end of lagging strand?

Answer 41

No issue due to circular chromosome

Question 42

What is the solution for the issue with the 5' end of the lagging strand in Eukaryotes?

Answer 42

Telomeres! - Multiple repetitions of one short nucleotide sequence (no genes!) - (ex. humans is TTAGGG)

Question 43

Issues with proofreading

Answer 43

- DNA polymerase III works from 5' to 3' - It proofreads (3' to 5') so each nucleotide against its template as soon as it is added to the growing strand (opposite direction)