L11: DNA Replication in Vivo - Inside Cells

Question 1

What is the function of DNA replication?

Answer 1

• DNA Encodes all of the proteins and RNA (rRNA & tRNA) in the cell to allow for proper cell function
• New cells will need a copy of the genome (all DNA)

Question 2

Explain the concept of semiconservative DNA replication?

Answer 2

• Each new DNA duplex consists of one strand that was originally part of the parental duplex and one newly synthesized strand (daughter strand).

Question 3

What is the enzyme that plays a big role in DNA replication?

Answer 3

• DNA polymerase

Question 4

What are some key properties of DNA polymerase?

Answer 4

• it “reads” the sequence on a template and links nucleotides together
• ex: reads the template 3’ to 5’ and synthesizes new DNA in the direction 5’ to 3’
• cannot start on its own; it must always start from an existing 3’ OH end of an existing template (a primer; RNA in the cell)

Question 5

What are the steps of DNA polymerization?

Answer 5

• Incoming nucleotides are accepted if they correctly base-pair w/ the template.
• The 3’ OH of the growing strand attacks the high-energy phosphate bond of the incoming nucleotide, providing energy to drive the reaction.

Question 6

What provides energy to the polymerization reaction?

Answer 6

• Activated monomers (triphosphates) are used to provide energy to drive the reaction

Question 7

What are the enzymes involved in DNA replication?

Answer 7

• Helicase
• Primase
• DNA Pol. I
• DNA Ligase
• Topoisomerase
• Single-Stranded Binding Proteins (SSBPs)
• DNA Pol. II

Question 8

What is the function of Helicase?

Answer 8

• Breaks H-bonds

- Unwinding DNA double helix

Question 9

What is the function of Primase?

Answer 9

• Synthesizes RNA primers on leading and laggings strands

Question 10

What is the function of DNA Pol. I?

Answer 10

• Replaces RNA primers with DNA nucleotides

Question 11

What is the function of DNA ligase?

Answer 11

• Catalyzes phosphodiester bond formation
• Join all Okazaki fragments
• joins leading and lagging strands at the origin of replication

Question 12

What is the function of Topoisomerase?

Answer 12

• Relaxes supercoiled DNA

- Relieves stress on the winding helix

Question 13

What is the function of Single-stranded binding proteins (SSBPs)?

Answer 13

• Coats single-stranded DNA

- Keeps the single-stranded DNA apart

Question 14

What is the function of DNA Pol. II?

Answer 14

• Synthesizes DNA 5’ to 3’ on leading and lagging strands

Question 15

What’s the difference between replication in PROKARYOTES and EUKARYOTES?

Answer 15

• Prokaryotes: Bacterial cells can replicate in a circle - both directions. The ends will meet up, and it usually has 1 origin of replication (OriR)
• Eukaryotes: DNA can be big and linear so need more than one origin of replication (OriR)

Question 16

What are the 3 problems DNA must solve in order to replicate DNA?

Answer 16

1. Separate the DNA strands only a little at a time
   
   • Can’t separate an entire genome - very messy
2. Make primers for DNA Polymerase
   
   • can’t make a DNA primer without a DNA primer
   • Cell makes an RNA primer - can start without a 3’ OH
3. Allow synthesis to happen simultaneously from the two template strands
   
   • DNA strands are anti-parallel yet DNA polymerization has to occur in the 5’ - 3’ direction
     - Synthesis has to happen in opposite directions

Question 17

Describe what happens as the replication fork opens.

Answer 17

• At first, all is awesome - both strands are synthesized at the replication fork (although in opposite directions)

Question 18

What is the problem encountered with the replication fork?

Answer 18

• As it opens further, the “top” strand has DNA polymerase moving (synthesizing) to the right and leaving a gap at the fork.

Question 19

What is the solution to this problem at the replication fork?

Answer 19

• Add another Okazaki fragment to join the first fragment to fill in the gap

Question 20

What will the cell have to do as DNA replication proceeds?

Answer 20

• Remove RNA primers and replace with nucleotides that are DNA
• Link all the fragments together to form one continuous strand

Question 21

Discontinuous vs. continuous synthesis.

Answer 21

• Discontinuous: lagging strands

- Continuous: leading strands

Question 22

Describe the leading strand.

Answer 22

• A daughter strand that has its 3’ end pointed toward the replication fork
• can be synthesized as one long, continuous polymer

Question 23

Describe the lagging strand.

Answer 23

• Grows away from the fork
• Daughter strand is synthesized in relatively short, discontinuous pieces
• 5’ end pointed toward the replication fork
• Okazaki fragments: the short pieces in the lagging strand.