Lecture 23

Question 1

What are the two possible mechanisms for DNA replication from a single origin

Answer 1

• replication is bidirectional
• unidirectional and bidirectional

Question 2

What does DNA synthesis require in vivo

Answer 2

• an RNA primer synthesized by an RNA polymerase
• DNA is unwound by enzymes - helicases - to expose the bases on each of the template strands, and replication is primed by short RNA primers that are synthesized by primase, an RNA polymerase. (The RNA primer is removed at a later stage of replication and replaced by DNA).
• DNA polymerases require a pre-existing DNA or RNA primer and template. They
  cannot synthesize DNA de novo. They must add incoming dNMPs to an existing
  primer with a 3’-OH. (RNA polymerases do not need a primer and can synthesize
  RNA from scratch.)

Question 3

Primosome

Answer 3

• primase - generates RNA primer
• helicase - unwinds DNA

Question 4

DNA pol 3 multimer

Answer 4

• main elongation enzyme
• highly processive: adds many thousands of nucleotides in 5’-3’ direction before falling off the DNA

Question 5

DNA pol 1 monomer

Answer 5

• “clean-up polymerase”
• replaces the RNA primer nucleotides (ribonucleotides) with deoxynucleotides in a process called nick translation
• proof reading function

Question 6

Which enzymes catalyze the mains steps in DNA polymerization

Answer 6

• single-stranded binding protein (SSB) - keeps DNA unwound
• Primase
• DNA polymerase 3 holoenzyme
• DNA polymerase 1
• DNA ligase

Question 7

DNA ligase

Answer 7

• ligates the lagging strand Okazaki fragments together

Question 8

DNA pol 3 holoenzyme

Answer 8

• 3 copies of the “core” DNA pol 3 enzyme
• each comprised of 3 subunits and one copy of the sliding clamp ladder
• sliding clamp loader includes 3 copies of the tau protein, each of which binds one DNA pol 3 core enzyme

Question 9

Explain why DNA pol 1 is essential in lagging strand DNA synthesis

Answer 9

1. Removes RNA primers one ribonucleotide at a time, due to its 5’ -> 3’ exonuclease activity
2. Adds deoxyribonucleotides to the 3’ end of the adjacent Okazaki fragment (lagging strand), due to its 5’ -> 3’ polymerase activity (recall that polymerization always occurs 5’ -> 3’) These processes together are called “nick translation,” because the gap (nick) between the Okazaki fragments seems to move (translate) as the RNA ends of one fragment are removed and dNMPs are added to the adjacent Okazaki fragment (see next slide).
3. Excises mismatched or defective nucleotides in the 3’ end of the Okazaki fragments: 3’ ® 5’
   exonuclease. This process is called “proof-reading,” or editing.

Question 10

Explain Nick translation to complete the synthesis of the lagging strand

Answer 10

• Each Okazaki fragment has its own RNA primer that must be removed. DNA pol I simultaneously polymerizes (extends) one end of an Okazaki fragment and excises the RNA primer of an adjacent Okazaki fragment one
  nucleotide at at time.
• As it adds dNMPs it checks that they are correct (proof-reads). If they are incorrect, it
  excises them (3’ -> 5’ exonuclease activity) and replaces them with the correct nucleotide.
• Because the dNMPs are added to the 3’ end of one Okazaki fragment and the ribonucleotides are removed from the 5’ end of the adjacent RNA primer, the gap (“nick”) between these two strands gets moved (“translated”) along the strand in the 5’ -> 3’ direction.
• The nick will be subsequently sealed by DNA ligase in an ATP-driven process. .

Question 11

How does 3’-5’ proofreading repair by DNA pol 1 work

Answer 11

• DNA pol 1 removes newly-added mismatched nucleotides from the 3’ end of the growing strand