Prokaryote Replication (Gelinas)

Question 1

DNA replication occurs during which phase of the cell cycle?

Answer 1

S (synthesis)

Question 2

Replicated DNA molecules contain:

Answer 2

• a parental strand and a newly synthesized strand
  
  • parental strand serves as template for new strand
• SEMI-CONSERVATIVE

Question 3

Replisome

Answer 3

• the complex molecular machine that carries out DNA replication

Question 4

Origin of replication:

Answer 4

• where DNA replication is initiated
• partial opening of double helix
• region rich in A:T base pairs (less H-bonds)
• DnaA recognizes and induces “melting” of the AT-rich origin in an ATP-dependent manner

Question 5

What protein recognizes and induces “melting” of the AT-rich origin in an ATP-dependent manner at the origin of replication?

Answer 5

DnaA

Question 6

DNA strand separation is catalyzed by:

Answer 6

• DNA helicase (DnaB) within the pre-priming complex
  
  • binds near the replication fork
  • uses ATP to force the DNA strands apart

Question 7

DNA helicase (DnaB):

Answer 7

• catalyzes DNA strand separation during replication
• ATP dependent
• in pre-preiming complex

Question 8

Singlestranded DNA-binding (SSB) proteins:

Answer 8

• bind cooperatively to DNA single strands to:
  
  1. keep them apart
  2. protect them from nucleases

Question 9

During DNA replication, what is the role of Topo II?

Answer 9

Works AHEAD of the replication fork to remove POSITIVE supercoiling induced by strand separation

Question 10

During DNA replication, what is the role of Topo I?

Answer 10

Works BEHIND of the replication fork to remove NEGATIVE supercoiling

Question 11

RNA primers are needed to:

Answer 11

• initiate DNA synthesis
  
  • DNA polymerases cannot initiate synthesis on a totally single-stranded template
• run in 5’ to 3’ direction

Question 12

RNA primers provide a ______ that serves as an acceptor of the first deoxyribonucleotide by DNA polymerase.

Answer 12

free 3ʼ-hydroxyl group

Question 13

Primers are continuously synthesized at the replication fork on:

Answer 13

• the lagging strand
  
  • only a few are needed on the leading strand

Question 14

DNA polymerases only synthesize DNA in what direction?

Answer 14

• 5’ to 3’
  
  • continuous on leading strand
  • fragmented on lagging strand
    
    • a lot of RNA primers
    • okazaki fragments

Question 15

Leading strand:

Answer 15

synthesized continuously in the 5ʼ to 3ʼ direction toward the replication fork

Question 16

Lagging strand:

Answer 16

synthesized discontinuously in short 5ʼ to 3ʼ Okazaki fragments directed away from the fork.

Question 17

How does chain elongation occur?

Answer 17

• DNA polymerase III catalyzes nucleophilic attack of the 3ʼ-OH terminus of the growing DNA chain on the innermost phosphate group of an incoming deoxyribonucleotide triphosphate.
• phosphodiester bond forms

Question 18

Proofreading:

Answer 18

• removal of erroneously introduced nucleotides that are not complementary to the template
• done by 3’ → 5’ exonuclease activity of DNA Pol I and DNA Pol III

Question 19

Mismatched nucleotides that escaped proofreading may be fixed using what pathway?

Answer 19

Mismatch Repair (MMR) pathway

Question 20

Two functions of DNA polymerase I and III in prokaryotes:

Answer 20

1. replication in 5’ to 3’ direction
2. exonuclease activity in 3’ to 5’ direction
   
   1. proofreading

Question 21

DNA chain elongation can be blocked by the incorporation of:

Answer 21

nucleoside analogs

(sugar group of the nucleotide has been modified to prevent further DNA chain elongation. )

Question 22

RNA primer excision:

Answer 22

• DNA Pol I synthesizes a complementary strand until it hits a RNA primer
• DNA Pol I then uses its 5’ to 3’ exonuclease activity to excise the RNA primer
• DNA Pol I then continues to synthesize the complementary strand
• DNA Pol I can then proofread its work using its 3’ to 5’ exonuclease capability

Question 23

What DNA polymerase in prokaryotes removes RNA primers?

Answer 23

DNA Pol I

has both 5’ to 3’ and 3’ to 5’ exonuclease activity

Question 24

DNA ligase:

Answer 24

• covalently joins Okazaki fragments by catalyzing the ATP-dependent formation of a phosphodiester bond between 5ʼ-phosphate group and 3ʼ-hydroxyl group

Question 25

The three DNA polymerases involved in prokaryote DNA replication:

Answer 25

• DNA Pol I
  
  • synthesize 5’ to 3’
  • repair both direction
• DNA Pol II
  
  • repair only 3’ to 5’
• DNA Pol III
  
  • synthesize 5’ to 3’
  • repair only 3’ to 5’