DNA Replication

Question 1

list the 5 components required for DNA synthesis

Answer 1

• All 4 dNTPs
• A fragment of DNA to act as template
• DNA polymerase
• Mg2+ ions, which are required for DNA pol activity
• A primer providing a free 3’OH group

Question 2

explain how chain elongation is catalyzed by DNA pol

Answer 2

1. DNA pol catalyzes the formation of a phosphodiester bond between the 3’OH group of the deoxyribose on the last nucleotide and the 5’ phosphate of the dNTP precursor
2. The deoxynucleoside 5’ triphophsate provides the energy source for the reaction
3. At each step in the lengthening the new DNA chain, DNA pol finds the correct precursor dNTP that can form a complementary base pair with the nucleotide on the template strand of the DNA

Question 3

explain the function of acycolvir

Answer 3

• Acyclovir gets converted to acyclovir triphosphate by the viral enzyme thymidine kinase, which can serve as a substrate for viral DNA pol
  
  • Since it lacks a 3’OH group, it causes chain termination when it gets incorporated during viral DNA replication

Question 4

explain how arabinosides function

Answer 4

• The planar configuration of arabinosides induces DNA damage, and inhibits the activity of DNA polymerase. These are anti-cancer agents

Question 5

describe the process of wrong bases by DNA pol

Answer 5

1. Rare tautomeric form of C (C*) happens to basepair with A and is thereby incorporated by DNA polymerase into the primer strand
2. Rapid tautomeric shift of C* to normal cytosine (C) destroys its basepairing with A
3. Unpaired 3’OH end of primer blocks further elongation of primer strand by DNA polymerase
4. 3’ to 5’ exonuclease activity attached to DNA pol chews back to create a basepaired 3’OH end on the primer strand
5. DNA pol continues the process of adding nucleotides ot the basepaired 3’OH end of the primer strand

Question 6

what’s the difference between prokaryote vs eukaryote replication

Answer 6

• Prokaryotes have circular genomes and therefore undergo Theta replication
  
  • single origin of replication
• Eukaryotes have a linear genome and therefore undergo linear replication
  
  • ​multiple origins of replication are required

Question 7

describe the function of initiator proteins (DNaA protein)

Answer 7

• binds to replication and breaks H-bonds between bases

Question 8

describe the function of DNA helicase (DNaB)

Answer 8

• DNA helicase opens the helix and binds primase to form primosome

Question 9

describe the function of helicase inhibitor (DNaC)

Answer 9

• Helicase inhibitor delivers helicase to DNA template

Question 10

describe the function of DNA primase

Answer 10

• DNA primase is an RNA polyererase that synthesizes an RNA primer on the lagging strand to enable DNA polymerase to synthesize DNA strand

Question 11

describe the function of DNA polyermase I

Answer 11

• DNA polymerase I removes the RNA primer and replaces it with DNA
• Synthesizes new DNA in the 5’ to 3’ direction, especially to extend Okizaki fragments
• 3’ to 5’ exonuclease activity to remove primers in lagging strand
• 3’ to 5’ proofreading activity

Question 12

describe the function of DNA polyermase III

Answer 12

• DNA polymerase III allows for the synthesis of leading and lagging strands
• Synthesizes new DNA in the 5’ to 3’ direction
• Exonuclease activity 3’ to 5’

Question 13

explain the function of single stranded binding protein (ssb)

Answer 13

• ssb binds to single stranded DNA in the replication bubble and prevents it from re-annealing or forming secondary structure

Question 14

describe the function of clamp protein

Answer 14

• Clamp protein tightly holds the DNA polymerase III onto the template for synthesis of long templates (increases expressivity) and releases DNA pol when it stalls at a region of ds DNA

Question 15

what problem does DNA polymerase encounter in eukaryotes?

Answer 15

• At the telomeres there are gaps at the 5’ end of the new DNA resulting from RNA primer removal
• DNA pol can only synthesize DNA in the 5’ to 3’ direction–therefore no new DNA synthesis can fill the gaps made by the missing primers

Question 16

how are the gaps filled?

Answer 16

• The gaps in the telomeres are filled by an enzyme called telomerase
• Telomerase is similar to reverse transcriptase in that it synthesizes DNA using an RNA template
• The telomerase itself carries with it an RNA primer
• Synthesis is completed by DNA polymerase containing a primase subunit

Question 17

describe the function of polymerase-alpha

Answer 17

• Synthesizes RNA primer on leading and lagging strand
• Subunit possesses primase activity and initiates DNA synthesis
• Low processivity–falls off after synthesizing a short DNA segment
• No exonuclease activity
• Equivalent to prokaryotic primase

Question 18

describe the function of polymerase-δ (delta)

Answer 18

• Polymerase-δ synthesizes DNA from leading and lagging strands
• Main replicative enzyme
• High processivity–can synthesize long stretches of DNA
• 3’ to 5’ exonuclease proofreading activity
• Equivalent to prokaryotic DNA pol III

Question 19

describe the function of camptothecin (anticancer drug)

Answer 19

• Camptothecin binds to and inhibits topoisomerase I activity
• The result is DNA breakage

Question 20

describe the function of etoposide (anticancer drug)

Answer 20

• Inhibits the activity of topoisomerase II

Question 21

describe the function of actinomycin D (Dactinomycin)

Answer 21

• Prevents DNA replication by:
  
  • Preventing the formation of regions of ss DNA
  • Inhibits RNA transcription (as it prevents strand separation)
• The phenoxazone ring (structure of drug) intercalates between adjacent G-C bases
  
  • The polypeptide chains extend along the minor groove of the helix

Question 22

describe the function of RNAseH and FEN-1

Answer 22

• Eukaryotes: remove primer in eukaryotes on the lagging strand

Question 23

describe the function and structure of telomerase

Answer 23

• telomerase is similar to reverse transcriptase in that it synthesizes DNA using an RNA template
  
  • the telomerase itself carries with it an RNA primer
• Synthesis is completed by DNA polymerase containing a primase subunit