Midterm #2: DNA Replication

Question 1

Flow of Genetic Information

Answer 1

• DNA Replication: refers to the synthesis of new DNA using the exisiting DNA as a template
• **Transcription: **refers to the synthesis of RNA using a gene sequence as a template
• **Translation: **refers to the synthesis of protein using mRNA as a template
• Note that:
  
  • Most human DNA is never transcribed
  • not all RNA molecules are translated
  • RNA is sometimes reverse transcribed back to DNA

Question 2

Name this structure

Answer 2

adenine

Question 3

Name this structure

Answer 3

Guanine

Question 4

Name this structure

Answer 4

Thymine

Question 5

Name this structure

Answer 5

Cytosine

Question 6

Name this structure

Answer 6

Uracil

Question 7

Function of DNA

Answer 7

storgage of genetic information

Question 8

Define genes

Answer 8

DNA sequences that specify the kinds of proteins made by cells

Question 9

What does complementary base pairing allow for

Answer 9

• AT, GC
• ensure redundancy
• allows for genetic information to be copied

Question 10

DNA polymerase

Answer 10

• ​carries out DNA replication
• step-by-step addition of deoxynucleoside monophosphates to a DNA chain (primer strand)

Question 11

Reaction of DNA polymerases

Answer 11

• nucleophilic attack by the 3’-hydroxy group of the primer strand on the alpha-phosphate of the incoming deoxynucleoside triphosphate.
• This results in the formation of a covalent phosphodiester bond in the transesterification reaction
• Note that polymerization proceeds in the 5’ to 3’ direction
• A driving force is the subsequent hydrolysis of PPi by inorganic pyrophosphatase

Question 12

All DNA Polymerases Require:

Answer 12

• Template strand
• Primer strand with free 3’ OH
• 2’-deoxynucleoside 5’-triphosphates
• Mg²⁺

Question 13

Prokaryotic DNA polymerase I

Answer 13

• First characterized is DNA I from E. coli
• single 103 kDa polypeptide
• Not essential for DNA replication, but important in DNA repair

Question 14

Pol I has 3 distinct catalytic activities

Answer 14

• Replication Function (DNA polymerase activity)
  
  • Moderate processivity (adds 20 bp per encounter with DNA)
  • Moderate rate (25 bp/sec)
  • Moderate fidelity (1 error in 10⁵ bp=10^-5error/bp)
• Editing Function (Exonuclease Acitivty 1, 3’→5’)
  
  • boosts fidelity to 10^-8error/bp
• Repair Function (Exonuclease Activity 2, 5’→3’)

Question 15

DNA polymerase III

Answer 15

• replicates DNA for cell replication
• Large, multiprotein complex composed of 8 different subunits
  
  • Very fast: ~1000 bp/sec
  • Very processive: 1000 bp/encounter
  • Possess 3’→5’ exonuclease activity, but not 5’→3’
  • polymerase fidelity: ~10^-5 error/bp, boosted to 10^-7 to 10^-8 by exonuclease proofreading

Question 16

Initiation Phase of DNA Replication in E. coli

Answer 16

• begins at discrete site known as the origin of replication (in E. coli known as oriC)
• Binding of dnaA protein to the oriC results in unwinding of the DNA duplex
• The duplex is further unwound with the binding of dnaB and dnaC proteins
• The unwound DNA is stabalized and protected by single stranded binding protein (SSB)
• A bubble forms at the oriC with the unwinding of DNA in both directions

Question 17

DNA Replication: Priming Step

Answer 17

• Primase is an RNA polymerase that binds to single-stranded DNA in a multiprotein complex known as a primosome (7 proteins)
• Primase synthesizes short (~5-10 base) RNA primers
• Primase dissociates and Pol III utilizes these RNA primers for the synthesis of DNA

Question 18

DNA Replication: Elongation Phase

Answer 18

• DNA pol III assembled onto DNA to form the replication fork
• Polymerase error rate 10^-5
• 3’→5’ exonuclease corrects >99% to make 10^-7 to 10^-8 error rate

Question 19

Leading vs. Lagging strand, supercoiling

Answer 19

• Leading Strand
  
  • template strand is oriented 3’→5’
• Lagging Strand
  
  • template strand is oriented 5’→’3
  • Loops out during replication
  • Okazaki fragments
• Positive supercoiling from unwinding DNA at replication fork is undone by DNA gyrase
  
  • ​Induces negative supercoils as it hydrolyzed ATP

Question 20

Termination of DNA Replication

Answer 20

• Replication of E. coli genome ends when replication forks meet at the termination region (ter)
• The “concatanes” are resolved by topoisomerase IV

Question 21

Name this structure

Answer 21

Ciprofloxacin

Question 22

Name this structure

Answer 22

Levofloxacin

Question 23

Fluroquinolone antibiotics

Answer 23

• Ciprofloxacin, Levofloxacin
• Inhibit both DNA gyrase (gram negative) and topo IV (gram positive) enzymes

Question 24

DNA Mismatch Repair: Overview

Answer 24

• This system fixes >99% of the rare errors made by DNA pol, boosting the overall fidelity to 10^-9 to 10^-10 error/bp
• E. coli genome: ~5x10⁶ bp
• Human genome: ~3x10⁹ bp

Question 25

Mismatch Repain: Features

Answer 25

• MutS recognizes mismatch
• Recruits MutL and MutH
• MutH nicks the replicated DNA
• UvrD helicase comes in and unwinds DNA
• Exonuclease removes DNA
• DNA pol holoenzyme fixes mistake
• Ligase ligates the strands together
• ATP is required

Question 26

DNA Damage Repair:

Answer 26

• UV lights, X-Rays, ROS, plant toxins, chemical carcingoens, and therapeutic alkylating agents can damage DNA
• Base Excision Repair
• Nucleotide Excision Repair

Question 27

Base Excision Repair

Answer 27

• DNA glycosylate removes damaged base from AP site (example, recognizes uracil in DNA)
• AP endonuclease removes some bases from the middle of the DNA
• DNA pol I puts in the DNA bases
• Ligase ligates the strand back together

Question 28

Nucleotide Excision Repair

Answer 28

• Distortions in DNA
• Uvr-A, Uvr-B, and Uvr-C excise out an area including the damage
• DNA pol and Ligase

Question 29

Bacterial Plasmids

Answer 29

• Circular, extra-chromosomal, double-stranded DNA molecules that replicate autonomously
  
  • the number of plasmid molecules per cell is determined by the “strength” of the replication origin (copy number concept)
• Plasmids may be transferred between bacteria and often carry genes responsible for antibiotic resistance and toxins

Question 30

DNA Replication in Eukaryotes: The players

Answer 30

• 13 different eukaryotic DNA pol (all with Greek letters)
• DNA pol alpha:primase complex synthesizes RNA primer and then adds ~15 bases of DNA. This primer is removed by FEN-1 nuclease.
• DNA pol delta and epsilon are the main DNA pol involved in chromosomal replication
• Many other polymerases appear to function primarily in DNA repair pathways (ex: pol beta), plus those found in mitochondria (pol gamma) and chloroplasts

Question 31

Name this structure

Answer 31

Camptothecin

Question 32

Name this structure

Answer 32

Etoposide

Question 33

Topoisomerase Poisons

Answer 33

• Etoposide (topo II) and Camptothecin (topo I)
• Trap topo:DNA complexes and ultimately causes single and double stranded DNA breaks in the duplex
• Used in cancer chemotherapy