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