Lecture 5 - Nucleic Acids, DNA Replication

Question 1

Nucleic Acids (4)

Answer 1

• Polymers specialized for storage and transmission of biological information.
• DNA: Deoxyribonucleic Acid
• RNA: Ribonucleic Acid
• Comprised of monomers called nucleotides.

Question 2

Nucleotides have three components (3):

Answer 2

1. nitrogen-containing base.
2. pentose sugar
3. phosphate group

Question 3

Nucleotide vs. Nucleoside:

Answer 3

Nucleoside is a molecule consisting of just the nitrogen-containing base and pentose sugar (two components).
Nucleotide is a nucleoside with a phosphate group (three components).

Question 4

Variations of Nitrogenous Bases:

Answer 4

Pyrimidines: single ring (T/U, C)
Purines: two fused rings (A, G)

Question 5

Variations of Pentose Sugars:

Answer 5

• RNA contains the sugar RIBOSE which has additional -OH group (more polar)
• DNA contains DEOXYRIBOSE

Question 6

Nucleotides can contain 1, 2, or 3 phosphates. Nucleotides that make up nucleic acids have _ phosphate group and are called…

Answer 6

1 phosphate group

nucleoside monophosphates.

Question 7

Other roles for nucleotides (3)

Answer 7

• ATP, GTP: energy source in cellular work
• cAMP (cyclic AMP): essential for relaying extracellular cues to intracellular processes
• act as coenzymes in metabolic group transfer reactions (ex. FAD)

Question 8

Formation of nucleic acids (3)

Answer 8

• phosphodiester linkage: formed when two nucleotides undergo condensation (anabolic) reaction.
• phosphate group of 5’ carbon of new nucleotide attaches to 3’ carbon of previous nucleotide. “grow in 5’-3’ direction”
• nucleic acids are also known as polynucleotides.
  
  • oligonucleotides are short (~20 monomers)

Question 9

Complementary base pairing is key to understanding ___ and ___ (4).

Answer 9

structure, function

• complementary base pairing: purines pair with pyrimidines by HYDROGEN bonds.
• DNA: A-T, G-C
• RNA: A-U, G-C
• A-T/A-U are double H-bonds, G-C are triple H-bonds and harder to break.

Question 10

RNA structure (3)

Answer 10

• single-stranded
• base pairing can occur between different regions of the molecule, leading to diverse three-dimensional structures (structure is determined by order of bases)
• complementary base pairing can occur between RNA and DNA.

Question 11

DNA structure (4 major points)

Answer 11

• double helix
• antiparallel strands
• bases exposed in major and minor groups
• right-handed

Question 12

DNA structure: The Helix (2)

Answer 12

• sugar-phosphate backbone
• chains held together by…
  
  1. H-bonds between base pairs
  2. Van der Waals between adjacent bases on same strand.

Question 13

DNA structure: Antiparallel strands (4)

Answer 13

• sugar-phosphate bonds determine strand direction.
• 5’ end has free 5’ phosphate group
• 3’ end has free 3’ -OH hydroxyl group
• 5’ end of one strand is paired with 3’ end of the other strand.

Question 14

DNA structure: Base exposure (2)

Answer 14

• Minor groove: backbone of strands closer together.

- Minor groove: backbone of strands further apart.

Question 15

DNA structure: Base exposure (4)

Answer 15

• Four possible configurations of base pairs in grooves
• Exposed base edges available for H-bonds with other molecules.
• Surfaces of A-T and G-C are chemically distinct.
• Proteins can recognize specific DNA sequences which is important for DNA replication and gene expression.

Question 16

All DNA molecules have the same ____. Diversity is determined by ___.

Answer 16

structure, difference in nucleotide base sequences.

Question 17

Why is DNA’s structure important? (4)

Answer 17

• stores genetic information: in the form of millions of nucleotides.
• susceptible to mutations: alterations in base sequences, genetic diversity.
• precisely replicated in cell division by complementary base pairing.
• genetic info is expressed as the phenotype: nucleotide sequence determines sequence of amino acids in proteins.

Question 18

DNA transmits information in 2 ways:

Answer 18

1. DNA can reproduce itself (replication)
2. Certain DNA sequences can be copied into RNA (transcription). This RNA can specify sequence of amino acids in a polypeptide (translation)

Question 19

Gene expression (defn)

Answer 19

is the process of transcription and translation.

Question 20

DNA structure was determined by… (1)

Answer 20

1. Physical evidence from X-ray cyrstallography.
   
   • Biophysicist Maurice Wilkins discovered way to prepare DNA for X-ray diffraction.
   • Rosalind Franklin analyzed DNA samples and found:
     - DNA to be a double helix
     - 10 nucleotides in each full turn (3.4 nm in length)
     
     • 2 nm diameter

Question 21

DNA structure was determined by… (2)

Answer 21

2. Chemical evidence from base composition.
   
   • known that DNA is a polymer of nucleotides, and four different DNA nucleotides differed only by their bases.
   • Erwin Chargraff reported that all DNA had the same about of purines and pyrimidines. CHARGAFF’S RULe (A=T, G=C)

Question 22

Watson and Crick’s Model incorporated lines of evidence and published structure of DNA in 1953. (2)

Answer 22

1. Antiparallel with sugar phosphate backbone.

2. Followed Chargaff’s rule, resulting in uniform width (OO=O, O=OO)

Question 23

How is DNA replicated? (3)

Answer 23

• Semiconservative: each parent strand serves as a template and new molecule has one new and one old strand.
• Conservative: original DNA serves as template and original is not incorporated into new molecule.
• Dispersive: fragments of original DNA incorporated into new DNA.

Question 24

The Meselson-Stahl Experiment (3)

Answer 24

• E-coli cultures grown with 15N then transferred to 14N
• Density gradient separation (centrifugation performed)
• Results explained semiconservative model. DNA is reproduced by semiconservative replication!

Question 25

DNA replication requires 2 steps:

Answer 25

1. Double helix is unwound, making two template strands.

2. New nucleotides form complementary base pairs with template DNA and are linked by phosphodiester bonds.

Question 26

Formation of new DNA strands (3)

Answer 26

• free monomers that form DNA polymers have three phosphate groups.
• new nucleotides added to the growing 3’ end lose two phosphates (pyrophosphate) during phosphodiester bond formation.
• energy released by hydrolysis of dNTP drives condensation reaction.

Question 27

6 Key players in DNA replication (6)

Answer 27

1. DNA polymerase
2. primase
3. DNA helicase
4. single-strand binding proteins
5. sliding DNA clamp
6. telomerase

Question 28

Origins of replication (2)

Answer 28

• DNA replication begins with binding of the pre-replication complex to the origin of replication (ori)
  
  • prokaryotes have 1
  • eukaryotes have >1
• DNA is unwound and replication forks move away from one another.

Question 29

DNA replication begins with a ___. (4)

Answer 29

Primer

• DNA polymerase requires a primer, a short “starter” strand - usually RNA
• Primase synthesizes a primer that is complementary to the DA template
• DNA polymerase adds nucleotides to the 3’ end of the primer
• Primer is degraded and DNA added in its place.

Question 30

DNA polymerase (3)

Answer 30

• DNA polymerase substrates: dNTPS and template DNA
• “palm” brings active site and substrates together
• “fingers” recognize nucleotide bases

Question 31

2 Proteins assist DNA polymerase

Answer 31

1. DNA helicase uses energy from ATP hydrolysis to unwind DNA.
2. Single-strand binding proteins keep the strands from reforming double helix.

Question 32

Two types of newly replicating strands (2)

Answer 32

1. Leading strand: grows continuously “forward” at its 3’ end as the form opens.
2. Lagging strand: grows in shorter “backward” discontinuous stretches, known as Okazaki fragments.
   
   • exposed 3’ end gets farther from the fork.
   • each fragment requires own primer, synthesized by primase.
   • segments linked by enzyme DNA ligase, resulting in a continuous strand of DNA.

Question 33

DNA polymerases are fast because.. (2)

Answer 33

• they are processive: catalyze many linkages each time they bind to DNA.
  
  • high processivity = greater number of nucleotides added before polymerase dissociates.
• polymerase-DNA complex is stabilized by a sliding DNA clamp - a protein that keeps the enzyme and DNA in close contact, increasing efficiency of polymerization.

Question 34

In eukaryotes, DNA is ___ and the polymerase/replication complex is ____.

Answer 34

mobile, stationary.

- DNA goes in as one double stranded molecule, and emerges as two double stranded molecules.

Question 35

Telomeres are not fully replicated (4)

Answer 35

• telomeres: repetitive sequences at the ends of eukaryotic chromosomes.
• in humans, sequence is TTAGGG, and repeated about 2,500 times.
• prevents DNA repair system from seeing chromosome end as a break.
• on lagging strand, terminal Okazaki primer is removed, no DNA can be synthesized to replace it.
  
  • short piece of DNA removed.
  • chromosome shortening with each replication; genes may be lost and cell dies.

Question 36

Telomerase (2)

Answer 36

• continuously dividing cells have telomerase, which catalyzes addition of lost telomeres by using its own RNA sequence as a template (bone marrow stem cells and gamete producing cells)
• telomerase expressed in most cancer cells (anti-cancer target)