DNA and Genes - Detailed Notes

DNA Structure and Properties

• Review DNA structure and properties independently.
• Practice discussion questions (DQs) and concept check questions independently.

The Structure of DNA

• Primary Structure: Each strand of DNA has:
  • A backbone consisting of sugar and phosphate groups of deoxyribonucleotides.
  • A series of nitrogen-containing bases projecting from the backbone.
• Review this section independently.

DNA Directionality

• DNA has directionality determined by the orientation of the sugar-phosphate backbone.
  • 3' end: Has an exposed hydroxyl (OH) group attached to the 3' carbon of deoxyribose.
  • 5' end: Has an exposed phosphate group attached to the 5' carbon.
• Review this section independently.

Secondary Structure of DNA

• Antiparallel: The two DNA strands run in opposite directions.
• Double Helix: DNA consists of two strands twisted around each other.
• Stabilization: The secondary structure is stabilized by complementary base pairing.
  • Adenine (A) forms hydrogen bonds with thymine (T).
  • Guanine (G) forms hydrogen bonds with cytosine (C).
• Review this section independently.

Significance of Complementary Base Pairing

• Existing DNA strands can serve as templates for the production of new strands due to complementary base pairing.
• Semiconservative Replication:
  • Parental strands separate.
  • Each parental strand serves as a template for a new daughter strand.
  • Each daughter strand consists of one old (parental) strand and one new strand.
• Review this section independently.

Discussion Questions

1. Practice drawing a nucleotide and label the major components.
2. List defining features of DNA in its double-stranded form:
   • Double-stranded helix.
   • Antiparallel strands.
   • Directional.
   • Stabilized by hydrogen bonds.
   • Hydrophilic overall.
   • Sugar-phosphate backbone faces the exterior.
   • Hydrophobic nitrogenous bases face the interior.

• Review these questions independently.

The Process of DNA Replication

• DNA Polymerase:
  • Catalyzes DNA synthesis.
  • Adds nucleotides to the 3' end of a growing strand.
  • DNA synthesis proceeds in the 5' → 3' direction.
• Initiation:
  • A replication bubble forms when DNA is being synthesized.
  • Formation occurs at a specific sequence called the origin of replication.

Origins of Replication

• Bacteria:
  • Have a single origin of replication.
  • Form one replication bubble.
• Eukaryotic Cells:
  • Have multiple origins of replication on each chromosome.
  • Each replication bubble has two replication forks because synthesis is bidirectional.

Opening and Stabilizing the DNA Helix

• DNA Helicase:
  • Breaks hydrogen bonds between the two DNA strands to separate them.
• Single-Strand DNA-Binding Proteins (SSBPs):
  • Attach to the separated strands to prevent them from closing back up.
• Topoisomerase:
  • Cuts and rejoins the DNA to relieve tension created by unwinding the helix.

Leading vs. Lagging Strand

• DNA Polymerase:
  • Works only in the 5' → 3' direction on a single-stranded template.
  • Requires a 3' end to extend from.
• This directionality causes DNA replication to occur differently on the two template strands because they are antiparallel.

Leading Strand Synthesis

1. DNA is opened, unwound, and primed.
   • Helicase opens the double helix.
   • Single-strand DNA-binding proteins (SSBPs) stabilize single strands.
   • Topoisomerase relieves twisting forces.
   • Primase synthesizes an RNA primer. Primase is an RNA polymerase enzyme that makes an RNA primer to help initiate DNA synthesis.
2. Synthesis of leading strand begins.

Role of Primase

• Primase synthesizes the RNA primer, which is an RNA strand about a dozen nucleotides long.
• The primer base-pairs with the DNA template strand.
• Provides a free 3' hydroxyl (OH) group that can combine with a dNTP to form a phosphodiester bond.
• Primase does not require a free 3' OH group, unlike DNA polymerase, enabling it to initiate nucleotide addition.

Completion of Leading Strand Synthesis

• DNA polymerase can now begin working after the primer is in place.
• DNA Polymerase Structure:
  1. A sliding clamp that forms a ring around the DNA.
  2. A part that grips the DNA strand.
• DNA polymerase adds dNTPs to the primer’s 3' end.
• The leading strand is synthesized continuously in the 5' → 3' direction.

Discussion Question #3

• Fill in the missing terms:
  • __ synthesizes RNA primer
  • __ opens double helix
  • __ stabilize single strands
  • __ holds DNA polymerase in place
  • __ synthesizes leading strand in 5' → 3' direction
  • _ relieves twisting forces

Discussion Question Answer

• Primase synthesizes RNA primer
• Helicase opens double helix
• Single-strand DNA-binding proteins (SSBPs) stabilize single strands
• Sliding clamp holds DNA polymerase in place
• DNA polymerase synthesizes leading strand in 5' → 3' direction
• Topoisomerase relieves twisting forces

Lagging Strand Synthesis

• The lagging strand (or discontinuous strand) is synthesized away from the replication fork.
• This occurs because DNA synthesis must proceed in the 5' → 3' direction.
• Primase synthesizes new RNA primers on the lagging strand as the replication fork opens.
• DNA polymerase synthesizes short fragments of DNA along the lagging strand.
• These fragments are then linked into a continuous strand.

Okazaki Fragments

• The lagging strand is synthesized as short, discontinuous fragments called Okazaki fragments.
• Primers are added by primase to the exposed 3' end near the replication fork.

Synthesis Steps of Lagging Strand

1. Primer added.
2. First fragment synthesized by DNA polymerase III.
3. Second fragment synthesized.
4. Primer replaced: DNA polymerase I removes the RNA primers and replaces them with DNA.
5. Gap closed: The enzyme DNA ligase joins the Okazaki fragments by forming a phosphodiester bond.

Discussion Question #4

Fill in the missing terms:

1. Primer added.
2. First fragment synthesized. __ fragment
3. Second fragment synthesized. _
4. Primer replaced. _
5. Gap closed. _

Discussion Question Answer

1. Primer added.
2. First fragment synthesized. Okazaki fragment
3. Second fragment synthesized. DNA polymerase III
4. Primer replaced. DNA polymerase I
5. Gap closed. DNA ligase

Summary Table of Proteins Required for DNA Synthesis in Bacteria

Replicating the Ends of Linear Chromosomes

• Telomeres:
  • Short, repeating stretches of bases at chromosome ends.
  • Do not contain genes.
• Replication of telomeres (ends of linear chromosomes) can be problematic.
• The leading strand is synthesized all the way to the end.
• On the lagging strand, primase adds an RNA primer close to the end of the chromosome.
• The final Okazaki fragment is made, and the primer is removed.

The End Replication Problem

• DNA polymerase cannot add to the end with no primer.
• A single-stranded (ss) DNA is left at the end of the lagging strand.
• ss-DNA is eventually degraded.
• This would shorten the chromosome by 50-100 nucleotides each time replication occurs.
• Over time, linear chromosomes would be degraded completely.

Telomerase: Solving the End Replication Problem

1. End is unreplicated, resulting in missing DNA on the lagging strand.
2. Telomerase extends the unreplicated end using its own RNA template.
3. Telomerase repeats this activity.
4. The extended single-strand DNA acts as a template for DNA polymerase and a sliding clamp.

Effect of Telomere Length on Cell Division

• Telomerase only functions in some cell types.
• Somatic cells normally lack telomerase.
• Chromosomes of somatic cells shorten as the individual ages.
• The number of cell divisions in somatic cells is limited by the initial telomere length.
• Once chromosomes are shortened to a threshold length, further divisions are shut down.

Discussion Question #5

• Why would a cell want to shut down cell division at some threshold length? What problem might there be if the cell continues dividing?
• Loss of essential genes.
• Will result in cell death.
• Also a safeguard against the development of cancer.

Correcting Mistakes in DNA Synthesis

• DNA replication is very accurate.
• The overall error rate is about one mistake per billion bases.
• DNA polymerase matches bases with high accuracy.
  • Correct bases are the most energetically favorable.
  • Have a distinct shape.
  • Inserts an incorrect base ~ 1/100,000 bases.
• DNA polymerase proofreads, reducing the error rate to ~ 1/10 million bases.
• Other repair enzymes remove defective bases and replace them with the correct one.

Mismatch Repair

• DNA polymerase sometimes leaves a mismatched pair behind.
• Mismatch repair occurs when mismatched bases are corrected after DNA synthesis is complete.
• Mismatch repair enzymes:
  • Recognize the mismatched pair.
  • Remove a section of the newly synthesized strand that contains the incorrect base.
  • Fill in the correct bases using the older strand as a template.

Repairing Damaged DNA

• DNA can be damaged by sunlight, X-rays, and many chemicals.
• Organisms have DNA damage-repair systems.
• UV light and some chemicals can cause thymine dimers to form.
• These dimers produce a kink in the DNA.
• This blocks DNA replication.

Nucleotide Excision Repair

• The nucleotide excision repair system recognizes damage.
• A protein complex recognizes the kink.
• Removes (excises) the damaged single-stranded DNA.
• Uses the intact strand as a template for new DNA.
• DNA ligase links the repaired strand to the original undamaged DNA.

Xeroderma Pigmentosum (XP)

• Rare autosomal recessive disease in humans.
• Causes extreme sensitivity to UV light.
• Increases chance of skin cancer by 1000−2000 times.
• Caused by mutations in nucleotide excision repair systems.
• Cannot repair DNA damaged by ultraviolet radiation.
• Can result from mutations in any of eight genes.

DNA Repair Defects and Cancer

• Defects in the genes required for DNA repair are frequently associated with cancer.
• If mutations in the genes involved in the cell cycle go unrepaired:
  • The cell may begin to grow in an uncontrolled manner.
  • This growth can result in the formation of a tumor.
• If the overall mutation rate in a cell is elevated because of defects in DNA repair genes:
  • The mutations that trigger cancer become more likely.

Chapter 15 Concept Check Questions

1. Which enzyme is incorrectly paired with its function?
   • A. DNA polymerase III—proofread DNA base pairs
   • B. DNA ligase—synthesize Okazaki fragments
   • C. DNA helicase—unwind DNA
   • D. primase—correct replication mistakes
2. Which of the following sequences would result from replication of this DNA template strand? 3′-AAGTCAGT-5′
   • A. 5′-AAGTCAGT-3′
   • B. 5′-TTCAGTCA-3′
   • C. 3′-TTCAGTCA-5′
   • D. 3′-AAGTCAGT-5′
3. DNA replication is semiconservative because replicated DNA molecules are composed of _.
   • A. two strands of old DNA
   • B. two strands of new DNA
   • C. one strand of old DNA and one strand of new DNA
   • D. one strand of new DNA
4. Hutchinson–Gilford progeria is a syndrome that results in the accelerated aging of infants and children. Balding, circulatory trouble, and fragile bone structure typically begin during infancy, and the children usually do not live through their teenage years. When scientists looked at the DNA of these individuals, they found the DNA had shorter _ than those of healthy individuals.
   • A. genes
   • B. primers
   • C. telomeres
   • D. DNA polymerase
5. Benzopyrene in cigarette smoke binds to DNA and distorts its shape, interfering with DNA replication. Which repair mechanism would most likely be used to repair the damage caused by this chemical?
   • A. proofreading
   • B. mismatch repair
   • C. telomere repair
   • D. nucleotide excision repair
6. DNA polymerase cannot replicate DNA unless an RNA primer is first attached to the template strand. This is because DNA polymerase can only _.
   • A. add onto existing 3′ hydroxyl groups
   • B. replicate in a 5′ → 3′ direction
   • C. replicate the leading strand
   • D. add onto ribonucleotides