Slide 1

Genetics and Evolution- Molecular Basis of Inheritance - Eukaryotic DNA Replication

• DNA replication: the process of copying a DNA molecule
• Occurs during the S phase of the cell cycle
• Ensures that genetic information is faithfully passed on to daughter cells
• Essential for growth, development, and cell division
• Involves many enzymes and proteins

Slide 2

DNA Replication Steps

1. Initiation:

• DNA helicase unwinds the double helix and breaks hydrogen bonds between the bases
• Creating a replication fork

2. Elongation:

• DNA polymerase adds complementary nucleotides to the template strands
• Leading strand synthesized continuously
• Lagging strand synthesized discontinuously in Okazaki fragments

3. Termination:

• DNA polymerase reaches the end of the DNA molecule
• DNA ligase joins Okazaki fragments on the lagging strand

Slide 3

Enzymes Involved in DNA Replication

1. DNA Helicase:

• Unwinds the double helix, separating the two strands

2. DNA Polymerase:

• Catalyzes the addition of nucleotides to the growing DNA strand
• Requires a primer to start synthesis

3. Primase:

• Synthesizes the RNA primer needed for DNA polymerase to initiate synthesis

4. DNA Ligase:

• Joins the Okazaki fragments on the lagging strand

Slide 4

DNA Replication: Initiation

• DNA replication begins at specific sites called origins of replication
• Multiple origins of replication in eukaryotic cells
• Proteins bind to the origin and initiate unwinding of the DNA double helix

Slide 5

DNA Replication: Elongation

• Leading strand is synthesized continuously in the 5’ to 3’ direction
• DNA polymerase adds nucleotides to the 3’ end of the growing strand
• Lagging strand is synthesized discontinuously
• Okazaki fragments are synthesized in the 5’ to 3’ direction away from the replication fork

Slide 6

DNA Replication: Elongation (cont.)

• Each Okazaki fragment requires a separate primer
• DNA polymerase adds nucleotides to the primer, elongating the fragment
• RNA primers are later replaced with DNA by DNA polymerase and DNA ligase

Slide 7

DNA Replication: Termination

• DNA replication continues bidirectionally from multiple origins of replication
• When the replication forks meet, DNA polymerase reaches the end of the DNA molecule
• DNA ligase joins the Okazaki fragments on the lagging strand, resulting in a complete DNA molecule

Slide 8

DNA Replication Errors

• DNA replication is a highly accurate process, but errors can still occur
• DNA polymerase has proofreading ability to correct errors
• Mismatch repair proteins also fix errors that escape proofreading

Slide 9

Telomeres and Telomerase

• Telomeres are repetitive DNA sequences at the ends of linear chromosomes

• Protect chromosomes from degradation and fusion

• Shorten with each round of DNA replication

• Telomerase is an enzyme that adds telomeric sequences to the ends of chromosomes

• Present in germ cells, stem cells, and some types of cancer cells

• Allows cells to maintain their telomeres and continue dividing

Slide 10

Significance of DNA Replication

• DNA replication is crucial for cell division and growth
• Errors in DNA replication can lead to genetic mutations and diseases
• Understanding the process of DNA replication helps scientists develop treatments for genetic disorders and design strategies for genome engineering

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Slide 11

• DNA replication is a highly regulated process

• Ensures that each cell receives a complete and accurate copy of the genome

• Regulation occurs at various levels:

  1. Initiation of replication
  2. Ensuring fidelity during replication
  3. Termination of replication

• Misregulation of DNA replication can lead to genomic instability and diseases

Slide 12

• Initiation of DNA replication involves the assembly of pre-replication complexes (pre-RC)

• Pre-RC formation restricts DNA replication to occur only once per cell cycle

• Regulation of pre-RC assembly prevents re-replication

• Key regulatory factors:

  1. Origin recognition complex (ORC)
  2. Cdc6 and Cdt1 proteins
  3. Cyclin-dependent kinases (CDKs)
  4. Checkpoint proteins

Slide 13

• Fidelity during DNA replication is maintained by proofreading and error correction mechanisms

• DNA polymerase has an intrinsic 3’ to 5’ exonuclease activity for proofreading

• Incorrectly paired nucleotides are excised and replaced with correct nucleotides

• Mismatch repair:

  • Corrects errors missed by proofreading
  • Detects and removes mispaired nucleotides post-replication
  • Relies on specific repair enzymes and proteins

Slide 14

• Telomeres play an essential role in maintaining genomic stability

• Telomeres, comprised of repetitive DNA sequences, prevent degradation and fusion

• Telomeres shorten with each round of DNA replication

• Telomerase helps counteract telomere shortening:

  • Adds additional telomeric repeats to the ends of chromosomes
  • Present in cells with high proliferative capacity (e.g., stem cells, germ cells)

Slide 15

• Telomerase structure:

  • Contains a catalytic subunit (TERT)
  • An RNA component (TERC) serves as a template for DNA synthesis

• Telomerase regulation:

  • Activity tightly controlled during development and in somatic cells
  • Reduced or absent in most adult cells to prevent uncontrolled cell division

Slide 16

• Telomerase and aging:

  • Telomere shortening is associated with cellular senescence and aging
  • Limited telomere length leads to replicative exhaustion and cellular dysfunction

• Telomerase and cancer:

  • Upregulated telomerase activity is a hallmark of many cancer cells
  • Sustained telomerase activity enables unlimited cell division

Slide 17

• Applications of DNA replication:
  1. DNA cloning: Amplification of DNA fragments using replication machinery
  2. Polymerase chain reaction (PCR): Rapid amplification of specific DNA sequences
  3. DNA sequencing: Determining the order of nucleotides in a DNA molecule
  4. Genome editing: Utilizing replication machinery to introduce specific changes in DNA sequences

Slide 18

• Techniques used to study DNA replication:

  1. DNA labeling with nucleotide analogs (e.g., BrdU)
  2. DNA fiber assay: Visualizing replication tracks on stretched DNA fibers
  3. Fluorescence microscopy: Imaging replication foci and dynamics in live cells

• These techniques aid in understanding replication dynamics, origins, and replication fork progression

Slide 19

• DNA replication and human diseases:

  • Mutations in DNA replication-associated genes can lead to genetic disorders
  • Examples of diseases associated with impaired DNA replication:
    1. Ataxia-telangiectasia (AT)
    2. Bloom syndrome
    3. Werner syndrome

• Research on DNA replication disorders provides insights into normal replication and disease mechanisms

Slide 20

• Key takeaways:
  1. DNA replication is a complex and highly regulated process
  2. Initiation, fidelity maintenance, and termination are crucial steps in DNA replication
  3. Telomeres and telomerase play vital roles in genomic stability and cellular lifespan
  4. Misregulation of DNA replication can lead to diseases and cancer
  5. DNA replication has numerous applications in research and biotechnology

Slide 21

• DNA replication is driven by a series of enzyme-catalyzed reactions.
• Each enzyme has a specific role in the process and contributes to the overall accuracy and efficiency of replication.
• The main enzymes involved in DNA replication include:
  1. DNA helicase: Unwinds the DNA double helix
  2. DNA polymerase: Catalyzes the addition of nucleotides to the growing DNA chain
  3. DNA ligase: Joins the Okazaki fragments on the lagging strand
  4. Topoisomerase: Relieves the supercoiling tension ahead of the replication fork
• These enzymes work together to ensure the faithful replication of the DNA molecule.

Slide 22

• DNA replication is a highly coordinated process that occurs in multiple steps.
• The steps of DNA replication include:
  1. Initiation: Specific sequences called origins of replication are recognized, and the replication machinery is assembled.
  2. Unwinding: DNA helicase separates the DNA strands by breaking the hydrogen bonds.
  3. Elongation: DNA polymerase adds complementary nucleotides to the template strands.
  4. Termination: DNA replication is completed, and the newly synthesized DNA molecules are separated.
• Each step is tightly regulated and ensures the accurate duplication of the DNA molecule.

Slide 23

• DNA replication is semiconservative, meaning that each DNA strand of the original molecule serves as a template for creating a new strand.
• The complementary bases are added according to the base pairing rules: adenine (A) pairs with thymine (T), and guanine (G) pairs with cytosine (C).
• The newly synthesized DNA molecule has one original strand and one newly synthesized strand.
• This mechanism ensures the preservation of genetic information during cell division.

Slide 24

• DNA replication occurs in different directions on the leading and lagging strands.
• The leading strand is synthesized continuously in the 5’ to 3’ direction, following the replication fork movement.
• The lagging strand is synthesized discontinuously in short fragments called Okazaki fragments.
• Each Okazaki fragment requires a primer for DNA polymerase to initiate synthesis.
• The fragments are later joined by DNA ligase to create a continuous strand.

Slide 25

• DNA replication is a highly accurate process, with an error rate of approximately 1 in 10^9 base pairs.
• The accuracy is achieved through multiple mechanisms, including:
  1. Proofreading: DNA polymerase has an intrinsic ability to correct errors by removing mismatched nucleotides.
  2. Mismatch repair: Specialized proteins detect and repair errors that escape proofreading.
  3. Checkpoints: Cell cycle checkpoints monitor DNA replication and halt the process if errors are detected.
• These mechanisms ensure the fidelity of DNA replication and prevent the accumulation of mutations.

Slide 26

• Telomeres are repetitive DNA sequences located at the ends of linear chromosomes.
• They protect the genetic material from degradation and prevent the loss of essential genes during replication.
• Telomeres shorten with each round of DNA replication due to the inability of DNA polymerase to fully replicate the ends.
• This shortening is associated with cellular aging and senescence.

Slide 27

• Telomerase is an enzyme that adds telomeric repeats to the ends of chromosomes.
• It contains an RNA component (TERC) that serves as a template and a catalytic subunit (TERT) that synthesizes the DNA.
• Telomerase is active in germ cells, stem cells, and some cancer cells.
• Its activity allows these cells to maintain their telomeres and undergo continuous cell division.

Slide 28

• Misregulation of DNA replication and telomere maintenance can lead to various diseases.
• Examples of diseases associated with DNA replication errors include:
  • Xeroderma pigmentosum: Defects in DNA repair mechanisms result in sensitivity to UV radiation and an increased risk of skin cancer.
  • Fanconi Anemia: Impaired DNA repair leads to bone marrow failure and an increased risk of cancer.
• Telomere shortening or dysregulation of telomerase activity is linked to aging and age-related diseases, such as cardiovascular disorders and neurodegenerative conditions.

Slide 29

• Understanding the molecular basis of DNA replication is crucial for various scientific fields, including:
  • Molecular genetics: Investigating the mechanisms underlying genetic diseases and disorders.
  • Biotechnology: Developing techniques for DNA amplification, sequencing, and genetic engineering.
  • Medicine: Developing targeted therapies for diseases caused by DNA replication errors.
• The study of DNA replication continues to advance our understanding of genetics and has practical applications in various sectors.

Slide 30

• In conclusion, DNA replication is a complex and highly regulated process that ensures the accurate duplication of the genetic material.
• Enzymes and proteins work together to unwind the DNA, synthesize new strands, and repair errors.
• Telomeres and telomerase play critical roles in maintaining genomic stability and cellular lifespan.
• Understanding DNA replication has broad implications in genetics, biotechnology, and medicine, impacting both basic research and practical applications.