Announcements and Upcoming Deadlines

• Lab Report Due: Sunday; one report per group on DIY experiments.

• Exam 5: Scheduled for Monday of next week.

• Topics for Next Week: Introduction to mitosis and the cell cycle.

  • Perusal Assignments:

    • Due Tuesday: Before mitosis discussion on Wednesday.

    • Due Thursday: Before cell cycle discussion on Friday.

• Important Dates:

  • Week 14: Exam 6 on the 25th.

  • Week 16: Final exam week.

DNA Packaging and Chromatin Structure

Background on DNA Packing

• DNA must pack tightly to fit within the nucleus in non-dividing cells.

• Exists as chromatin, a complex of DNA and proteins.

• Histone Proteins: DNA wraps around histones to form nucleosomes, which further coil into chromatin fibers.

  • Can be further compacted to form mitotic chromosomes.

• Transcription: Does not occur during mitosis.

Chromatin Structure

• Heterochromatin: Condensed regions that are transcriptionally inactive.

• Euchromatin: Less condensed regions where transcription occurs, allowing access to transcription machinery.

Introduction to DNA Replication

Importance of DNA Replication

• Essential for cell division in both normal and cancer cells; occurs before mitosis.

• Focus on how DNA is replicated and understanding its role in cancer biology.

Learning Outcomes for DNA Replication

1. Mechanism of DNA replication.

2. Key proteins involved and their functions.

3. The overall process and directionality of DNA replication.

Overview of DNA Structure

Strands and Directionality

• DNA consists of two anti-parallel strands.

  • Top strand: 5’ to 3’ (left to right).

  • Bottom strand: 5’ to 3’ (right to left).

Key Bonds

• Hydrogen Bonds:

  • Cytosine (C) to Guanine (G): 3 hydrogen bonds.

  • Adenine (A) to Thymine (T): 2 hydrogen bonds.

Mechanism of DNA Replication

Separation of Strands

• Strands must separate to allow replication; hydrogen bonds between nucleotides are broken.

Role of DNA Polymerase

• DNA Polymerase: Main enzyme involved in synthesizing new DNA strands.

  • Uses base-pairing rules to match nucleotides to the template.

• DNA replication is semi-conservative: one parental strand and one new strand per DNA molecule.

Origin of Replication

• High A-T content makes it easier to separate strands due to lower energy requirement to break 2 hydrogen bonds (vs. 3 for G-C).

Proteins involved in DNA Replication

Key Proteins

• Initiator Proteins: Recognize the origin of replication and initiate DNA unwinding.

• Helicase: Unwinds the DNA strands at replication forks.

• Single-Stranded Binding Proteins (SSBPs): Bind to unwound DNA to prevent re-annealing of strands.

Leading vs. Lagging Strands

• Leading Strand: Synthesized continuously in the direction of the replication fork's opening (5’ to 3’).

• Lagging Strand: Synthesized in short segments (Okazaki fragments) in the opposite direction (5’ to 3’) from the replication fork.

DNA Polymerases in Action

DNA Polymerase Types

• DNA Polymerase III: Main enzyme for elongating new DNA strands; works with existing template strands and requires a free 3’ hydroxyl group.

• Primase: Starts DNA replication by laying down short RNA primers (5-10 nucleotides) on the template strand.

• DNA Polymerase I: Removes RNA primers and replaces them with DNA nucleotides.

• DNA Ligase: Joins the Okazaki fragments on the lagging strand by forming the final phosphodiester bond.

Summary of the DNA Replication Process

1. Origin of Replication Opens: DNA strands are unwound.

2. Primase Initiation: Lays down RNA primers to allow DNA polymerases to start.

3. DNA Polymerase Action: DNA Polymerase III synthesizes the leading strand continuously and the lagging strand in fragments.

4. Primer Replacement: DNA Polymerase I removes RNA primers and replaces them with DNA.

5. Sealing Strands: DNA Ligase fuses any remaining gaps between newly synthesized DNA fragments.

Announcements and Upcoming Deadlines

• Lab Report Due: Sunday; one report per group on DIY experiments which should analyze the methods used and results obtained, reflecting on the experimental design and errors encountered.

• Exam 5: Scheduled for Monday of next week; ensure to review all covered materials including DNA packaging, chromatin structure, and the DNA replication process.

• Topics for Next Week: Introduction to mitosis and the cell cycle, emphasizing the importance of each stage in cellular division and its implications in growth and development.

Perusal Assignments:

• Due Tuesday: This assignment should be completed before the mitosis discussion on Wednesday. It will include reading material and practice questions to reinforce learning.

• Due Thursday: Prepare this before the cell cycle discussion on Friday. Focus on key stages of the cycle and their regulatory mechanisms.

Important Dates:

• Week 14: Exam 6 scheduled for the 25th; this will cover advanced topics including cancer biology and its relation to cell cycle dysregulation.

• Week 16: Final exam week; be sure to revise all topics discussed throughout the course.

DNA Packaging and Chromatin Structure

Background on DNA Packing

• Tight DNA Packaging: DNA must pack tightly to fit efficiently within the nucleus of non-dividing cells. This complex organization is necessary to enable proper gene expression and regulation.

• Chromatin Structure: Exists as chromatin, a dynamic complex of DNA and histone proteins. Histones are critical for the formation of nucleosomes and further compaction into fibers, allowing for the condensation needed for cell division.

• Mitotic Chromosomes: During mitosis, DNA can be further compacted into distinct mitotic chromosomes, making it easier to segregate the genetic material. Notably, transcription does not occur during this phase.

Chromatin Structure

• Heterochromatin: These are condensed regions that are transcriptionally inactive, playing roles in structural support and regulation of gene expression.

• Euchromatin: This less condensed form allows for transcription to occur, facilitating accessibility for transcription factors and let RNA polymerase perform its role in gene expression.

Introduction to DNA Replication

Importance of DNA Replication

• Cell Division: DNA replication is essential for cell division, occurring before mitosis in both normal and cancerous cells; its fidelity is crucial for maintaining genetic integrity.

• Cancer Biology: Understanding DNA replication is key to unraveling its role in cancer biology—irregularities can lead to uncontrolled cell growth.

Learning Outcomes for DNA Replication

1. Mechanism of DNA Replication: Understanding the step-by-step process involved.

2. Key Proteins: Recognizing the main proteins and their specific functions in replication.

3. Process & Directionality: Grasping the overall process and the significance of directionality, ensuring accurate replication.

Overview of DNA Structure

Strands and Directionality

• DNA is composed of two anti-parallel strands, a critical feature for its replication and function.

• Top strand: Runs in a 5’ to 3’ direction (left to right).

• Bottom strand: Also runs in a 5’ to 3’ direction but opposite (right to left).

Key Bonds

• Hydrogen Bonds: Understanding the bond strengths is vital.

  • Between Cytosine (C) and Guanine (G): 3 hydrogen bonds enhance stability.

  • Between Adenine (A) and Thymine (T): 2 hydrogen bonds present less stability than G-C pairs.

Mechanism of DNA Replication

Separation of Strands

• The strands must separate to initiate replication; this involves breaking the hydrogen bonds connecting nucleotides, a process facilitated by helicase.

Role of DNA Polymerase

• Main Function: DNA Polymerase is the principal enzyme responsible for synthesizing new DNA strands.

• Base-Pairing Rules: It matches nucleotides with the template strand, ensuring accuracy.

• Semi-Conservative Nature: Each new DNA molecule comprises one old (parental) strand and one newly synthesized strand, maintaining genetic fidelity.

Origin of Replication

• High A-T Content: Regions with a high adenine-thymine (A-T) content are easier to open due to the lower energy needed to break just 2 hydrogen bonds compared to 3 in guanine-cytosine (G-C) pairs.

Proteins Involved in DNA Replication

Key Proteins

• Initiator Proteins: They recognize the origin of replication, initiating the unwinding process.

• Helicase: Responsible for unwinding the two strands of DNA at the replication forks, allowing access.

• SSBPs (Single-Stranded Binding Proteins): Bind to the unwound DNA to prevent the strands from re-annealing, maintaining openness for replication.

Leading vs. Lagging Strands

• Leading Strand: Synthesized continuously in the direction of the opening replication fork from 5’ to 3’.

• Lagging Strand: Synthesized in short segments known as Okazaki fragments, moving in the opposite direction (5’ to 3’), necessitating additional processing for proper linkage.

DNA Polymerases in Action

DNA Polymerase Types

• DNA Polymerase III: The chief enzyme elongating new strands; it operates on existing template strands requiring a free 3’ hydroxyl group to function.

• Primase: Initiates replication by providing short RNA primers (5-10 nucleotides) for DNA polymerases to build upon.

• DNA Polymerase I: Responsible for removing RNA primers from the newly synthesized strand and replacing them with DNA nucleotides, ensuring complete replication.

• DNA Ligase: Joins Okazaki fragments on the lagging strand, creating the final phosphodiester bond to ensure continuity.

Summary of the DNA Replication Process

1. Opening: Begins at the origin of replication where DNA strands are unwound.

2. Primer Initiation: Primase lays down RNA primers, necessary for DNA polymerases to start replication.

3. DNA Polymerase Action: Polymerase III synthesizes the leading strand continuously and the lagging strand in fragments via Okazaki segments.

4. Primer Replacement: The RNA primers are removed by DNA Polymerase I, which replaces them with DNA nucleotides.

5. Sealing Strands: Finally, DNA Ligase fuses any remaining gaps, ensuring the newly synthesized DNA fragments are connected, effectively completing the replication process.