Week 8 - Oct 29

DNA Structure and Replication

Introduction to DNA Structure

DNA (deoxyribonucleic acid) is the hereditary material in all living organisms, composed of two antiparallel strands forming a double helix. Each strand consists of nucleotides, which include a phosphate group, a deoxyribose sugar, and a nitrogenous base (adenine, thymine, cytosine, or guanine).

• Double Helix: The two strands are held together by hydrogen bonds between complementary bases (A-T, C-G).

• Antiparallel Orientation: One strand runs 5' to 3', the other 3' to 5'.

• Phosphodiester Bonds: Nucleotides are joined by phosphodiester bonds between the 3' hydroxyl and 5' phosphate groups.

DNA Replication Overview

DNA replication is the process by which a cell copies its DNA before cell division. Watson and Crick's discovery of base pairing suggested a mechanism for copying genetic material.

• Semi-Conservative Replication: Each new DNA molecule consists of one parental strand and one newly synthesized strand.

• Template Mechanism: Each strand serves as a template for the synthesis of a complementary strand.

Key Features of DNA Synthesis

• Speed: DNA synthesis occurs rapidly (10–1000 base pairs per second).

• Human Genome: 2 meters of DNA per cell, 23 chromosome pairs, billions of base pairs; replication takes ~8 hours.

• Bacterial DNA: Replicates in less than an hour due to smaller genome and single origin of replication.

• Enzymatic Complexity: Over a dozen enzymes are involved in DNA replication.

Chemistry of DNA Synthesis

DNA synthesis involves the addition of nucleotides via a condensation reaction, forming a phosphodiester bond between the 3' end of the growing strand and the 5' end of the incoming nucleotide.

• Energy Source: Hydrolysis of the incoming deoxynucleoside triphosphate (dNTP) provides energy for bond formation.

• Equation: $(dNMP)_{n} + dNTP \rightarrow (dNMP)_{n+1} + PP_i$

DNA Polymerase: The Central Enzyme

DNA polymerase catalyzes the addition of nucleotides to the growing DNA strand.

• Types: Prokaryotes have multiple DNA polymerases (e.g., Polymerase III for replication), eukaryotes have even more (e.g., Polymerase ε, δ).

• Directionality: Can only add nucleotides to the 3' end of a primer; cannot initiate synthesis de novo.

• Primer Requirement: DNA synthesis requires a short RNA primer synthesized by primase.

Steps in DNA Replication

• Unwinding: Helicase unwinds and separates the DNA strands at the replication fork.

• Stabilization: Single-strand binding proteins (SSBPs) stabilize the separated strands.

• Relieving Tension: Topoisomerase relieves supercoiling ahead of the replication fork by breaking, twisting, and rejoining DNA.

• Primer Synthesis: Primase synthesizes a short RNA primer (~10 nucleotides).

• Elongation: DNA polymerase extends the primer, synthesizing new DNA in the 5' to 3' direction.

Origin of Replication and Replication Bubbles

Replication begins at specific sequences called origins of replication (OR).

• Bacteria: Typically have a single origin of replication.

• Eukaryotes: Have hundreds to thousands of origins, allowing rapid replication of large genomes.

• Replication Bubble: As the origin opens, a bubble forms with two replication forks, allowing bidirectional replication.

Leading and Lagging Strand Synthesis

DNA polymerase synthesizes new DNA continuously on the leading strand and discontinuously on the lagging strand.

• Leading Strand: Synthesized continuously toward the replication fork (requires one primer).

• Lagging Strand: Synthesized discontinuously away from the fork as short fragments called Okazaki fragments, each requiring a new primer.

• Discontinuous Synthesis: Okazaki fragments are later joined by DNA ligase.

Enzymes Involved in Lagging Strand Synthesis

• Primase: Synthesizes RNA primers for each Okazaki fragment.

• DNA Polymerase: Extends each primer with DNA nucleotides.

• DNA Ligase: Joins Okazaki fragments to form a continuous strand.

The Replisome Complex

The replisome is a multi-protein complex that coordinates DNA synthesis at the replication fork.

• Includes helicase, primase, DNA polymerase, sliding clamp, SSBPs, topoisomerase, and DNA ligase.

• Ensures efficient and accurate replication of both leading and lagging strands.

End-Replication Problem in Eukaryotes

Linear chromosomes face a unique challenge: the 5' end of the lagging strand cannot be fully replicated, leading to progressive shortening of chromosomes with each cell division.

• Cause: DNA polymerase requires a primer and cannot fill in the very end of the lagging strand.

• Result: Loss of DNA at chromosome ends after each replication cycle.

Telomeres and Telomerase

Telomeres are repetitive, non-coding DNA sequences at the ends of eukaryotic chromosomes (e.g., 5' TTAGGG 3' in humans, repeated 300–8000 times).

• Function: Protect genes from being lost during replication.

• Telomerase: An enzyme that extends telomeres by adding telomeric repeats using its own RNA template and reverse transcriptase activity.

How Telomerase Works

• Telomerase binds to the 3' end of the lagging strand and extends it using its RNA template.

• DNA polymerase then fills in the complementary strand, ensuring chromosome ends are maintained.

Telomerase Activity in Humans

• Active in: Germline cells, embryonic cells, and some stem cells.

• Inactive in: Most somatic cells; telomeres shorten over time, contributing to cell aging (senescence).

Cancer and Telomeres

• Reactivation of telomerase genes in cancer cells allows them to maintain telomere length, contributing to cellular immortality and uncontrolled division.

Comparison Table: DNA Replication in Prokaryotes vs. Eukaryotes

Feature	Prokaryotes	Eukaryotes
Genome Structure	Circular DNA	Linear DNA
Origin of Replication	Single	Multiple
Replication Speed	Fast (less than 1 hour)	Slower (several hours)
End-Replication Problem	No (circular DNA)	Yes (linear DNA)
Telomerase	Not required	Required for telomere maintenance

Summary

• DNA replication is a highly coordinated, semi-conservative process involving multiple enzymes and protein complexes.

• Leading and lagging strand synthesis ensure complete and accurate copying of genetic material.

• Telomeres and telomerase are essential for maintaining chromosome integrity in eukaryotes, with implications for aging and cancer.

Additional info: The notes above expand on the original slides by providing definitions, mechanisms, and context for each enzyme and process, as well as a comparison table for prokaryotic and eukaryotic replication.