Biochem Nov. 10th

Instructors reveal the topic of discussion: DNA replication.
Use of a comic featuring Calvin to illustrate the precision needed for DNA replication, similar to counterfeiting money.
Objective: thoroughly and accurately replicate DNA in living organisms.

Key players in DNA synthesis include:
- DNA Polymerase: An enzyme that extends a DNA strand by adding nucleotides, but it cannot initiate DNA synthesis de novo (from scratch). It requires an existing 3'-hydroxyl group to add to.
- Primase: A specialized RNA polymerase responsible for synthesizing a short RNA primer (typically 5-10 nucleotides long) that is complementary to the DNA template strand. This RNA primer provides the necessary 3'-hydroxyl group, allowing DNA polymerase to begin synthesis. The RNA primer is later removed and replaced with DNA.
- Sliding Clamp: A ring-shaped protein complex (e.g., in E. coli, it's a dimer known as the $\beta$ -clamp; in eukaryotes, it's a trimer called PCNA) that encircles the DNA and binds to DNA polymerase. It acts as a processivity factor, preventing DNA polymerase from dissociating from the template strand, thereby greatly increasing the efficiency and speed of DNA synthesis.

DNA polymerase synthesizes DNA by:
- Always extending a new strand in the 5' to 3' direction, adding nucleotides to the 3'-hydroxyl end of the growing DNA chain.
- Its structure resembles a right hand, with distinct domains:
- The palm domain contains the active site where the template strand binds and catalysis occurs. It monitors the accuracy of base pairing.
- The fingers domain is responsible for picking up incoming deoxynucleoside triphosphates (dNTPs) and guiding them into the active site, ensuring correct base pairing with the template.
- The thumb domain helps maintain contact between DNA polymerase and the DNA template, contributing to processivity.
Nucleotide Selection: DNA polymerase exhibits high specificity through a "shape-sensing" mechanism. It primarily incorporates correct nucleotides because the geometry of a correct A-T or G-C base pair fits precisely into the active site. Incorrectly paired nucleotides disrupt this geometry, leading to their exclusion or removal by proofreading.

Arthur Kornberg: Won Nobel Prize in 1959 for discovering DNA polymerase.
Initial unintentional isolation of repair polymerase instead of the genome-copying enzyme.
Thomas Kornberg: Arthur's son later isolated the replicative DNA polymerase.

DNA synthesis is an energy-intensive process driven by the hydrolysis of incoming deoxynucleoside triphosphates (dNTPs: dATP, dGTP, dCTP, dTTP).
The reaction involves a nucleophilic attack by the 3'-hydroxyl group of the growing DNA strand on the innermost (alpha, $\alpha$ ) phosphate of the incoming dNTP.
This attack releases a pyrophosphate group ( $PP<em>i$ ). The subsequent hydrolysis of pyrophosphate into two inorganic phosphates ( $2P</em>i$ ) by pyrophosphatase is highly exergonic, effectively "pulling" the DNA synthesis reaction forward by making it energetically favorable and irreversible.

DNA polymerase’s action likened to trial-and-error for nucleotide incorporation based on geometry of base pairing.
Importance of substrate specificity, the consequences of mismatches.
Efficacy of polymerase in incorporating the correct nucleotide on first attempt.

Discussion of an anti-HIV drug (AZT):
- Structurally mimics nucleosides but has an azide group instead of an -OH group at the three-prime position.
- Functions as a chain terminator in viral DNA replication, preventing ongoing synthesis.
- Please note that the antiviral action is specific to viral DNA polymerases, which are often less discriminating than human versions.

Characteristics of replication fork:
- Extending bubble due to the action of helicase.
- Leading strand synthesizes continuously, while lagging strand synthesizes in fragments (Okazaki fragments).

Each fragment requires a primer and involves repeated synthesis processes by DNA polymerase.
Named after Tuneko and Reiji Okazaki, first to discover these fragments.

DNA polymerase possesses an intrinsic proofreading mechanism that significantly enhances replication accuracy.
When an incorrect nucleotide is incorporated, the polymerase stalls, and its 3' to 5' exonuclease activity is activated. This exonuclease domain removes the mismatched nucleotide from the 3' end of the growing strand.
After excision, the polymerase re-initiates synthesis, incorporating the correct nucleotide. This proofreading function reduces the error rate from approximately 1 in $10^5$ bases to about 1 in $10^7$ bases, achieving high fidelity in DNA replication.

After the synthesis of Okazaki fragments on the lagging strand, there are still gaps and nicks that need to be processed:
- DNA Polymerase I (E. coli) / RNase H and DNA Polymerase $\delta$ (Eukaryotes): This enzyme removes the RNA primers. In E. coli, DNA Polymerase I uses its 5' to 3' exonuclease activity to degrade the RNA primer and simultaneously synthesizes DNA to fill the gap using its 5' to 3' polymerase activity.
- DNA Ligase: Once the RNA primer is removed and the gap is filled with DNA, a single-strand break (nick) remains between the newly synthesized DNA and the adjacent DNA fragment. DNA ligase seals this nick by forming a new phosphodiester bond between the 3'-hydroxyl end of one fragment and the 5'-phosphate end of the next. This reaction requires energy, typically derived from ATP hydrolysis in eukaryotes (and T4 DNA ligase) or NAD $^+$ hydrolysis in bacteria.

The end replication problem arises in linear chromosomes because DNA polymerase cannot synthesize DNA without a primer, and when the RNA primer at the very 5' end of the nascent lagging strand is removed, there's no available 3'-OH group upstream to fill that gap. This leads to a progressive shortening of the chromosome ends with each round of replication.
This shortening eventually exposes coding genes, which can trigger cellular senescence (a state of irreversible growth arrest) or apoptosis.
Telomeres are specialized structures at the ends of eukaryotic chromosomes, consisting of repetitive, non-coding nucleotide sequences (e.g., TTAGGG in humans). They act as protective caps, buffering against the loss of genetic information during replication by providing a disposable region that can be shortened