Nucleotide Metabolism and DNA Replication Study Notes

Synthesis of Nucleotides: Nucleotides are synthesized through two main pathways: de novo synthesis and salvage pathways.
Functions of Nucleotides Beyond DNA and RNA:
- Carrying chemical energy (e.g., ATP).
- Serving as building blocks for coenzymes (e.g., NAD+, FAD).
- Acting as signaling molecules (e.g., cAMP, cGMP).
Enzymes in DNA Replication:
- Key enzymes include DNA polymerases, helicases, primases, and ligases.
Differences in Replication:
- Eukaryotic replication is complex due to multiple chromosomal structures and origins of replication, while bacterial replication is simpler with one circular chromosome and a single origin.

In salvage pathways, preformed bases are recovered and re-attached to ribose units.

Structure of PRPP:
- Formula: $C<em>5H</em>{10}N<em>5O</em>{14}P_5$ or depicted as a ribose-phosphate backbone with two phosphate groups.
PRPP is central to both de novo and salvage pathways of nucleotide synthesis.
PRPP Synthetase catalyzes the formation of PRPP from ribose 5-phosphate.

Ribonucleotides are predominantly formed through the de novo pathways.
Deoxyribonucleotides:
- Formed by the reduction of ribose to deoxyribose in fully formed nucleotides.
- Methylation adding the distinguishing methyl group occurs at the last step for thymine.

Reactions:
- Orotate reacts with PRPP to form orotidylate (pyrimidine nucleotide).
- Orotidylate (OMP) is decarboxylated to form Uridine Monophosphate (UMP).
Amination of UMP:
- UTP is generated, which further converts to CTP through amination.

Bases from degraded nucleic acids are salvaged:
- Thymine forms thymidine via thymidine phosphorylase, then converted to a nucleotide by thymidine kinase.
Viral Considerations:
- Viral thymidine kinase can be a therapeutic target as seen with Acyclovir, which is selectively activated in infected cells.

The purine ring is built directly on ribose phosphate from PRPP through nine additional steps.
Final product: Inosine Monophosphate (IMP).
Conversion:
- AMP and GMP are derived from IMP through various pathways.

Thymidylate Synthase Activity:
- Converts deoxyuridylate (dUMP) to thymidine monophosphate (TMP) using N5,N10-methylenetetrahydrofolate as a methyl donor.
Drug Implications:
- Fluorouracil and Methotrexate target enzymes involved in nucleotide synthesis, impacting cancer cell growth.

Adenosine Deaminase Deficiency (SCID):
- Deficiency leads to accumulation of dATP, impairing DNA synthesis.
- Symptoms include severe immunodeficiency due to loss of T cell function.
Hyperuricemia and Gout:
- Elevated urate levels can lead to gout through inflammation caused by urate crystals in joints.

Competitive inhibitor of xanthine oxidase, results in lowered urate levels and increases hypoxanthine and xanthine concentrations, reducing gout symptoms.

Base-Pairing Mechanism:
- DNA replication relies on complementary base pairing to ensure accurate genome duplication.
- Each DNA strand serves as a template for replication, resulting in a semiconservative mode of replication.

Semiconservative Model:
- Each strand serves as a template for a new strand (Watson and Crick).
Meselson-Stahl Experiment:
- Use of isotopes to distinguish between heavy and light DNA confirmed semiconservative replication.
- Techniques involved centrifugation and density gradient analysis.

Replication Origins:
- Specific sequences where initiator proteins bind to begin strand separation.
Characteristics of E. coli:
- Origin of replication (oriC) is 245 bp with specific DnaA binding sequences.

Two replication forks form at each origin, moving in opposite directions at approximately 1000 nucleotides per second in bacteria.
Helicases unwind DNA strands using ATP energy.
Topoisomerases relieve supercoiling tension created ahead of replication forks.

Polymerization:
- DNA polymerase requires a primer for synthesizing DNA in a 5′ to 3′ direction.
- The addition of nucleotides occurs by forming phosphodiester bonds, utilizing the energy from nucleotide triphosphates.

Exonuclease Activity:
- DNA polymerases have proofreading capabilities to correct errors, maintaining fidelity.
Common Features of All DNA Polymerases:
- Structural configuration akin to a right hand, facilitating base pairing and catalysis.

Continuous vs. Discontinuous Synthesis:
- Leading strand synthesized smoothly while the lagging strand has to be synthesized in short fragments (Okazaki fragments).
Joining of Fragments:
- DNA ligase seals nicks between Okazaki fragments, forming a continuous strand.

Multiple DNA polymerases (α, δ, ε) are involved in eukaryotic replication with specific roles for initiation and elongation.
Replication occurs from multiple origins to accommodate the larger genome size.

Telomeres:
- Repetitive sequences at the end of chromosomes, protect genetic information from degradation during replication.
Telomerase Activity:
- Adds repeats to telomeres, countering shortening during cell division, crucial for maintaining long-term cellular proliferation, especially in cancer cells.