Study Notes on Nucleotide Metabolism and DNA Replication

Nucleotides can be synthesized via two major pathways: de novo synthesis and salvage pathways.
- De novo pathways: Nucleotide bases are assembled from simpler compounds.
- Pyrimidine Synthesis: The framework is assembled first and then attached to ribose.
- Purine Synthesis: The framework is synthesized piece by piece directly onto a ribose-based structure.
Both pathways primarily produce ribonucleotides.
- Deoxyribonucleotides are synthesized by the reduction of ribonucleotides.

Beyond being building blocks of DNA and RNA, nucleotides have several critical functions:
- Carrier of chemical energy within cells (e.g., ATP).
- Serve as coenzymes (e.g., NAD+, Coenzyme A).
- Function as regulatory molecules (e.g., cAMP, cGMP).

Eukaryotic vs. Bacterial Replication:
- In eukaryotes, DNA replication occurs at multiple origins of replication spread across linear chromosomes.
- In bacteria, DNA replication begins at a single origin on a circular chromosome, specifically at the oriC locus.
Both processes involve the formation of replication forks, where new DNA strands are synthesized based on the template strands.

Pyrimidine Nucleotide Synthesis:
- Orotate reacts with PRPP to form orotidylate, which is then decarboxylated to form UMP.
- UMP can be converted to UTP, then aminated to form CTP using ammonia and ATP.
Purine Nucleotide Synthesis:
- Begins with the displacement of pyrophosphate from PRPP by ammonia, resulting in 5-phosphoribosyl-1-amine, then undergoes a sequence of steps to form IMP.
- From IMP, both AMP and GMP are formed.

Recycling Pyrimidine Bases:
- Degraded DNA and RNA can be salvaged to produce nucleotides. For instance, thymine is converted to thymidine by thymidine phosphorylase, which is then phosphorylated to form TMP by thymidine kinase.

Adenosine Deaminase Deficiency: Leads to SCID; excess dATP inhibits ribonucleotide reductase, thus affecting DNA synthesis and causing immune deficiencies.
Cancer Therapeutics:
- Agents like Fluorouracil target thymidylate synthase, while Methotrexate inhibits dihydrofolate reductase, interfering with nucleotide synthesis particularly in rapidly dividing cancer cells.
Gout: Associated with high levels of urate due to purine degradation, leading to painful inflammation when urate crystals form in joints.

DNA is synthesized using each strand as a template, resulting in one conserved (parent) and one newly synthesized strand.
The Meselson-Stahl experiment demonstrated that DNA replication is semiconservative using density gradient centrifugation of heavy (15N) and light (14N) labeled DNA.

Initiator proteins bind to replication origins, breaking hydrogen bonds to separate DNA strands, facilitated in bacteria by the DnaA protein.
Replication forks are formed at each origin, moving in opposite directions and requiring helicases and topoisomerases for unwinding DNA.

DNA Polymerases: Enzymes that synthesize DNA by adding nucleotides to the 3' end of growing strands.
- They cannot start synthesizing new strands without a primer but can extend existing strands in a 5' to 3' direction.

Leading Strand: Synthesized continuously, requiring only one RNA primer.
Lagging Strand: Synthesized in short segments (Okazaki fragments), needing multiple RNA primers due to its antiparallel nature.

DNA polymerase possesses intrinsic proofreading capabilities through 3' to 5' exonuclease activity, allowing the removal of mismatched nucleotides, achieving a replication fidelity of less than $10^{-8}$ errors per base pair.

Eukaryotic cells utilize multiple DNA polymerases (α, δ, ε) with polymerase switching occurring after the priming step, transitioning from primase activity to a more efficient polymerase for elongation.
Eukaryotic replication origins are approximately 30,000 in total, managing the entirety of the human genome, highlighting the complexity of eukaryotic replication compared to prokaryotic processes.

Telomeres: Protect the ends of linear chromosomes from degradation during replication, with the enzyme telomerase adding repetitive sequences to chromosome ends, counteracting length loss during cell division. In cancer cells, telomerase is often reactivated, allowing for replication without the loss of telomeres.