DNA Replication and Synthesis Overview

DNA Replication General Features

• DNA strands are complementary, each acting as a template for its partner
• Replication is semi-conservative
• Replication is often bidirectional
• Replication requires RNA primers

DNA Synthesis Chemistry

• DNA synthesis uses deoxynucleoside triphosphates (dNTPs)
• dNTPs have three phosphate groups
• The primer provides a 3'-OH group
• DNA synthesis extends the 3' end, releasing pyrophosphate
• Pyrophosphate is quickly hydrolyzed into phosphate molecules by pyrophosphatase
• DNA strand orientation (template strand is opposite the growing strand)
• The reaction is favored thermodynamically

DNA Polymerase Mechanism

• DNA polymerases use a single active site to catalyze DNA synthesis
• Correct base pairing is essential for catalysis
• Steric constraints prevent use of rNTPs
• DNA polymerase has a three-domain structure: palm, fingers, and thumb
• Metal ions in the active site are essential for catalysis.
• There are multiple DNA polymerases; Pol ε and Pol δ are prominent in eukaryotic replication

DNA Polymerase Function and Activity

• DNA polymerase monitors ability of incoming nucleotides to form correct base pairs (A-T or G-C)
• Incorrect base pairing is quickly corrected by exonuclease activity
• DNA polymerase activity is measured by nucleotide incorporation assay
• Two metal ions bound to the enzyme catalyze nucleotide addition.

DNA Replication Processes

• DNA polymerase I is responsible for removing RNA primers
• Klenow Fragment polymerase is responsible for proofreading and removal of errors during DNA replication
• DNA Replication is semi-discontinuous
• The leading strand replicates continuously in the direction of the replication fork
• The lagging strand replicates discontinuously in fragments called Okazaki Fragments
• DNA pol III and accessory proteins like the sliding clamp ensure high processivity

DNA Polymerase Processivity

• Sliding clamp proteins dramatically increase DNA polymerase processivity
• The sliding clamp is a ring-shaped structure that enhances DNA polymerase processivity by preventing it from dissociating from the template strand
• Clamp loaders are essential for loading sliding clamps onto DNA

DNA Replication Initiation

• The replicon model describes initiation of replication
• E. coli contains the origin of replication (oriC) where DNA replication initiates
• Initiation requires proteins to unwind the DNA double helix which includes DnaA proteins.
• Initiation is tightly regulated by the cell cycle, to ensure duplication only occurs once per cycle.
• Proteins that load helicases onto the origin of replication, including Cdc6/Cdt1 and Mcm2-7, have roles in coordinating this initiation step

Eukaryotic DNA Replication

• Eukaryotic DNA synthesis is similar but more complex than prokaryotic replication because of larger genome size and linear chromosomes
• Replicators are specific regions where initiation begins; Origin Recognition Complexes (ORCs)
• There are multiple origins of replication in eukaryotic chromosomes

Telomerase

• Telomeres are repetitive sequences at the ends of linear chromosomes and shorten during replication
• Telomerase is an enzyme that extends telomeres, preventing further shortening
• Telomerase contains an RNA component guiding DNA synthesis
• Shelterin proteins protect telomeres from the cell's DNA repair machinery by binding to the telomere region, thus preventing it from being recognized as a break

DNA Replication Errors and Proofreading

• DNA polymerase has proofreading capability to reduce replication errors
• DNA is scanned for errors after base pairing
• Base-pairing mismatches and incorrect nucleotides are removed before replication proceeds further
• This ensures high fidelity of replication, minimizing mutations in the genome

This quiz explores the general features, chemistry, and mechanisms involved in DNA replication and synthesis. It covers topics such as the semi-conservative nature of replication, the role of RNA primers, and the action of DNA polymerase. Test your knowledge about these essential biological processes!