DNA Replication - Chapter 7 Flashcards

DNA Replication Overview

• DNA strands are antiparallel; replication occurs in the 5' to 3' direction.
• DNA polymerases synthesize DNA strictly in the 5' to 3' direction by adding deoxynucleotide triphosphates (dNTP) to the 3' OH of a growing DNA strand.
• RNA polymerase can initiate synthesis de novo, while DNA polymerases require a primer.

Replication Fork and Synthesis

• Daughter strands are synthesized at the replication fork, a region of active DNA synthesis.
• Continuous synthesis occurs on the leading strand, while the lagging strand is synthesized in short, discontinuous pieces known as Okazaki fragments.

Lagging Strand Synthesis

• Lagging strand synthesis initiates with RNA primers, produced by primase, which are later replaced by DNA.
• DNA polymerase I removes RNA primers in bacteria and replaces them with DNA nucleotides.

Enzymatic Roles

• In eukaryotic cells, RNA primers are removed through the action of RNase H and 5' to 3' exonucleases, with gaps filled by DNA polymerase delta.
• Leading and lagging strands are synthesized by different DNA polymerases in eukaryotes: DNA polymerase epsilon for the leading strand and a complex of primase with polymerase α for the lagging strand.

Sliding Clamp and Stability

• Sliding-clamp proteins (like PCNA in eukaryotes) enhance the association of DNA polymerase with the template DNA, ensuring continuous DNA synthesis.
• Clamp-loading proteins (RFC in eukaryotes) load sliding clamps onto DNA using ATP hydrolysis.

Helicase and Single-Stranded Stabilization

• Helicases unwind the DNA ahead of the replication fork, while single-stranded DNA-binding proteins stabilize the unwound strands for template use.
• Topoisomerases relieve strain caused by DNA unwinding, allowing smooth replication without twisting.

Fidelity of DNA Replication

• Fidelity is enhanced through complementary base pairing, proofreading by DNA polymerase, and mismatch repair mechanisms.
• DNA polymerases possess 3' to 5' exonuclease activity for proofreading, improving replication accuracy by excising mismatched bases.

Origins of Replication

• DNA replication initiates at specific sites called origins of replication (ORI).
• E. coli has a single ORI, while eukaryotic cells have many ORIs, allowing simultaneous replication at multiple sites.

Initiation in E. coli vs. Eukaryotes

• In E. coli, initiation begins with an initiator protein binding to the ORI, leading to unwinding of the DNA and loading of replication machinery.
• Eukaryotes utilize the origin recognition complex (ORC) to identify ORIs and recruit proteins necessary for DNA replication initiation.

Experimental Insights

• Identification of ORIs in yeast involved demonstrating autonomous replication sequences (ARS) capable of supporting plasmid replication.
• The activity of telomerase is crucial for eukaryotic chromosome ends; its inhibition leads to loss of linear chromosome integrity, but bacteria are unaffected due to their circular genomes.

Role of Clamp Loading and Primase

• RFC acts as a clamp loader for DNA polymerase and helicase, essential for coupling synthesis processes.
• Primase, while not directly bound to RFC, works closely with helicase to facilitate lagging strand synthesis by creating RNA primers for DNA polymerase III to extend.

Conclusion

• DNA replication is a complex, tightly regulated process involving numerous enzymes and protein complexes to ensure accurate and efficient synthesis across different organisms.