DNA Replication - Chapter 7 Flashcards
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Questions and Answers

How would you test a sequence of DNA in a yeast cell to determine whether it contains an origin of replication?

You would insert the DNA sequence into a plasmid that lacks an origin of replication and determine whether the recombinant plasmid is able to transform mutant yeast that require a plasmid gene for their growth.

How would an inhibitor of telomerase affect replication of eukaryotic cells? Would it affect bacteria?

Inhibition of telomerase would block synthesis of the ends of linear chromosomes, which would be lost as eukaryotic cells replicate. Bacteria would be unaffected since they have a circular genome.

What is the RFC clamp loading protein bound to?

Polymerase enzymes and helicase.

Is RNA primase attached to the RFC clamp loading protein?

<p>False</p> Signup and view all the answers

How does DNA polymerase III initiate DNA synthesis of the lagging strand?

<p>It binds to the 3' end of an RNA primer, synthesizes a DNA copy in a 5' to 3' direction until it reaches another Okazaki fragment where it dissociates but remains attached to the clamp-loading protein.</p> Signup and view all the answers

What is the overall direction for both strands in DNA replication?

<p>5' to 3'</p> Signup and view all the answers

What are two main fundamental properties all DNA polymerases have?

<p>Add new dNTPs only to primer strand</p> Signup and view all the answers

What enzyme can start from scratch without needing a primer?

<p>RNA polymerase</p> Signup and view all the answers

Where are the daughter strands synthesized?

<p>Replication fork</p> Signup and view all the answers

How is the synthesis of the lagging strand initiated?

<p>Using short RNA fragments as primers</p> Signup and view all the answers

What enzyme removes RNA primers in bacteria?

<p>DNA polymerase I</p> Signup and view all the answers

How are RNA primers removed in eukaryotic cells?

<p>Combined action of RNase H and 5' to 3' exonucleases</p> Signup and view all the answers

What fills the resulting gaps after RNA primers are removed in eukaryotic cells?

<p>DNA polymerase delta</p> Signup and view all the answers

What synthesizes the leading strand in eukaryotic cells?

<p>DNA polymerase epsilon</p> Signup and view all the answers

What synthesizes the leading strand in bacterial cells?

<p>DNA polymerase III</p> Signup and view all the answers

What maintains the association of DNA polymerase with template DNA?

<p>Sliding-clamp proteins</p> Signup and view all the answers

How are sliding-clamp proteins loaded onto DNA?

<p>By clamp-loading proteins</p> Signup and view all the answers

Is helicase ahead of the replication fork?

<p>True</p> Signup and view all the answers

What follows helicases in the replication process?

<p>Single-stranded DNA-binding proteins</p> Signup and view all the answers

What is the function of topoisomerases in DNA replication?

<p>To act as swivels to allow DNA to rotate</p> Signup and view all the answers

Do eukaryotic cells require topoisomerases?

<p>True</p> Signup and view all the answers

What happens to the two strands of template DNA as they unwind?

<p>They become twisted around themselves</p> Signup and view all the answers

How does the lagging strand maintain the overall direction of replication?

<p>It folds at the replication fork</p> Signup and view all the answers

What happens to DNA polymerase III when it reaches the end of an Okazaki fragment?

<p>It dissociates from the DNA</p> Signup and view all the answers

How does DNA polymerase III reassociate to another Okazaki fragment?

<p>It is bound to the clamp loader</p> Signup and view all the answers

What happens to nucleosomes during DNA replication?

<p>Nucleosomes are disrupted</p> Signup and view all the answers

What increases the fidelity of DNA replication?

<p>3’ to 5’ exonuclease activity</p> Signup and view all the answers

How does DNA polymerase increase the fidelity of replication in terms of base selection?

<p>By actively discriminating against mismatched bases</p> Signup and view all the answers

What is proofreading in terms of DNA polymerase III?

<p>The ability to excise mismatched bases during synthesis</p> Signup and view all the answers

How is proofreading important in increasing fidelity of DNA replication?

<p>It selectively excises mismatched bases</p> Signup and view all the answers

Give an example of proofreading.

<p>G is incorporated instead of A due to mispairing with T.</p> Signup and view all the answers

Where does replication occur for both prokaryotic and eukaryotic DNA?

<p>At sites called origin of replication (ORI)</p> Signup and view all the answers

How was the first ORI discovered?

<p>By genetic analysis of E. coli</p> Signup and view all the answers

How is DNA replication initiated in E. coli bacterial cells?

<p>By the binding of an initiator protein to specific DNA sequences within the origin</p> Signup and view all the answers

How many ORIs are in E. coli?

<p>There is only one</p> Signup and view all the answers

How many replication forks are formed per ORI?

<p>Two replication forks</p> Signup and view all the answers

How does replication begin in eukaryotes?

<p>Replication proceeds in both opposite directions</p> Signup and view all the answers

How many ORIs do eukaryotic cells have?

<p>Many ORIs</p> Signup and view all the answers

How were the ORIs of eukaryotic chromosomes first studied?

<p>In yeasts by identifying autonomously replicating sequences (ARS)</p> Signup and view all the answers

What is ARS in yeast?

<p>Autonomously replicating sequences, or origins of replication for yeasts</p> Signup and view all the answers

How is ORI recognized in eukaryotes?

<p>Using the origin recognition complex (ORC)</p> Signup and view all the answers

How does the origin recognition complex (ORC) work?

<p>It recruits other proteins necessary for initiation of replication</p> Signup and view all the answers

Are ORC conserved across eukaryotes?

<p>True</p> Signup and view all the answers

What is the role of DNA polymerase I in E. coli?

<p>Degrading RNA primers</p> Signup and view all the answers

What would be the effect of an RNA polymerase inhibitor on DNA synthesis?

<p>It would block synthesis of the lagging strand</p> Signup and view all the answers

What is the function of 3′ to 5′ exonuclease activity in DNA polymerases?

<p>Excision of mismatched bases during proofreading</p> Signup and view all the answers

Study Notes

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.

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Test your knowledge on DNA replication with these flashcards from Chapter 7. Explore key concepts such as the direction of DNA strands in replication and fundamental properties of DNA polymerases. Perfect for students looking to enhance their comprehension of genetics.

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