DNA Replication Mechanisms Quiz

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Questions and Answers

What role does DNA polymerase III play in relation to RNA primers on the lagging strand?

  • Synthesizes RNA primers necessary for initiation.
  • Extends RNA primers by incorporating deoxynucleotides. (correct)
  • Unwinds the double helix to facilitate replication.
  • Removes RNA primers and replaces them with DNA.

Which statement accurately describes the function of DNA polymerase I during DNA replication?

  • It synthesizes RNA primers to begin replication.
  • It removes RNA primers from Okazaki fragments and fills the gap with dNTPs. (correct)
  • It replaces DNA polymerase III in the elongation phase.
  • It facilitates the loop formation of the lagging strand.

What characteristic of the lagging strand during replication allows simultaneous activity of two DNA polymerases?

  • It is synthesized continuously in a single direction.
  • It forms a looped structure facilitating coordination. (correct)
  • It requires fewer RNA primers compared to the leading strand.
  • It has multiple origins of replication.

How does DNA polymerase III behave when it encounters an Okazaki fragment?

<p>It detaches from the lagging strand template. (A)</p> Signup and view all the answers

What aspect of primase's function is specific to the lagging strand during DNA replication?

<p>It creates multiple RNA primers in a discontinuous fashion. (B)</p> Signup and view all the answers

What is the key process in DNA replication that ensures each daughter duplex contains one strand from the parent structure?

<p>Semi-conservative replication (D)</p> Signup and view all the answers

During which phase of the cell cycle does DNA replication take place in eukaryotic cells?

<p>S Phase (C)</p> Signup and view all the answers

What structure is formed at the site where the parental double helix is undergoing strand separation?

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

How many origins of replication can be found in human cells?

<p>10,000-100,000 (B)</p> Signup and view all the answers

What term describes the small portions of the eukaryotic genome that are replicated simultaneously during DNA replication?

<p>Replicons (B)</p> Signup and view all the answers

What type of DNA polymerase is essential for synthesizing daughter strands in prokaryotes?

<p>DNA Polymerase III (A)</p> Signup and view all the answers

Approximately how many active replication forks can be found within a single replication focus in eukaryotic cells?

<p>10-100 (A)</p> Signup and view all the answers

Which of the following factors is NOT involved in DNA replication in prokaryotes?

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

What is the function of a replication foci in the eukaryotic nucleus during DNA replication?

<p>To concentrate multiple replicons for simultaneous replication (A)</p> Signup and view all the answers

What mechanism allows the gradual separation of the two strands of the double helix during DNA replication?

<p>Hydrogen bond breaking (B)</p> Signup and view all the answers

What is the role of DNA ligase during DNA replication?

<p>It covalently joins 3’ dNT to 5’ end of Okazaki fragments. (D)</p> Signup and view all the answers

Which statement about eukaryotic DNA replication is accurate?

<p>Human cells have 10,000-100,000 different origins of replication. (A)</p> Signup and view all the answers

What happens to nucleosomes during DNA replication in eukaryotic cells?

<p>Replication machinery displaces them but they quickly reassemble. (B)</p> Signup and view all the answers

What is the primary function of the 3'->5' exonuclease activity in DNA Polymerases?

<p>To proofread and remove mispaired nucleotides. (B)</p> Signup and view all the answers

Which of the following accurately describes the spontaneous mutation rate during DNA replication?

<p>It is influenced by the geometric fit of nucleotides. (B)</p> Signup and view all the answers

What is the significance of histone molecules during DNA replication?

<p>They use parental histones for quick reassembly during replication. (C)</p> Signup and view all the answers

What mutation can lead to various genetic diseases related to DNA replication?

<p>Deficiencies in the DNA polymerase proofreading ability. (C)</p> Signup and view all the answers

How do eukaryotic cells ensure accuracy during DNA repair?

<p>Through the mismatch repair system employing various proteins. (C)</p> Signup and view all the answers

What is the primary function of DNA Polymerase I during DNA replication?

<p>To replace RNA primers with DNA (D)</p> Signup and view all the answers

In prokaryotic cells, what is the role of DnaA during DNA replication initiation?

<p>It recognizes the origin of replication (A)</p> Signup and view all the answers

What characterizes the lagging strand during DNA replication?

<p>It requires multiple RNA primers for synthesis (B)</p> Signup and view all the answers

Which of the following is true about eukaryotic DNA replication initiation?

<p>Helicase binds to the origin after the pre-replication complex forms (A)</p> Signup and view all the answers

What are Okazaki fragments?

<p>Discontinuous DNA segments synthesized away from the replication fork (A)</p> Signup and view all the answers

Which enzyme is primarily responsible for unwinding DNA during replication in prokaryotes?

<p>DnaB helicase (B)</p> Signup and view all the answers

What is the role of single-stranded DNA-binding (SSB) proteins in DNA replication?

<p>To stabilize unwound DNA strands during replication (C)</p> Signup and view all the answers

Which type of topoisomerase relaxes DNA supercoiling in eukaryotic cells?

<p>Type I topoisomerases (C)</p> Signup and view all the answers

What is the direction of new DNA synthesis by DNA Polymerase during replication?

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

In eukaryotes, when is the formation of new pre-replication complexes synchronized?

<p>During the M and G1 phases of the cell cycle (B)</p> Signup and view all the answers

What role does DNA Gyrase play in prokaryotic DNA replication?

<p>It introduces or removes supercoils. (B)</p> Signup and view all the answers

Which statement accurately describes the function of the γ-clamp loading complex?

<p>It loads the sliding clamp onto DNA. (A)</p> Signup and view all the answers

How many core DNA polymerases are present in the DNA Polymerase III holoenzyme?

<p>Two (D)</p> Signup and view all the answers

During DNA replication in prokaryotes, what is the role of helicase?

<p>To unwind the DNA double helix. (D)</p> Signup and view all the answers

In the context of DNA replication, what does 'replisome' refer to?

<p>The entire complex of active proteins at the replication fork. (B)</p> Signup and view all the answers

What is the primary function of primase in prokaryotic DNA replication?

<p>To synthesize RNA primers. (A)</p> Signup and view all the answers

What occurs to the DNA strands during the elongation phase of prokaryotic DNA replication?

<p>Both strands are looped simultaneously. (B)</p> Signup and view all the answers

Which enzyme is primarily responsible for incorporating deoxynucleotides during DNA replication on the lagging strand?

<p>DNA Polymerase III (A)</p> Signup and view all the answers

Flashcards

DNA Replication

The process of copying DNA, essential for cell division.

Semi-Conservative Replication

The theory that each new DNA molecule contains one original strand and one newly synthesized strand.

Diploid Cell

A type of cell that contains two sets of chromosomes (one from each parent).

Haploid Cell

A type of cell that contains only one set of chromosomes, found in sperm and egg cells.

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S-Phase

A stage in the cell cycle where DNA is replicated.

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Replicon

A small portion of the eukaryotic genome that is replicated independently.

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Origin

The starting point for DNA replication within a replicon.

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Replication Fork

The site where the DNA double helix is being separated and new strands are being synthesized.

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Replication Foci

Specialized regions within the nucleus where replication occurs.

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DNA Polymerase

The enzyme responsible for synthesizing new DNA strands during replication.

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Type I Topoisomerase

A topoisomerase that allows rotation around the intact strand of DNA to relieve strain during transcription or replication. It does not require ATP.

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Type II Topoisomerase

A topoisomerase that changes DNA topology by breaking and rejoining double-stranded DNA. They introduce or remove supercoils in DNA, which is critical for untangling and separating intertwined DNA strands during replication and cell division. This process requires ATP.

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DNA Gyrase

A prokaryotic Type II topoisomerase that introduces negative supercoils into DNA, making it easier to unwind during replication.

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DNA Polymerase III Holoenzyme

The primary enzyme responsible for replicating DNA in prokaryotes. It is a complex enzyme containing two core DNA polymerase III units, multiple β clamps, and a γ-clamp loading complex. The two core polymerase units replicate DNA by extending the RNA primers on both the leading and lagging strands.

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β-Clamps

The ring-shaped proteins that allow DNA polymerase to remain attached to the DNA during replication. They ensure that the polymerase doesn't detach from the DNA strand, allowing for continuous synthesis.

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β-Clamps on Leading and Lagging Strands

A clamp on the leading strand allows for continuous DNA synthesis. On the lagging strand, there is one clamp for each Okazaki fragment

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γ-Clamp Loading Complex

The component of the DNA polymerase III holoenzyme responsible for loading the β-clamp onto the DNA. It is also bound to the helicase.

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Replisome

The entire complex of active proteins that operate at the replication fork. Includes DNA polymerase III holoenzyme, helicase, single-stranded binding proteins (SSBs), and primase. It's essentially the 'replication machine' that produces two new DNA molecules from one original molecule.

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5→ 3 direction

A new strand of DNA is made in the 5' to 3' direction. This means that the new nucleotides are added to the 3' end of the growing strand. At the same time, the old strand is being separated (unwound) so that the new strand can be built.

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DNA Polymerase III

DNA polymerase III is the primary polymerase for replication in prokaryotes. It is responsible for the majority of DNA synthesis.

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DNA helicase

An enzyme called DNA helicase unwinds the DNA helix, breaking the bonds between the two strands. The unwinding process uses energy from ATP.

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Single-stranded DNA-binding (SSB) proteins

These proteins bind to the single-stranded DNA, preventing it from re-annealing (coming back together) so that the new strand can be built.

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RNA primer

The RNA primer is a short stretch of RNA nucleotides that is required to initiate DNA synthesis. DNA polymerase III can only add nucleotides to an existing 3' hydroxyl group.

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Lagging strand

The lagging strand is synthesized in short fragments, called Okazaki fragments, because it is built in the opposite direction of the replication fork. Each fragment requires a separate RNA primer.

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Leading strand

The leading strand can be synthesized continuously in the 5' to 3' direction, following the movement of the replication fork.

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Origin of replication

The origin of replication is a specific sequence within the genome where DNA replication begins.

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What is DNA Polymerase III's role in DNA replication?

DNA polymerase III extends the RNA primers laid down by primase on the lagging strand during DNA replication. It continuously adds deoxynucleotides to the primer, resulting in a growing DNA strand.

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Why is the lagging strand looped in DNA replication?

The lagging strand is looped around DNA polymerase III to ensure both DNA polymerase III and DNA polymerase I, which removes the RNA primers, work in tandem during DNA replication.

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What happens when DNA Polymerase III reaches a previously synthesized Okazaki fragment?

DNA Polymerase III releases the lagging strand once it encounters a 5' end of a previously synthesized Okazaki fragment.

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What happens after DNA Polymerase III releases the lagging strand?

DNA Polymerase III continues its journey down the lagging strand template, binding to the template further along its length and elongating DNA from the next RNA primer.

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What roles does DNA Polymerase I play in DNA replication?

DNA Polymerase I has both exonuclease and polymerase activities. It removes the RNA primers of the Okazaki fragments using its 5' to 3' exonuclease activity and then fills the gaps generated by the RNA primers with dNTs using its polymerase activity.

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DNA ligase

The enzyme responsible for joining Okazaki fragments during DNA replication.

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Study Notes

HSS2305: Molecular Mechanisms of Disease

  • Lecture 13 covered DNA replication, DNA damage, and DNA repair.
  • A diagram of a cell's components was shown, labeling various organelles and structures.
  • DNA Replication: Reproduction relies on copying genetic material. DNA is copied during mitosis and meiosis. This copying is done by replication machinery, also used for repair. Human diploid cells have 46 chromosomes; gametes (haploid) have 23.

DNA Replication

  • Semi-Conservative Replication: Watson and Crick described gradual strand separation of the double helix via breaking hydrogen bonds. Daughter strands are synthesized as complementary pairs to the parental templates. Each daughter duplex contains one strand from the parent.
  • Bacterial DNA Replication: Temperature sensitive bacterial mutants are heavily studied; these allow researchers to quickly turn gene expression on/off. In vitro culture systems, and more than 30 proteins are involved in the prokaryotic replication process. Bacterial and eukaryotic cell replication mechanisms are similar.
  • Eukaryotic DNA Replication: "Synthesis" phase (S-phase) of the cell cycle where DNA replicates. Many small portions of the eukaryotic genome (replicons) are replicated all at once. Human cells have 10,000 - 100,000 different replication origins. 10-15% of replicons are actively involved in S-phase.
  • Eukaryotic Replication: DNA replication proceeds in both directions (bi-directionally). Replication forks are areas where the double helix separates and new nucleotides are incorporated. 2 replication forks move in opposite directions

DNA Replication: Nuclear Structure

  • Replication foci are localized sites within the nucleus. Several replicons become active within these foci. A typical replicating nucleus has ~50-250 foci. ~10-100 active replication forks (replicons)/replication foci occur in a cell.

DNA Replication: Prokaryotes: DNA Polymerase

  • DNA polymerase holoenzyme is responsible for DNA synthesis. DNA Polymerase III dimer synthesizes daughter strands simultaneously with other proteins. DNA replication in prokaryotes requires a template DNA strand, primers, and appropriate nucleotides.

DNA Replication: Semi-discontinuous

  • DNA replication is semi-discontinuous. This means the new DNA strands are not created continuously in a single direction; the leading strand is continuous, while the lagging strand is made in small fragments (Okazaki fragments) that require RNA primers to initiate.

DNA Replication: Prokaryotes: Initiation

  • DnaA proteins recognize the origin of replication (OriC). DnaB helicase unwinds the DNA strands, and single-stranded DNA binding (SSB) proteins coat the unwound DNA. Primase synthesizes RNA primers, a necessary step before DNA polymerase can act.

DNA Replication: Eukaryotes: Initiation

  • The origin recognition complex (ORC) recognizes replicon origins. Licensing factors (Cdc6 and Cdt1) and helicase are recruited to the origin.
  • Helicase is an unwinding protein comprised of minichromosome maintenance proteins (MCM2-7)

DNA Replication: Eukaryotes: Pre-replication complex (pre-RC)

  • The pre-RC involves ORC, licensing factors, and helicase binding during the G1 phase. Protein kinases (Cdk and DDK) phosphorylate and activate the pre-RC. High activity in S-phase. The pre-RC is also responsible for inhibiting the formation of new complexes. Primase synthesizes RNA primers.

DNA Replication: Supercoiling

  • DNA supercoiling arises during DNA unwinding; tension builds up. Topoisomerases relieve this tension. Type I topoisomerases relax DNA by nicking and closing one strand of the duplex. Type II topoisomerases change topology by breaking and rejoining double-stranded DNA to introduce or remove supercoils.

DNA Replication: Prokaryotes: Elongation

  • Two core polymerases (DNA III) replicate DNA, assisted by at least two beta clamps. One clamp is associated with the leading strand, while one is associated with each lagging strand fragment. y-Clamp loading complex loads the sliding clamp onto DNA, also associated with helicase. The replisome refers to the replication fork complex.

DNA Replication: Eukaryotes: Elongation

  • Eukaryotic replication fork proteins are homologous to related proteins in bacterial replication, but have slightly different nomenclature.

DNA Repair: High Fidelity Mutation Rate

  • An error rate of incorporating incorrect nucleotides during DNA replication is low. DNA polymerases have extremely high fidelity.Only one correct orientation is geometrically proper to fit within the active site, insuring accurate incorporation of nucleotides. Proofreading capacity maintains accuracy.

DNA Repair: Prokaryotes: Mismatch repair

  • DNA polymerases I and III have a 3'-5' exonuclease activity that removes wrongly incorporated nucleotides during DNA replication. Prokaryotic polymerases use a proofreading mechanism.

DNA Repair: DNA Damage

  • DNA is susceptible to damage from ionizing radiation, chemicals, UV light, thermal energy from metabolism and from spontaneous lesions. Cells have mechanisms to repair damage. DNA repair mechanisms include:
  • accurate selection of nucleotides during replication
  • immediate proofreading
  • post-replicative mismatch repair

DNA Repair: Nucleotide Excision Repair-Eukaryotes

  • Removes bulky lesions(pyrimidine dimers). Includes cut-and-patch repair.
    • Global genomic pathway (corrects lesions throughout genome)
    • Transcription-coupled pathway (repairs lesions on the DNA strand currently being transcribed by RNA polymerase II). Genes expressed most frequently are most likely to be repaired.
  • There are XPC and TFIIH complexes involved in repairing these lesions.

DNA Repair: Base Excision Repair and Eukaryotes

  • Removes altered nucleotides(reactive chemicals/free radicals). Different types of alterations: Uracil, 8-oxoguanine, 3-methyladenine. Specific glycosylases identify and excise the base.
  • The AP endonuclease then cuts the sugar-phosphate backbone. DNA polymerase B adds the correct nucleotide and DNA ligase III seals the gap.
  • Alzheimer's disease is associated with increased oxidative DNA damage and deficiencies in base excision repair enzymes, leading to less effective repair which may contribute to disease development.

DNA Repair: Double-strand Breakage Repair- Eukaryotes

  • Double-strand breaks occur from ionizing radiation and certain chemicals (such as those in chemotherapy). Nonhomologous end joining (NHEJ) repairs breaks quickly but may introduce errors. Homologous recombination (HR) is a more accurate method that requires an identical template strand, but is slower.

Summary: DNA Replication, Damage and Repair

  • DNA replication is crucial for cell reproduction. DNA damage occurs frequently, and repair mechanisms play a key role in maintaining cell health and preventing disease.
  • Various types of errors or damage can be repaired.

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