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Flow Of Genetic Information > DNA Replication > Flashcards

DNA Replication Flashcards

Learn the process of DNA replication

1
Q

Where does DNA replication start in prokaryotes, and what initiates it?

A

DNA replication begins at the single origin of replication called OriC in prokaryotes like E. coli.

DnaA proteins bind to OriC, creating a replication bubble by unwinding the DNA, which allows for the recruitment of other replication enzymes.

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2
Q

What role does DNA helicase play in prokaryotic DNA replication?

A

DNA helicase (DnaB) unwinds the double-stranded DNA at the replication fork by breaking hydrogen bonds between nucleotides.

This creates two single-stranded templates needed for synthesis and requires DnaC to help load helicase onto the DNA.

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3
Q

How are the leading and lagging strands synthesized in prokaryotic DNA replication?

A

Leading Strand: Synthesized continuously by DNA polymerase III in the 5’ to 3’ direction toward the replication fork.

Lagging Strand: Synthesized discontinuously as Okazaki fragments by DNA polymerase III moving away from the replication fork, requiring new primers for each fragment.

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4
Q

What are Okazaki fragments, and how are they processed in prokaryotic cells?

A

Okazaki fragments are short DNA segments on the lagging strand.

DNA polymerase I removes RNA primers, replacing them with DNA nucleotides.

DNA ligase then seals these fragments by forming phosphodiester bonds, creating a continuous strand.

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5
Q

What is the main function of DNA polymerase III, and how does it ensure accuracy?

A

DNA polymerase III is the main enzyme for adding nucleotides to the new strand.

It has 3’→5’ exonuclease activity, which enables proofreading, excising of mismatched nucleotides during replication to maintain high fidelity.

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6
Q

How does termination occur in prokaryotic DNA replication?

A

In E. coli, replication terminates at ter sites opposite the origin.

Tus proteins bind these ter sites, halting replication forks and ensuring both replication forks meet and complete DNA synthesis.

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7
Q

How is DNA supercoiling resolved during prokaryotic replication?

A

As DNA unwinds, supercoiling tension accumulates ahead of the replication fork.

Topoisomerase II (DNA gyrase) cuts the DNA strands to relieve tension, allowing replication to proceed smoothly.

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8
Q

In what direction does DNA synthesis occur during prokaryotic replication, and what enzyme is responsible for this synthesis?

A

DNA synthesis occurs in the 5’ to 3’ direction.

This is carried out primarily by DNA polymerase III, which adds nucleotides to the growing strand.

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9
Q

What is the function of primase during DNA synthesis in prokaryotic cells?

A

Primase (DnaG) synthesizes short RNA primers that provide a 3’ hydroxyl group for DNA polymerase III to initiate synthesis.

Primers are essential for both leading and lagging strand synthesis.

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10
Q

What role do nucleotide triphosphates (dNTPs) play in DNA synthesis?

A

Nucleotide triphosphates (dATP, dTTP, dGTP, dCTP) are the building blocks of DNA.

During synthesis, pyrophosphate (PPi) is released when a dNTP is added, providing energy for the formation of phosphodiester bonds between nucleotides.

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11
Q

How does the synthesis of the leading strand differ from that of the lagging strand in prokaryotic DNA replication?

A

Leading Strand: Synthesized continuously towards the replication fork.

Lagging Strand: Synthesized in short segments (Okazaki fragments) away from the replication fork, requiring multiple RNA primers.

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12
Q

What are Okazaki fragments, and how are they formed during DNA synthesis?

A

Okazaki fragments are short DNA sequences synthesized on the lagging strand during DNA replication.

They form as DNA polymerase III synthesizes DNA away from the replication fork, requiring new RNA primers for each fragment.

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13
Q

What is the role of DNA ligase in prokaryotic DNA synthesis?

A

DNA ligase connects Okazaki fragments by forming phosphodiester bonds, ensuring the lagging strand is a continuous DNA molecule after synthesis.

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14
Q

How does DNA polymerase III ensure fidelity during DNA synthesis?

A

DNA polymerase III has a 3’→5’ exonuclease proofreading activity that allows it to remove incorrectly paired nucleotides during synthesis, enhancing the accuracy of DNA replication.

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15
Q

What is the typical topology of DNA in prokaryotes, and how does it affect replication?

A

Prokaryotic DNA is typically circular and exists in a supercoiled form.

Supercoiling helps compact the DNA and plays a crucial role in facilitating the unwinding necessary for replication at the replication fork.

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16
Q

How does DNA supercoiling affect the movement of the replication fork during prokaryotic DNA replication?

A

As the DNA unwinds at the replication fork, it induces positive supercoiling ahead of the fork, which can create tension.

This tension can impede replication fork progression if not resolved.

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17
Q

What are the roles of topoisomerases in prokaryotic DNA replication?

A

Topoisomerase I relaxes supercoils by making single-strand cuts and allowing rotation, thereby relieving tension.

Topoisomerase II (DNA gyrase) introduces negative supercoils by making double-strand cuts, allowing for unwinding and enabling smoother progression of the replication fork.

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18
Q

What is negative supercoiling, and why is it important in prokaryotic DNA replication?

A

Negative supercoiling refers to the under-winding of DNA, which helps facilitate DNA unwinding during replication.

This form of supercoiling increases the efficiency of replication by making the DNA more accessible to the replication machinery.

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19
Q

How does DNA topology influence the activity of enzymes involved in DNA replication?

A

Enzymes such as DNA helicase and DNA polymerase rely on a certain DNA topology to function effectively.

Proper topological state ensures that helicase can unwind the DNA efficiently, allowing DNA polymerase to synthesize new strands without excessive tension.

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20
Q

What can occur if supercoiling is not managed during prokaryotic DNA replication?

A

If supercoiling is not addressed, it can lead to replication fork stalling or breakage.

Persistent positive supercoiling can cause physical stress on the DNA, potentially leading to mutations or replication errors.

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21
Q

What is the typical topology of DNA in eukaryotic cells, and how does it differ from prokaryotic DNA?

A

Eukaryotic DNA is linear and associated with histone proteins, forming chromatin.

This structure allows for higher levels of organization compared to the circular supercoiling seen in prokaryotes.

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22
Q

How does chromatin structure affect DNA replication in eukaryotes?

A

The chromatin structure must be modified (e.g., euchromatin versus heterochromatin) to allow replication machinery access to DNA.

Nucleosome remodeling occurs, enabling the replication machinery to proceed efficiently.

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23
Q

What types of topoisomerases are involved in eukaryotic DNA replication, and what are their functions?

A

Topoisomerase I: Relieves positive supercoiling by cutting one strand of DNA.

Topoisomerase II: Relieves torsional strain ahead of the replication fork by cutting both strands of DNA, allowing the DNA to unwind.

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24
Q

What is the effect of supercoiling on the progression of the replication fork in eukaryotes?

A

Supercoiling ahead of the replication fork can create tension, which may slow down or stall replication if not adequately managed.

Eukaryotic cells utilize topoisomerases to maintain optimal supercoiling levels during replication.

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25
Q

How do origins of replication differ in eukaryotes compared to prokaryotes?

A

Eukaryotes have multiple origins of replication on each chromosome, allowing for simultaneous replication of different regions.

This contrasts with prokaryotes, which typically have a single origin of replication due to their circular DNA structure.

26
Q

What are telomeres, and how do they relate to DNA topology in eukaryotes?

A

Telomeres are repetitive nucleotide sequences at the ends of linear chromosomes that protect them from degradation.

They play a crucial role in maintaining chromosomal stability and are involved in the replication process to prevent loss of genetic information.

27
Q

What specific challenges does the linear structure of eukaryotic DNA present during replication?

A

Linear DNA must resolve issues related to the ends of chromosomes, leading to potential end replication problems.

Eukaryotic cells use telomerase to extend telomeres and counteract the shortening that occurs during replication.

28
Q

What is the function of Type I topoisomerases?

A

Type I topoisomerases introduce single-strand breaks in the DNA, allowing one strand to pass through another.

This action relieves torsional strain without requiring ATP.

Example: Topoisomerase I in eukaryotes relaxes supercoiled DNA.

29
Q

How do Type II topoisomerases differ from Type I?

A

Type II topoisomerases introduce double-strand breaks in DNA, allowing two strands to pass through each other.

They require ATP to function.

Example: Topoisomerase II (DNA gyrase) in bacteria introduces negative supercoils, facilitating DNA replication.

30
Q

What are the key steps in the mechanism of Topoisomerase I?

A
  1. Binding: Topoisomerase I binds to the DNA at a specific site.
  2. Cleavage: It introduces a single-strand break in the DNA by breaking the phosphodiester bond of one strand, forming a tyrosyl-DNA covalent intermediate.
  3. Passage: The unbroken strand rotates around the cleaved strand, relieving torsional strain and supercoiling.
  4. Resealing: The enzyme then reseals the break by restoring the phosphodiester bond, allowing the DNA to return to its original state.

This mechanism reduces the supercoiling of DNA, facilitating replication and transcription.

31
Q

What role does the active site play in Topoisomerase I’s mechanism?

A

The active site of Topoisomerase I contains a catalytic tyrosine residue, which is critical for the cleavage and resealing of the DNA strand.

Upon binding, the hydroxyl group of the tyrosine attacks the phosphodiester bond in the DNA, creating a transient break.

The enzyme’s structure allows for the DNA to pass through the break, a crucial step in relieving supercoiling without consuming ATP.

32
Q

What are the key steps in the mechanism of Topoisomerase II?

A
  1. Binding: Topoisomerase II binds to DNA, forming a complex that wraps around the enzyme.
  2. Cleavage: It introduces double-strand breaks in the DNA, resulting in the formation of two separate segments.
  3. Passage: A second segment of DNA passes through the break in the first segment, effectively changing the linking number and relieving torsional stress.
  4. Resealing: After the passage, Topoisomerase II reseals both strands, restoring the integrity of the DNA.

This process is vital for managing DNA tangles during replication and is particularly important during mitosis

33
Q

What is the role of the active site in Topoisomerase II’s mechanism?

A

The active site of Topoisomerase II contains multiple critical residues, including two tyrosine residues, which are essential for the cleavage of both strands of DNA.

It forms a cleavage complex, where the enzyme temporarily binds to the DNA and creates a double-strand break.

The ATP binding and hydrolysis are required for conformational changes that facilitate the passage of the second DNA strand through the break.

34
Q

How do drugs interact with Topoisomerase II?

A

Drugs like etoposide and doxorubicin inhibit Topoisomerase II, trapping the enzyme in a state where it cannot reseal the DNA after cleavage.

This leads to the accumulation of DNA breaks, triggering cellular apoptosis, particularly in rapidly dividing cancer cells.

35
Q

What is the general process of initiation in DNA replication?

A

DNA replication begins at a specific site called the origin of replication (oriC in prokaryotes).

Key initiation proteins bind to this region, causing the DNA to unwind and form a replication bubble.

This process prepares the DNA for the synthesis of new strands

36
Q

What role does DnaA play in the initiation of DNA replication in prokaryotes?

A

DnaA is an initiator protein that binds to the DnaA boxes within the origin of replication.

This binding causes the DNA to wrap around the DnaA complex, inducing localized unwinding of the DNA helix at the AT-rich regions.

This unwound region serves as the initial replication bubble.

37
Q

How are helicases loaded during the initiation of DNA replication in prokaryotes?

A

DnaC acts as a helicase loader, helping to load the DnaB helicase onto the single-stranded DNA at the replication bubble.

Once DnaB is loaded, DnaC is released, and DnaB begins unwinding the DNA, expanding the replication bubble.

38
Q

What role do Single-Strand Binding Proteins (SSBs) play in the initiation of DNA replication?

A

Single-Strand Binding Proteins (SSBs) bind to the single-stranded DNA generated by helicase.

SSBs prevent the single strands from reannealing and protect the DNA from nucleases.

They stabilize the unwound DNA and ensure a smooth template for replication.

39
Q

How is a primer synthesized in the initiation of DNA replication?

A

Primase synthesizes a short RNA primer on each DNA strand within the replication bubble.

The RNA primer provides a 3’-OH group for DNA polymerase to initiate synthesis.

In prokaryotes, primase is part of the Primosome complex, which travels along the DNA with helicase.

40
Q

What is the replisome complex, and how does it form during initiation?

A

The replisome is a large protein complex responsible for DNA synthesis, composed of DNA polymerase, primase, helicase, and additional factors.

After primer synthesis, DNA polymerase III assembles at the primer-template junction.

This complex initiates the elongation phase, synthesizing new DNA strands continuously (leading strand) and discontinuously (lagging strand).

41
Q

What is the role of DNA Polymerase III during elongation in prokaryotic DNA replication?

A

DNA Polymerase III is the main enzyme responsible for synthesizing new DNA strands during elongation.

It adds nucleotides in the 5’ to 3’ direction using the RNA primer’s 3’-OH as the starting point.

DNA Polymerase III has high processivity, allowing it to synthesize long stretches of DNA without dissociating.

42
Q

How is the leading strand synthesized during elongation?

A

The leading strand is synthesized continuously in the same direction as the replication fork.

DNA Polymerase III adds nucleotides continuously to the 3’-OH end of the primer, following the unwinding by helicase.

This results in a smooth, uninterrupted strand as the fork progresses.

43
Q

How is the lagging strand synthesized in elongation, and what are Okazaki fragments?

A

The lagging strand is synthesized discontinuously in short sections called Okazaki fragments.

As the replication fork opens, primase synthesizes a new RNA primer for each fragment.

DNA Polymerase III then extends from these primers, creating short DNA segments that will be joined later.

44
Q

What happens to the RNA primers on the lagging strand during elongation?

A

DNA Polymerase I removes the RNA primers by its 5’ to 3’ exonuclease activity.

It then fills in the resulting gaps with DNA, synthesizing in the 5’ to 3’ direction.

This step is essential to ensure a continuous DNA strand on the lagging side.

45
Q

What is the role of DNA ligase in elongation?

A

DNA Ligase seals the gaps between Okazaki fragments on the lagging strand after DNA Polymerase I replaces RNA primers with DNA.

It forms a phosphodiester bond between the 3’-OH of one fragment and the 5’-phosphate of the adjacent one, creating a continuous DNA strand.

46
Q

What role do the sliding clamp and clamp loader complex play in elongation?

A

The sliding clamp (β-clamp in prokaryotes) encircles the DNA and holds DNA Polymerase III tightly to the template strand, enhancing its processivity.

The clamp loader complex helps load the sliding clamp onto DNA at the primer-template junctions, especially essential for starting new Okazaki fragments on the lagging strand.

47
Q

How do helicase and topoisomerase facilitate elongation?

A

Helicase continues to unwind the DNA double helix ahead of the replication fork, allowing DNA polymerase access to single-stranded DNA.

Topoisomerase prevents supercoiling and tension buildup by making transient cuts in the DNA, ensuring smooth progression of the replication machinery.

48
Q

What happens in eukaryotic cells after the DNA is fully replicated?

A

DNA ligase seals any remaining nicks in the DNA backbone, ensuring a continuous strand.

Newly synthesized DNA is then packaged into chromatin, with histone chaperones assisting in nucleosome assembly.

This process helps restore chromatin structure, allowing the cell to enter the next cell cycle phase with fully organized chromosomes.

49
Q

How does termination of DNA replication occur in prokaryotes?

A

In prokaryotes like E. coli, replication terminates at specific sequences known as ter sites.

Tus proteins bind to ter sites and block the progression of replication forks, allowing them to meet and prevent over-replication.

The replication forks eventually converge, completing replication.

50
Q

What role does the Tus protein play in termination?

A

The Tus protein binds to ter sequences and acts as a physical block to the DNA helicase approaching from one direction.

This directional blocking ensures that replication forks terminate at the designated termination site.

Tus-ter complexes effectively control the progression and meeting point of replication forks in prokaryotes.

51
Q

What is decatenation, and why is it important in prokaryotic DNA termination?

A

Decatenation is the process of separating interlinked circular DNA molecules after replication.

Topoisomerase IV resolves these links by making transient cuts, allowing the two newly replicated circular DNA molecules to separate.

This step ensures that each daughter cell receives a complete and separate chromosome.

52
Q

How does DNA replication terminate in eukaryotic cells?

A

In eukaryotes, replication terminates when replication forks meet and merge within large chromosomal regions, rather than at specific sequences.

Topoisomerase II helps relieve supercoiling and resolve any remaining links between sister chromatids.

The process is generally controlled by the completion of the synthesis and meeting of replication bubbles along the chromosome.

53
Q

What is the role of telomerase in eukaryotic DNA replication termination?

A

Telomerase extends the ends of linear chromosomes by adding repetitive DNA sequences (telomeres) to prevent loss of genetic material.

This is crucial in eukaryotes as the lagging strand cannot fully replicate the chromosome end.

Telomerase helps ensure the full length of the chromosome is maintained across cell divisions.

54
Q

How are telomere gaps on the lagging strand resolved during eukaryotic termination?

A

After telomerase extends the leading strand, primase synthesizes an RNA primer near the end of the lagging strand.

DNA polymerase then extends from this primer, filling in the gap.

Any remaining overhangs are trimmed or protected by shelterin protein complexes, stabilizing chromosome ends.

55
Q

What are the major DNA repair mechanisms in cells?

A
  1. Direct Repair: Reverses chemical damage directly (e.g., photoreactivation repairs UV-induced thymine dimers).
  2. Base Excision Repair (BER): Corrects small, non-helix-distorting base lesions. DNA glycosylase removes the damaged base, followed by endonuclease cutting, polymerase filling, and ligase sealing.
  3. Nucleotide Excision Repair (NER): Repairs bulky, helix-distorting lesions. A complex excises a segment containing the damage, and DNA polymerase and ligase restore the strand.
  4. Mismatch Repair (MMR): Fixes errors from DNA replication, like base mismatches. Recognizes the mismatch, excises the error-containing section, and resynthesizes the segment.
  5. Homologous Recombination (HR): An error-free repair of double-strand breaks using a sister chromatid as a template for accurate repair.
  6. Non-Homologous End Joining (NHEJ): Repairs double-strand breaks by directly ligating broken ends without a template, making it more error-prone.
56
Q

What is the primary enzyme required for DNA replication?

A

Polymerase III

57
Q

What are the three components needed along with DNA polymerase III for DNA replication?

A
  • Primase
  • Template
  • dNTPs
58
Q

Describe the function of helicase in DNA replication.

A

Helicase during replication uses ATP to separate DNA locally

59
Q

How many subunits does helicase have, and how do they work together?

A

3 types 6 units all work together with each unit having a core structure that includes a P-loop NTPase domain aswell as two loops that extend to the centre of the ringed structure

60
Q

What is the role of primase in DNA replication?

A

Primase attaches an RNA primer to that is used to start the process of DNA synthesis

61
Q

Explain the function of DNA ligase in the replication process.

A

DNA ligase removes the RNA primer from the Okazaki

62
Q
A

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