DNA Replication Mechanisms: L5-6

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Questions and Answers

What is the primary function of DnaC in the initiation of DNA replication?

To cleave RNA primers

To bind to DnaA and stabilize the origin of replication

To load DnaB helicase onto single-stranded DNA (correct)

To synthesize RNA primers

What characterizes the RNA primers synthesized during DNA replication initiation?

They are typically 20 nucleotides long and contain no uracil.

They have a 5′ to 3′ directionality, synthesized on a 5′ to 3′ DNA strand.

They are complementary to the leading strand and synthesized continuously.

They are approximately 10 nucleotides long and lack editing functions. (correct)

Which process occurs after DnaA products bind to the origin of replication?

The RNA primer is synthesized

DnaB helicase is detached from the single-stranded DNA

The duplex is unwound at the 13bp repeats (correct)

Single-stranded binding proteins bind to re-annealed DNA

What is the role of single-stranded binding protein (SSB) during DNA replication?

To stabilize single-stranded DNA and prevent re-annealing Signup and view all the answers

Which statement about the primase involved in replication initiation is correct?

Primase activity is enhanced by its own presence and helicase. Signup and view all the answers

What are pseudo-Okazaki fragments associated with during DNA replication?

Replication of the leading strand Signup and view all the answers

What happens if enzyme 4 is mutated during dTTP synthesis?

More U incorporated and shorter fragments formed Signup and view all the answers

Why is the presence of U in DNA considered problematic?

It can lead to mutations due to its pairing properties Signup and view all the answers

What is the primary function of Uracil-N-glycosylase (ung) in DNA repair?

To remove U from DNA Signup and view all the answers

What role does DNA ligase play in the context of DNA replication?

It seals nicks in the DNA Signup and view all the answers

During DNA replication, at which stage does elongation occur?

Between initiation and termination Signup and view all the answers

What is the likely consequence of ung mutants in DNA replication?

No removal of U, leading to incorrect nucleotides Signup and view all the answers

Which nucleotide can be incorporated instead of dTTP due to errors in synthesis?

dUTP Signup and view all the answers

What is the function of the Ter sequences in the DNA replication process?

To prevent the DNA forks from overshooting Signup and view all the answers

How does Topoisomerase IV contribute to DNA replication termination?

By relieving positive supercoiling through decatenation Signup and view all the answers

Why is it important that initiation of DNA replication only occurs once per cell cycle?

To prevent multiple origins from forming Signup and view all the answers

What role does the SeqA protein play in DNA replication?

It binds to hemimethylated GATC sequences, preventing DnaA binding Signup and view all the answers

What happens to hemimethylated plasmids in dam- strains during replication?

They are unable to replicate Signup and view all the answers

Which of the following is a significant difference in DNA replication between eukaryotes and prokaryotes?

Eukaryotes replicate their DNA at a rate significantly slower than prokaryotes. Signup and view all the answers

What is one of the major structural differences in DNA of eukaryotes compared to prokaryotes?

Eukaryotic DNA is linear, while prokaryotic DNA is typically circular. Signup and view all the answers

In DNA replication of eukaryotes, what is the nature of Okazaki fragments in comparison to those in prokaryotes?

Eukaryotic Okazaki fragments are generally shorter than prokaryotic ones. Signup and view all the answers

How do the origins of replication differ between eukaryotic and prokaryotic cells?

Eukaryotic cells can have tens to thousands of origins per chromosome. Signup and view all the answers

Which of the following statements about eukaryotic DNA polymerases is true?

They lack 5’-3’ exonuclease activity. Signup and view all the answers

What role does single stranded binding protein (SSB) play in DNA replication?

It prevents the DNA strands from re-annealing. Signup and view all the answers

Which protein complex is primarily responsible for the loading of the β clamp onto DNA?

Clamp loader Signup and view all the answers

How does the β clamp enhance the activity of Pol III during DNA replication?

By imparting processivity to the enzyme. Signup and view all the answers

What triggers the arrival of the Pol III holoenzyme at the 3’ end of the primer?

The first primer and SSB. Signup and view all the answers

What is the result of ATP hydrolysis during the clamp loader's function in DNA replication?

It detaches the clamp loader from the β clamp. Signup and view all the answers

What is the significance of the Pol III holoenzyme residing in the replisome?

It synthesizes the leading strand as it progresses. Signup and view all the answers

Which process is NOT part of the DNA replication steps?

Selection Signup and view all the answers

What is the primary purpose of the primase in DNA replication?

To synthesize RNA primers. Signup and view all the answers

What role does the β clamp play during lagging strand synthesis?

It allows Pol III to bind to the DNA strand. Signup and view all the answers

What happens after the first Okazaki fragment is completed?

Primase re-binds helicase to add a second primer. Signup and view all the answers

During the elongation phase of replication, what occurs to the lagging strand?

It loops out to facilitate replication. Signup and view all the answers

What is the function of DNA ligase during DNA replication?

To seal the nicks between Okazaki fragments. Signup and view all the answers

Which of the following correctly describes the process of primer replacement in DNA replication?

Pol I replaces RNA primers with DNA. Signup and view all the answers

What triggers the process of DNA replication to terminate?

Detachment of replication machinery from the DNA. Signup and view all the answers

Which enzyme is responsible for pulling the DNA during replication?

DNA helicase Signup and view all the answers

In the context of Okazaki fragments, what is a key feature of their formation?

They require multiple primers at different locations. Signup and view all the answers

Study Notes

DNA Replication Overview

Lectures 5-6 of BIOL2010 cover DNA replication.
The lectures cover the composition/structure of DNA, DNA synthesis vs. DNA replication, problems and solutions in E. coli during the process, steps of replication in E. coli, and differences in eukaryotes/viruses.
Mutations/errors during synthesis can lead to incorrect nucleotides, RNA nucleotides instead of DNA nucleotides, and nicks in the backbone.

DNA Replication Problems

Incorrect nucleotide addition is a problem during DNA synthesis.
Shape discrimination, binding affinity, and induced fit are mechanisms to prevent incorrect nucleotide addition.
Shape discrimination and steric collisions prevent incorrect dNTPs from fitting.
Binding affinity checks the fit in the minor groove.
Induced fit causes a conformational change in the polymerase active site to ensure correct fit of the nucleotide.
The accuracy of Pol III is ~1 per 100,000 nucleotides in E. coli,
Proofreading activity reduces this error rate to ~ 1 per 10,000,000.

DNA Replication - Proofreading

Pol III has 3'-5' exonuclease activity.
It removes incorrect nucleotides added at the 3' end.
This action allows the polymerase to stall further incorporation until the correct nucleotide is in place.
The incorrect nucleotide is cleaved by the exonuclease active site, releasing a dNMP.
The shortened strand re-engages the polymerase active site, and synthesis resumes.
DNA polymerase I (Pol I) has 5'-3', 3'-5', and 5'-3' exonuclease activities.
The 5'-3' exonuclease activity removes and replaces incorrect nucleotides (nick translation).

DNA Replication - RNA in DNA

rNTPs can't be directly incorporated in growing DNA strands because of the extra OH in ribose, causing steric clash.
Uracil-N-glycosylase (ung) and AP(apyrimidinic) endonuclease (xthA) remove baseless nucleotides and cleaves phosphodiester backbones.

DNA Replication - Okazaki Fragments

Okazaki fragments are found on the lagging strand.
The leading strand is also made of fragments joined together (pseudo-Okazaki fragment) that need joining.
Pol I will incorporate dUTP instead of dTTP in the leading strand fragments ~1 out of every 300 times.
Uracil must be removed, leading to nicks that need to be sealed by the DNA ligase.

DNA Replication - Leading Strand Fragments

The presence of U is problematic in DNA; it suggests damage potentially leading to mutations.
Uracil-N-glycosylase and AP(apyrimidinic) enzymes remove the uracil.
Poly I binds to the nick, removes the baseless nucleotide, and replaces it with the correct nucleotide.
DNA ligase seals the nick.

Replication Steps

The steps of DNA replication are initiation, elongation, and termination—starting, progressing, and ending the process.

DNA Replication - Initiation

Circular chromosomes and plasmids have a single origin of replication (ori).
The ori is a region of repetitive dsDNA rich in A-T base pairs.
The oric sequence in the E. coli chromosome is 245bp long and contains 13-bp and 9-bp repeats.
DnaA protein binds to 9-bp repeats to trigger unwinding at 13-bp repeats.
A DnaC protein binds ssDNA and loads a DnaB helicase onto one strand, facing the 3' direction.
DnaC detaches, and helicase moves to the fork.
After ~65 nt are unwound by the helicases, DnaG primase enzymes bind to form a primosome.
Primase synthesizes a ~10nt RNA primer.
Single-stranded binding protein (SSB) prevents re-annealing of the exposed ssDNA.
Pol III holoenzyme with accessory proteins binds to primers, loading of the beta clamp, and assembling the replisome.

DNA Replication - Elongation

The lagging strand syntheses with primase re-binding to create a new primer.
Replication forks with ẞ clamp added to the primer; Pol III core binds to the ẞ clamp.
Primer 1 is bound, with correct polarity, and the first Okazaki fragment begins. The DNA strand is pulled by helicase and Pol III, and the lagging strand loops out.
Okazaki fragment lengthens, delaying the looping process for primer 2.
The first Okazaki fragment is finished; Primase re-binds to helicase and add a second primer.
The Pol III core and ẞ clamp detach, releasing the completed fragment.
Pol I binds the end of the first Okazaki fragment replacing the RNA with DNA. DNA ligase seals the nick.

DNA Replication - Termination

To prevent fork overshooting, there are 23 bp sequences (Ter) that bind the Tus protein.
Tus protein can be displaced by the fork only in one direction.
As the forks get within 200bp of each other, DNA gyrase can no longer bind.
Positive supercoiling is relieved by topoisomerase IV.

DNA Replication - Eukaryotes

Eukaryotic DNA replication uses helicases to create replication forks, SSB to hold ssDNA apart, RNA primers, and several DNA polymerases.
It occurs in the nucleus, uses multiple origins per chromosome (~10s-1000s), and uses much slower DNA polymerases (~50 nt/sec) compared to prokaryotes.
There are no DNA polymerases with 5'-3' exonuclease activity.
Okazaki fragments are much shorter (~165 nt).
Eukaryotic DNA polymerases involved in both leading and lagging strand synthesis are not physically linked.

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Description

This quiz explores the key mechanisms and functions involved in DNA replication, including the roles of proteins like DnaC, primase, and DNA ligase. It also covers the synthesis of RNA primers and the consequences of mutations during the replication process. Test your knowledge on the intricate details of how DNA is replicated and repaired.