DNA Replication Mechanisms

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Questions and Answers

What initiates the binding of primase to the ssDNA during DNA replication?

DnaB binding to ssDNA

SSB proteins coating the unwound DNA

RNA primer synthesis completion

DnaC/B complex interacting with DnaA (correct)

Which region of the DNA polymerase is responsible for ensuring accuracy during DNA replication?

Active site of SSB proteins

Palm region (correct)

Thumb region

Fingers domain

What happens when a correct dNTP enters the active site of DNA polymerase?

The polymerase disintegrates

The fingers domain opens

DNA synthesis stops immediately

Base pairing occurs with the template (correct)

What triggers the release of the fingers domain in DNA polymerase?

Release of pyrophosphate Signup and view all the answers

Which protein complex is responsible for starting DNA synthesis?

DNA polymerase Signup and view all the answers

How does DNA polymerase interact with the DNA template?

Using minor and major grooves Signup and view all the answers

What is the role of SSB proteins during DNA replication?

They coat unwound DNA to stabilize it. Signup and view all the answers

If a wrong nucleotide is incorporated during DNA replication, what is the likely outcome?

DNA polymerase may correct itself. Signup and view all the answers

What is the overall accuracy rate of DNA replication?

1 mistake per ~10^10 bp synthesized Signup and view all the answers

What happens when the template base is in a rare tautomeric form?

It pairs improperly, leading to a mismatch. Signup and view all the answers

How does the proofreading 3’-5’ exonuclease activity enhance DNA replication accuracy?

By increasing accuracy approximately 100 times. Signup and view all the answers

What is the first step in the incorporation assay to measure DNA Pol activity?

Filter free dNTPs from the DNA. Signup and view all the answers

What typically happens if a wrong nucleotide is incorporated during DNA synthesis?

It has a higher affinity for the exonuclease active site. Signup and view all the answers

What is a limitation of the filter binding method in assaying DNA Pol activity?

It does not yield information on the length of the product. Signup and view all the answers

What is the consequence of a mismatch during DNA replication?

The geometry of the helix does not fit properly. Signup and view all the answers

What is the purpose of the separation on a gel during the incorporation assay?

To separate the products on a denaturing gel. Signup and view all the answers

What is the primary enzyme responsible for adding nucleotides during DNA replication?

DNA polymerase Signup and view all the answers

Which process describes how each new DNA molecule contains one original strand and one newly synthesized strand?

Semi-conservative replication Signup and view all the answers

What must happen at the replication fork to allow DNA replication to proceed?

DNA strands must be unwound Signup and view all the answers

What is the role of helicase in DNA replication?

It unwinds the DNA strands Signup and view all the answers

Why does DNA polymerase require a primer for replication?

It can only extend from an existing 3' end Signup and view all the answers

Which type of DNA strand is synthesized continuously during replication?

Leading strand Signup and view all the answers

What is the function of topoisomerases during DNA replication?

To resolve supercoiling ahead of the replication fork Signup and view all the answers

What is removed after RNA primers are laid down during replication?

RNA primers Signup and view all the answers

At what sites does DNA replication initiate?

Origins of replication Signup and view all the answers

Which protein functions as the helicase during bacterial DNA replication?

DnaB Signup and view all the answers

What is synthesized in short segments known as Okazaki fragments during DNA replication?

Lagging strand Signup and view all the answers

What are the building blocks of DNA added by DNA polymerase during replication?

dNTPs Signup and view all the answers

During replication, which phase involves the actual addition of nucleotides to the growing DNA chain?

Elongation Signup and view all the answers

What is a key characteristic of repair polymerases in terms of processivity?

They have low processivity, typically 10-20 bp. Signup and view all the answers

How often does DNA replication have a mistake in nucleotide synthesis?

1 mistake in 10 billion bp Signup and view all the answers

Which enzyme is responsible for sealing the nicks between Okazaki fragments?

DNA ligase Signup and view all the answers

What is the role of the sliding clamp in DNA replication?

To keep DNA polymerase attached to the DNA. Signup and view all the answers

What is the objective of using chemotherapeutic drugs in relation to DNA replication?

To inhibit replication in cancer cells Signup and view all the answers

In the helicase assay, what does a successful reaction indicate?

The active helicase has displaced the primer. Signup and view all the answers

How does DNA Pol I contribute to the replication process?

It replaces RNA primers with DNA and fills gaps. Signup and view all the answers

What is the primary role of the tau complex at the replication fork?

To load and stimulate the helicase. Signup and view all the answers

During replication, how is the lagging strand structured?

It is folded back to facilitate the replication process. Signup and view all the answers

What does the Template Challenge Assay measure?

The processivity of DNA polymerases. Signup and view all the answers

What is the function of RNase H in DNA replication?

To remove RNA primers from the DNA. Signup and view all the answers

What distinguishes replicative polymerases from repair polymerases?

Replicative polymerases have significantly higher processivity. Signup and view all the answers

Study Notes

DNA Replication

DNA replication is the process by which a DNA molecule is copied to produce two identical DNA molecules.
DNA replication is essential for cell division, as it ensures that each new cell receives a complete set of genetic information.
Replication is a concerted effort of many enzymes.

Mechanisms of DNA Replication

DNA replication is semi-conservative: Each new DNA molecule contains one strand from the original helix and one newly synthesized complementary strand.
DNA replication is semi-discontinuous: DNA polymerase can only work in the 5' to 3' direction, leading to continuous synthesis on the leading strand and discontinuous synthesis on the lagging strand, which is synthesized in fragments called Okazaki fragments.
DNA replication is bidirectional: Two replication forks move in opposite directions from the origin of replication.

Key Enzymes Involved in Replication

Helicase: Unwinds the DNA double helix.
Topoisomerases: Resolve supercoiling ahead of the replication fork.
Primase: Synthesizes short RNA primers for both leading and lagging strands.
DNA Polymerase: Adds nucleotides to the 3' end of the primer.
DNA Ligase: Joins Okazaki fragments on the lagging strand.

Accuracy of Replication

DNA polymerase has a proofreading 3' to 5' exonuclease activity, allowing it to correct mismatched bases.
Proofreading increases the accuracy of replication ~100-fold.
The overall accuracy of replication is remarkable: 1 mistake per ~10^10 base pairs synthesized.

Initiation of Replication

Replication begins at specific sites called origins of replication.
Origins of replication are AT-rich regions, as A-T base pairs have fewer hydrogen bonds, making the DNA easier to separate.
In bacteria, the initiator protein DnaA binds to the origin of replication, initiating the unwinding of DNA.

Essential Replication Proteins

DnaA: Initiator protein in bacteria, binds to the origin and separates the strands.
DnaB: Helicase, unwinds the DNA during replication.
DnaC: Helicase loader, positions the helicase on single-stranded DNA (ssDNA) and inhibits its activity until needed.
DnaG: Primase, synthesizes the RNA primer.
SSB (Single-Stranded Binding Proteins): Coat unwound ssDNA, preventing it from reannealing.

DNA Polymerase Function

DNA polymerase interacts with DNA through non-covalent interactions with the major/minor groove and the DNA backbone.
The palm region of DNA polymerase interacts with the primer-template junction (PTJ) and newly synthesized DNA.
DNA polymerases have high processivity, meaning they can add many nucleotides without dissociating from the DNA template.

DNA Polymerase Activity Assay

Incorporation Assay: Measures the incorporation of labeled nucleotides into DNA. Can be used to study different polymerases.
Filter Binding Assay: Positively charged paper binds DNA; can measure signal but doesn't provide information on product length.
Gel Electrophoresis (Separation Assay): Separates products based on size; provides information on product length but is slower and less quantitative.
Primer Extension Assay: Uses a labeled primer; provides information on product length and quantitative data.

Processivity of DNA Polymerase

Template Challenge Assay: Measures the number of nucleotides added per single binding to the primer-template junction, providing insight into processivity.

Different DNA Polymerases in Bacteria

DNA Pol III Holoenzyme: This complex is the primary replicative enzyme in bacteria and possesses high processivity.
- Core Enzyme: The catalytic subunit that adds nucleotides.
- Sliding DNA Clamps: Ring-shaped structures that encircle DNA, increasing processivity.
- Sliding Clamp Loader (Tau Complex): Facilitates loading and unloading of sliding clamps.

Resolving Okazaki fragments

RNAse H: Removes RNA primers, leaving a single ribonucleotide.
DNA Pol I: Fills in the gap between Okazaki fragments and removes the last ribonucleotide. It has both 3' to 5' (proofreading) and 5' to 3' exonuclease activity.
DNA Ligase: Seals the nick between Okazaki fragments, creating a continuous DNA strand.

Replication of Leading and Lagging Strand

The leading and lagging strands are synthesized simultaneously at the replication fork.
The lagging strand is looped back to allow continuous movement of the replication fork.
Proteins at the replication fork interact and regulate each other, ensuring efficient and coordinated replication.

Clinical Applications of Replication

Chemotherapeutic Drugs: Target DNA replication to inhibit cancer cell proliferation.
Antivirals: Target viral DNA replication.
Some Antibiotics: Inhibit bacterial DNA replication.
CDK4/6 Inhibitors: Used in cancer treatment to block cell cycle progression.

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Description

Explore the fascinating process of DNA replication through this quiz. Learn about the semi-conservative nature, the roles of key enzymes like helicase and topoisomerases, and the bidirectional replication process. This quiz provides insights into the critical mechanisms that ensure accurate cell division.