DNA Replication Problems in E. coli: L3-4

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Questions and Answers

Which DNA polymerase is primarily responsible for DNA replication in Escherichia coli?

Pol IV

Pol I

Pol II

Pol III (correct)

What is the main role of Helicase during DNA replication in E.coli?

To repair damaged DNA

To unwind the DNA double helix (correct)

To synthesize RNA primers

To join Okazaki fragments

In PCR, which step follows the denaturing of DNA at 95°C?

Heating to 95°C again

Cooling to 72°C

Cooling to 55°C (correct)

Heating to 100°C

What is the main function of DNA ligase in DNA replication?

To join Okazaki fragments Signup and view all the answers

Which of the following statements regarding DNA polymerases in E.coli is true?

Pol III can replicate the entire chromosome quickly (40 minutes). Signup and view all the answers

What is the primary role of helicases in DNA replication?

To separate and unwind DNA strands Signup and view all the answers

How fast can DNA polymerase III synthesize nucleotides during replication?

1600 nt/s Signup and view all the answers

What phenomenon occurs due to unwinding of the DNA duplex by helicases?

Torsional strain and supercoiling Signup and view all the answers

In the equation Lk = T + W, what does 'W' represent?

Writhe or number of self-crossings Signup and view all the answers

What is a function of the linking number (Lk) in circular DNA?

It is a fixed value Signup and view all the answers

What condition describes positive supercoiling in DNA?

Lk > Lk0 Signup and view all the answers

If the twist (T) increases, what happens to the linking number (Lk)?

Lk must increase Signup and view all the answers

What does a negative superhelical density (σ) indicate?

DNA is underwound Signup and view all the answers

In the equation σ = (Lk-Lk0)/Lk0, what does a σ value of 0 imply?

DNA is in the relaxed state Signup and view all the answers

Which of the following values corresponds to relaxed DNA supercoiling?

W = 0 Signup and view all the answers

What is the primary function of topoisomerase type I?

Cleave the backbone of one DNA strand Signup and view all the answers

How does type II topoisomerase differ from type I?

It requires ATP for its mechanism Signup and view all the answers

What role does DNA gyrase play in bacterial DNA replication?

It introduces negative supercoils to assist in replication fork progression Signup and view all the answers

What happens to the linking number (LK) after type II topoisomerase's action?

LK decreases by 2 Signup and view all the answers

Which of the following statements is true regarding the topoisomerase type I mechanism?

A gap is formed that allows one strand to pass through Signup and view all the answers

What happens to circular DNA molecules during replication?

They form interlocked daughter rings known as catenanes. Signup and view all the answers

Which process allows the separation of interlocked DNA rings?

Topoisomerase IV action. Signup and view all the answers

What is the significance of antiparallel strands in DNA replication?

They allow simultaneous synthesis of both DNA strands. Signup and view all the answers

How do DNA polymerases synthesize DNA?

By adding nucleotides to the 3' end of an existing region. Signup and view all the answers

What is a consequence of semi-discontinuous replication?

Lagging strand remains unfinished with nicks. Signup and view all the answers

What is formed on the lagging strand during DNA replication?

Okazaki fragments due to multiple primers. Signup and view all the answers

What contributes to an increased mutation rate during DNA replication?

Extra genes required for replication. Signup and view all the answers

What happens during the unwinding of the DNA duplex during replication?

The duplex must fully unwind before synthesis can start. Signup and view all the answers

What is the primary function of topoisomerase IV in DNA replication?

To facilitate decatenation of interlocked rings. Signup and view all the answers

What is the reason for having two polymerases during DNA replication?

To ensure leading and lagging strands can be synthesized simultaneously. Signup and view all the answers

What is the primary role of Pol I during DNA replication?

To remove RNA primers and fill gaps Signup and view all the answers

What is the nature of Okazaki fragments?

They consist of short segments of DNA with RNA primers Signup and view all the answers

What happens at the 3’ end of Okazaki fragments after RNA removal?

A nick is left that needs to be sealed Signup and view all the answers

How does Pol III differ from Pol I in relation to nicks?

Pol I binds to nicks while Pol III does not Signup and view all the answers

Which enzyme is responsible for sealing nicks in the DNA?

DNA ligase Signup and view all the answers

What is the significance of using 3H-TTP in Okazaki's investigation?

To label new DNA strands Signup and view all the answers

What describes the sizes of radioactive ssDNA as observed in Okazaki's experiments?

They do not increase smoothly, showing variable sizes Signup and view all the answers

How does RNAse H contribute to the process of DNA replication?

By degrading RNA in DNA-RNA hybrids Signup and view all the answers

Study Notes

DNA Replication Overview

DNA replication is a complex process, unlike simple DNA synthesis.
There are multiple problems that need to be overcome to replicate circular DNA.

DNA Replication Problems and Solutions in E. coli

Problem 1: DNA Supercoiling: Coiled DNA strands need to be unwound; circular chromosomes have torsional strain. Unwinding at one point creates strain elsewhere leading to supercoiling.
Solution:
- Helicases separate and unwind the DNA double helix, using ATP hydrolysis (3 base pairs/ATP).
- Helicases move towards the 3' end of the strand, requiring rotations of 10,000 rpm.
- Replication bubble creation occurs, with replication forks forming on the unwound section.
- Uncoiling requires the removal of positive supercoiling, which is done by Topoisomerases.
Problem 2: Antiparallel Strands: Two DNA polymerases are needed - one for the leading strand and another for the lagging strand. This is to address the directionality of DNA replication.
Solution:
- DNA polymerase III (Pol III) is responsible for the leading strand synthesis and the lagging strand.
- Synthesis occurs in the 5'→ 3' direction.
- The lagging strand requires multiple primers and segments (Okazaki fragments)
Problem 3: Circular DNA molecules (topology): Circular DNA forms catenanes during replication
Solution: Topoisomerase IV. This enzyme separates the interlocked daughter DNA rings

DNA Replication Details

DNA polymerases: Bacteria contain multiple DNA polymerases, named in order of discovery, not importance.
- Pol I: repair and replication
- Pol II: repair
- Pol III: principal replication enzyme
- Pol IV: repair
- Pol V: repair
- All polymerases synthesize DNA in the 5' to 3' direction.
DNA Polymerase III (Pol III): The principal DNA replication enzyme in E. coli.
- Requires 22 genes, high processivity (~~1600 nt/s), large number of nucleotides at a time (~~>50,000).
- Pol III is very fast (~40 mins to replicate entire E. coli genome)
Pol I: Repair and replication, much slower than Pol III (10 nt/s)
- Requires only 1 gene; low processivity; lower amount of nucleotides at a time (~20-100 nt/time)
Topoisomerases (I and II): Enzymes that change the linking number (Lk) of DNA.
- Topoisomerase I: Cleaves one strand of DNA, allowing rotation, resealing the strand.
- Topoisomerase II: Cleaves both strands of DNA, then passed through the gap and rejoining, which changes the linking number by 2.
  - E Coli have a unique type II topoisomerase called DNA gyrase -DNA gyrase helps in supercoil removal and plays an important role in replication fork progressions and is a good antibiotic target.
Okazaki Fragments: Short fragments of DNA synthesized on the lagging strand during replication..
- These fragments are joined by DNA ligase.
- DNA primase synthesizes short RNA primers that are required by the DNA polymerase for initiation.
RNA primers: Short RNA sequences used to initiate DNA synthesis on the lagging strand during DNA replication, removed after synthesis.
- RNAse H: degrades RNA primers
- Pol I: fills in gaps where RNA was formerly.
- DNA ligase: seals nicks, joins Okazaki fragments
Evidence for Okazaki fragments: Experiments using radioactive thymidine (3H-TTP) and density centrifugation.

DNA Replication Mechanisms of E. coli

Mechanisms used:
- DNA polymerase III (Pol III) is a high-processivity enzyme; it maintains DNA strand separation for simultaneous synthesis on the leading and lagging strand. -DNA polymerase I (Pol I) removes RNA primers and fills in the gaps created. -DNA ligase joins Okazaki fragments on the lagging strand.

Summary of DNA Replication Problems

Problems in DNA replication stem from its anti-parallel structure and circular DNA structure, which creates supercoiling and catenanes.

DNA Polymerases (in Bacteria) Summary

Bacteria have several DNA polymerases. Pol III is the primary replicative enzyme, with high processivity.. Pol I is involved in repair and synthesis.

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Description

This quiz explores the challenges of DNA replication, specifically in E. coli. It covers issues like DNA supercoiling and the need for antiparallel strands, alongside the solutions provided by helicases and topoisomerases. Test your understanding of the key concepts and processes involved in DNA replication.