Lecture 9 - DNA Replication and Okazaki Fragments Overview

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Questions and Answers

What direction is new DNA synthesized during replication?

3’ → 5’

3’ → 3’

5’ → 3’ (correct)

5’ → 5’

What are Okazaki fragments primarily associated with?

DNA proofreading

Primase activity

Leading strand synthesis

Lagging strand synthesis (correct)

How long are Okazaki fragments in eukaryotes?

Approximately 300-500 nucleotides

Approximately 50-100 nucleotides

Approximately 1000-2000 nucleotides

Approximately 100-200 nucleotides (correct)

Why is there no synthesis of new DNA in the 3’ → 5’ direction?

DNA polymerase can only add nucleotides in one direction Signup and view all the answers

In E. coli, how long are Okazaki fragments typically?

Approximately 1000-2000 nucleotides Signup and view all the answers

What is the role of RNA primase in DNA replication?

It synthesizes an RNA primer to initiate replication. Signup and view all the answers

Why does rifampicin prevent the replication of M13 phage DNA?

It inhibits E.coli RNA polymerase. Signup and view all the answers

What are Okazaki fragments primarily made of?

Little pieces of RNA, approximately 10-12 bases long. Signup and view all the answers

What remains after DNase attempts to destroy Okazaki fragments?

Short RNA segments. Signup and view all the answers

What is true about RNA polymerases in the context of primer synthesis?

They can synthesize RNA without a primer. Signup and view all the answers

What is the role of DNA ligase in the joining of Okazaki fragments?

It releases AMP while forming covalent bonds. Signup and view all the answers

What happens after DNA ligase attaches AMP to the 5' phosphate of the downstream Okazaki fragment?

A phosphodiester bond is formed. Signup and view all the answers

Why is the simplest model for DNA polymerase III binding considered incorrect?

It suggests Pol III operates independently. Signup and view all the answers

What remains released from the reaction when DNA ligase utilizes ATP?

AMP Signup and view all the answers

How are the two DNA polymerase III molecules positioned relative to each other?

They face the same direction while held together. Signup and view all the answers

What is the energy source used by DNA ligase during the joining of Okazaki fragments?

ATP Signup and view all the answers

Which statement describes the role of the clamp holder in DNA replication?

It stabilizes the DNA polymerase during replication. Signup and view all the answers

Which incorrect feature is highlighted in the simplest model of DNA polymerase III activity?

Two distinct Pol III complexes acting independently. Signup and view all the answers

What is the primary function of Type II topoisomerases in bacteria?

To convert positively supercoiled DNA into negatively supercoiled DNA Signup and view all the answers

How does Type I topoisomerase relax negatively supercoiled DNA?

By making a nick in one DNA strand Signup and view all the answers

What occurs when the two replication forks meet during bacterial DNA replication?

Topoisomerase IV separates the catenated daughter chromosomes Signup and view all the answers

What is a characteristic of bacterial DNA polymerisation?

It involves simultaneous leading and lagging strand synthesis Signup and view all the answers

Which statement accurately describes the action of Type II topoisomerases?

They create a double-stranded cut to relieve supercoiling Signup and view all the answers

What is the primary function of the β clamp during DNA replication?

It increases the processivity of DNA Pol III. Signup and view all the answers

Which statement about lagging strand synthesis is correct?

The lagging strand template forms a loop during synthesis. Signup and view all the answers

What role does DNA primase play in DNA replication on the lagging strand?

It synthesizes the RNA primer. Signup and view all the answers

What happens to the Okazaki fragments during the replication process?

They are pulled back to DNA Pol III for further processing. Signup and view all the answers

What is the relationship between helicase and the lagging strand during replication?

Helicase unwinds the DNA while Pol III synthesizes the leading strand. Signup and view all the answers

What orientation does the RNA primer face on the lagging strand template?

It faces away from the replication fork. Signup and view all the answers

What processivity change occurs when DNA Pol III is clamped?

It increases, allowing longer DNA segments to be replicated. Signup and view all the answers

What is the first step of lagging strand synthesis?

DNA primase manufactures an RNA primer. Signup and view all the answers

What is a primary reason for the complexity of lagging strand synthesis?

DNA Pol III has low processivity to release Okazaki fragments. Signup and view all the answers

What role do DNA Pol I and DNA ligase play in lagging strand synthesis?

They repair gaps left after Okazaki fragment synthesis. Signup and view all the answers

What is the significance of the trombone model in DNA replication?

It shows how the lagging strand is synthesized in loops. Signup and view all the answers

How does the processivity of DNA Pol I compare to that of DNA Pol III?

DNA Pol III requires higher processivity. Signup and view all the answers

What is the approximate speed of polymerisation for eukaryotic DNA polymerases?

50 nucleotides/s Signup and view all the answers

What happens when DNA Pol III synthesizes a new Okazaki fragment?

It detaches from the DNA strand. Signup and view all the answers

Why would it take a longer time to replicate a human chromosome compared to an E.coli chromosome?

Eukaryotic DNA polymerases are slower than prokaryotic DNA polymerases. Signup and view all the answers

What is a characteristic feature of Okazaki fragments during lagging strand synthesis?

They are short segments of newly synthesized DNA. Signup and view all the answers

Study Notes

LF130 Cellular and Molecular Biology: DNA Replication

Lecture 9, 2024, Part 1: Okazaki Fragments
- DNA replication is semi-conservative
- DNA strands are anti-parallel
- Watson-Crick base pairing is fundamental
- New DNA is synthesized in the 5' to 3' direction
- DNA synthesis is semi-continuous, involving leading and lagging strands
- DNA polymerase possesses proofreading (3' to 5') exonuclease activity
Lecture 9, 2024, Part 1: Okazaki Fragments (1968)
- DNA replication requires a free 3'-OH group on a primer
- Leading strand is synthesized continuously, following the replication fork in the 5' to 3' direction
- Lagging strand is synthesized discontinuously, moving away from the replication fork
- Okazaki fragments are short DNA segments (~100-200 nucleotides in eukaryotes, ~1000-2000 in E. coli)
- New DNA is built in the 5' to 3' direction
- No 3' to 5' strand synthesis of new DNA
Lecture 9, 2024, Part 1: Okazaki Fragments, Mechanism and Errors
- DNA primase synthesizes RNA primers for lagging strand synthesis
- DNA polymerase extends the primers with dNTPs
- Removal of RNA primers and filling gaps are carried out by DNA polymerase I
- DNA ligase seals the gaps in lagging strand
- Errors in replication, such as misincorporation, are corrected by proofreading
- No 5' triphosphates available for hydrolysis: no energy for polymerization in 3' 5'.
M13 Bacteriophage
- M13 bacteriophage has a single-stranded DNA genome
- Infected E. coli cells release M13 phage.
M13 Life Cycle
- Infection via pilus, horizontal gene transfer
- Replicative form (RF)
- Single-stranded DNA genomes
- Viral SS DNA genomes, packaged
DNA Replication Start
- Arthur Kornberg (1971) showed that replication of M13 phage DNA, from single-stranded to double-stranded form (RF) in E. coli, is blocked by rifampicin, which inhibits bacterial RNA polymerase.
- Okazaki's discovery
  - DNA polymerase can't start replication, RNA primers are needed
  - It left little pieces of RNA, 10-12 bases long.
RNA Primer Synthesis
- DNA primase creates RNA primers, and is a DNA-directed RNA polymerase.
- RNA primer synthesis is needed to begin DNA synthesis on the lagging strand.
- RNA polymerase need no primer.
Lagging Strand Synthesis (Steps 1-5)
- DNA primase synthesizes RNA primers on the lagging strand
- DNA polymerase III (Pol III) extends the primers with dNTPs, synthesizing Okazaki fragments
- Pol I removes RNA primers
- DNA Pol I replaces RNA with DNA
- DNA ligase seals the gaps between Okazaki fragments
- The lagging strand is synthesized discontinuously, but in the direction of the replication fork movement
Leading Strand Synthesis (Steps 1-3)
- DNA helicase unwinds DNA
- DNA primase creates RNA primers on the leading strand template
- Primed duplex is captured by Pol III and clamped
Leading Strand Synthesis (Steps 2-3)
- Processivity of DNA pol III: makes long stretches of DNA continuously, ~1000 bases per second.
- DNA polymerase III remains attached to the template.
Lagging Strand Synthesis (Complexity 1-5)
- DNA primase starts RNA primers
- Primed duplex is captured by Pol III
- Helicase unwinds and Pol III extends the new primers on the lagging strand
- Exonuclease activity removes RNA primers
- DNA Pol I replaces RNA with DNA
- DNA ligase seals fragments
Trombone Model
- The lagging strand synthesis resembles a looping trombone
Electron micrograph of replication fork
- Electron micrograph shows the newly synthesized Okazaki fragments, parental DNA, and the replication complex
Eukaryotic DNA Replication Differences
- Eukaryotic DNA polymerase is much slower (~50 nucleotides/second) than bacterial DNA polymerase
- Eukaryotic DNA replication proceeds from multiple replication origins
Eukaryotic DNA Replication - Telomeres and Telomerase
- Telomere are DNA sequences at end of chromosomes
- DNA polymerase can't fully copy lagging strand
- The primers are erased
- The gap in the lagging strand is filled by DNA polymerase and repaired by DNA ligase
- Chromosomes shorten with each replication
- Telomerase replicates telomeres
- Telomerase is active in some germline cells, epithelial cells, and hematopoietic cells, and often in > 90% of cancer cell lines.
Supercoiling
- DNA supercoiling occurs as replication proceeds, causing torsional problems
- Topoisomerases relax/change supercoiling.
Topoisomerases
- Type I topoisomerase relaxes negatively supercoiled DNA by making a single-stranded break
- Type II topoisomerase, like DNA gyrase (in bacteria), relaxes positively supercoiled DNA by making a double-stranded break, enabling DNA strands to pass through
- Both enzymes help manage the torsional stress during DNA replication.

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Description

This quiz explores key concepts of DNA replication, focusing on the formation and role of Okazaki fragments, RNA primase, and DNA ligase. Questions cover the synthesis direction of DNA, the duration of Okazaki fragments, and the functionality of various enzymes involved in the process. Test your knowledge on how these processes contribute to accurate DNA replication.