BS1030 Topic 3 Lecture 3 DNA Polymerases and other enzymes The Replication Fork

Who proposed the model of DNA replication?

James Watson and Francis Crick, 1953.

What key insight did Watson and Crick note about DNA structure?

The specific base pairing suggests a copying mechanism for the genetic material.

What are the three proposed models of DNA replication?

Conservative, semi-conservative, and dispersive.

Which DNA replication model is correct?

Semi-conservative replication.

What happens in semi-conservative replication?

Each new DNA molecule has one parental strand and one newly synthesised strand.

What enzyme catalyses the joining of DNA fragments?

DNA ligase.

What reaction does DNA ligase perform?

Recreates a phosphodiester bond between a 3′-OH and a 5′-phosphate using ATP.

What is the function of DNA polymerase?

Adds nucleotides to a growing DNA strand complementary to the template.

Who discovered DNA polymerase I?

Arthur Kornberg in 1958.

What are the substrate requirements for DNA polymerase?

A template strand and a primer with a free 3′-OH group.

What direction does DNA polymerase synthesize DNA?

In the 5′→3′ direction.

What is meant by processivity in DNA polymerases?

The number of nucleotides added before the enzyme dissociates.

How many nucleotides per second does DNA polymerase I add?

Approximately 10 nucleotides per second.

What is the proofreading function of DNA polymerase I?

3′→5′ exonuclease activity removes mismatched nucleotides for error correction.

What are the three main enzymatic activities of DNA polymerase I?

5′→3′ polymerase, 3′→5′ exonuclease (proofreading), and 5′→3′ exonuclease (nick translation).

What was the polA1 mutant used for?

To identify DNA polymerase II and III.

Who discovered DNA polymerases II and III?

Thomas Kornberg in 1971.

What is the molecular weight of DNA polymerase I?

Approximately 103,000 Da.

How many domains does DNA polymerase I have?

Three distinct functional domains, containing Zn²⁺.

What is the molecular weight of DNA polymerase III?

Approximately 900,000 Da.

How many subunits make up DNA polymerase III?

At least 10 subunits.

What are the key subunits of DNA polymerase III?

α, ε, and θ (core enzyme), plus β clamp subunits.

What is the rate of DNA polymerase III?

>1000 nucleotides per second.

What is the processivity of DNA polymerase III?

Very high (>500,000 nucleotides added before dissociation).

What is the structure of the E. coli chromosome?

A single circular DNA molecule that replicates bidirectionally.

Where does E. coli DNA replication start?

At a unique origin of replication (oriC).

Where does E. coli replication terminate?

At the termination site (terC), where the two replication forks meet.

What is a replication fork?

The Y-shaped region where DNA strands are unwound and replicated.

What are the two DNA strands at the replication fork?

Leading strand (continuous) and lagging strand (discontinuous).

What are Okazaki fragments?

Short DNA fragments synthesised on the lagging strand.

What enzyme joins Okazaki fragments?

DNA ligase.

What is the primer for DNA synthesis?

Short RNA segments synthesised by primase.

Why are RNA primers needed?

DNA polymerase cannot initiate synthesis; it can only extend from an existing 3′-OH.

What enzymes are involved in unwinding DNA?

Helicase and single-stranded DNA-binding (SSB) proteins.

What does helicase do?

Unwinds the DNA double helix using ATP.

What do SSB proteins do?

Bind to single-stranded DNA to prevent reannealing.

What are DNA topoisomerases?

Enzymes that control DNA supercoiling and relieve torsional strain during replication.

What is DNA gyrase?

A bacterial topoisomerase that introduces negative supercoils using ATP.

Why is DNA gyrase important?

It prevents excessive positive supercoiling ahead of the replication fork.

Why is DNA gyrase a drug target?

It differs significantly from the mammalian enzyme and is inhibited by certain antibiotics.

What are restriction endonucleases?

Enzymes that cut DNA internally at specific palindromic sequences.

Why do restriction enzymes recognise palindromes?

Because they function as dimers binding to symmetric DNA sequences.

What are sticky ends?

Single-stranded overhangs created by restriction enzyme cuts that can base pair with complementary sequences.

What are blunt ends?

Ends with no overhangs produced by some restriction enzymes.

How can sticky ends be used in molecular biology?

DNA fragments with matching sticky ends can be ligated to form recombinant DNA, the basis of gene cloning.