General features of DNA replication

What is DNA replication?

The copying of double-stranded DNA to produce DNA for daughter cells before cell division.

Why is DNA replication necessary?

To generate genetic material for daughter cells during cell reproduction.

What cell cycle phase does DNA replication occur in?

S phase (Synthesis phase).

What is meant by “DNA synthesis” vs “DNA replication”?

DNA synthesis = molecular events assembling new DNA strands

DNA replication: A more complex term including:

DNA chain synthesis

Initiation, elongation and termination

Accuracy

Daughter strand separation

Chromosome distribution

What replication model did Watson and Crick propose?

Semi-conservative replication

Define semi-conservative DNA replication.

Each daughter DNA molecule contains 1 old (parental) strand + 1 new strand.

What happens after TWO rounds of semi-conservative replication?

Two molecules are entirely new, and two molecules still contain one old + one new strand.

What is conservative replication?

The original parental DNA stays fully intact (two old strands) and a completely new double helix is made.

What is dispersive replication?

Parental DNA is mixed/distributed into both daughter DNA molecules in fragments.

What was the purpose of the Meselson–Stahl experiment (1958)?

To determine whether DNA replication is conservative, semi-conservative, or dispersive.

What organism did Meselson and Stahl use?

E. coli

What isotope did they first grow E. coli in?

¹⁵N (“heavy” nitrogen)

What happened after shifting bacteria into ¹⁴N medium?

Cells replicated using ¹⁴N, producing DNA with different densities.

How did they measure DNA density?

Using salt density gradient centrifugation.

What was observed after ONE generation in ¹⁴N?

DNA was “half-heavy”
Consistent with semi-conservative and dispersive models.

What was observed after TWO generations in ¹⁴N?

Two DNA types: “light” and “half-heavy”
Only consistent with semi-conservative replication.

What is an origin of replication (ORI)?

A unique DNA sequence where DNA replication is initiated.

What is a replicon?

A unit of DNA replicated from one origin.

What is a replication fork?

The site where DNA is unwound and new DNA is synthesized as the fork moves along the template.

DNA replication usually proceeds in what direction from the ORI?

Bidirectionally

How many ORIs are found in most bacterial (circular) DNA?

Usually one ORI

How does replication proceed in circular bacterial DNA?

Bidirectionally around the chromosome until the “eye/bubble” covers the whole chromosome.

How many ORIs are found in eukaryotic (linear) chromosomes?

Multiple ORIs across the chromosome

Why do eukaryotes need multiple ORIs?

Because chromosomes are long, so multiple replicons allow faster replication.

Why is DNA replication described as “semi-discontinuous”?

One strand is made continuously, the other discontinuously.

What is the leading strand?

The new DNA strand synthesized continuously in the same direction as the replication fork moves.

Why is leading strand synthesis continuous?

DNA polymerase can continuously extend it as the fork opens

What is the lagging strand?

The new DNA strand synthesized discontinuously in short fragments opposite the fork direction.

Why is lagging strand synthesis more complicated?

Because synthesis occurs opposite the direction of replication fork movement.

What enzyme makes the RNA primer on the lagging strand?

Primase

What is the role of the RNA primer?

Provides a starting point for DNA polymerase to extend DNA.

What are Okazaki fragments?

Short DNA segments formed during lagging strand replication.

What enzyme extends Okazaki fragments?

DNA polymerase (extends from RNA primers)

Where does replication terminate in E. coli?

When the two replication forks meet in the terminus region (about 180° opposite the ORI).

What are ter sites?

Termination DNA sequences in E. coli that bind Tus protein and stop fork progression.

What does Tus protein do?

Binds ter sites and blocks replication forks in specific directions.

What is a “replication fork trap”?

Multiple termination sites cause forks to pause and fuse in a controlled terminus region.

Why are multiple ter sites useful?

Backup system to ensure forks terminate properly even if one ter site mutates.

How does termination work in eukaryotes?

Less understood; forks likely meet at random and termination involves ligation of new DNA ends.

DNA replication is _____

Semi-conservative

DNA replication begins at ____ and proceeds _____.

ORI: bidirectionally

DNA replication is semi-discontinuous because of ____ and ____ strands.

Leading and lagging

Lagging strand is made of _____.

Okazaki fragments