5.2 DNA Replication

Cell Cycle

Life cycle of a cell

Cells can reproduce

Daughter cells receive an

exact copy of DNA from

parent cell

DNA replication happens

during the S phase

Proposed Models of DNA Replication

Conservative Model

Semi-Conservative Model

Dispersive model

1. Conservative Model

Parental DNA makes two new

daughter strands

After first replication cycle:

• New daughter strands join to form

double helix

• Parental DNA remain as original

double helix Newly formed DNA shown as blue

2. Semi-conservative Model

Parental DNA makes two new

daughter strands

After first replication cycle:

• Each new daughter strand binds to a

parental strand to form double helix

Newly formed DNA shown as blue

3. Dispersive Model

During replication, parental DNA

broken into small fragments

After first replication cycle:

• Each small fragment binds to pieces

of the newly copied DNA to form

double helix

Newly formed DNA shown as blue

Proposed Models of

DNA Replication

Results

Conservative Model One new molecule and

one old molecule

Semi-Conservative Model Two hybrid molecules of

one old and one new

strands

Dispersive model Two hybrid molecules of

mixture of old and new

strands

Experiment to Determine Replication Model

Matthew Meselson and Franklin Stahl

Distinguish between parent and daughter strands

of DNA by using two isotopes of nitrogen:

“light” 14N

“heavy” 15N

Experiment to Determine Replication Model

Tag DNA with isotopes

Separate content by centrifuge

DNA containing the denser

isotope 15N forms a band near

the bottom of the test tube

DNA containing the lighter

isotope 14N forms a band near

the top of the test tube

Experiment to Determine Replication Model

Take a sample mix DNA with cesium chloride centrifuge density gradient

E. Coli grown in 15N medium

Transfer to 14N medium

After 20 minutes

(one cycle)

After 40 minutes

(two cycles)

And the Winner is .... Semi-Conservative Model

DNA Replication

3 main phases:

1. Initiation

double helix is unwounded; base pairs are exposed

2. Elongation

parental DNA is used as template to assemble the new strand; final DNA has one parental and one new strand

3. Termination

the two new DNA strands are separated

Phase 1: Initiation

Specific nucleotide sequence indicates the origin of replication

Circular prokaryotic DNA has 1 origin

linear eukaryotic DNA often has thousands

Phase 1: Initiation

1. Initiator proteins bind to DNA at the origin of replication

2. Helicase (enzyme) unwinds double helix by breaking the hydrogen bonds that link the complementary base pairs.

3. Single-strand-binding proteins stabilize the unwound strands.

4. Topoisomerase II (enzyme) relieves strain on the double helix that is generated from unwinding.

A replication bubble

forms with a Y-shaped

replication fork at

each end

Phase 1: Initiation

Replication begin at many origin of replications.

The replication bubbles expand laterally as DNA replication continues on both strands.

All of the replication bubbles eventually fuse together.

Phase 2: Elongation (Priming)

DNA polymerase cannot start incorporating nucleotides on its own.

Needs an existing 3’ end of a nucleic acid.

RNA primase lays down RNA primer onto template strand

The primer (10-60 nucleotides long) provides that 3’ end.

Phase 2: Elongation

DNA polymerase III then

binds to parental strand

adds complementary

nucleotides using parental

DNA as a template

5’ to 3’ direction towards

the replication fork

Phase 2: Elongation

Free bases are floating in the nucleoplasm as deoxyribonucleoside triphosphates.

The energy required for DNA synthesis is provided by hydrolyzing the bond between the 1st and 2nd phosphates of the deoxyribonucleoside.

Phase 2: Elongation

Elongation proceed in two

direction, outwards from the origin

of replication.

The two DNA strands are

antiparallel.

DNA polymerase III only

replicates in the 5’ to 3’ direction.

Elongation is semi-discontinuous

Phase 2: Elongation (Semi-discontinuous)

Leading strand

uses the 3’ to 5’ template strand as its guide.

Is built continuously towards the replication fork.

Lagging strand

uses the 5’ to 3’ template strand as its guide.

Is built discontinuously in short fragments.

RNA primase constantly adds new RNA primers

along the template strand.

The fragments are known as Okazaki

fragments.

Phase 2: Elongation (Semi-discontinuous)

DNA polymerase I

Removes the RNA primers

Replaces them with the

proper

deoxyribonucleosides

DNA ligase

Joins the Okazaki

fragments together

(phosphodiester bonds)

Phase 3: Termination

Eventually two replication forks will fuse together and form a

continuous strand of newly synthesized DNA.

The two new daughter DNA molecules will contain a newly

synthesized copy of DNA and the parental DNA.

Important Enzymes in DNA Replication