7.1 DNA structure and replication Understanding: -DNA structure suggested a mechanism for DNA replication -Nucleosomes help to supercoil the DNA -DNA replication.

7.1 DNA structure and replication

Understanding:- DNA structure suggested a mechanism for

DNA replication- Nucleosomes help to supercoil the DNA- DNA replication is continuous on the

leading strand and discontinuous on the lagging strand

- DNA replication is carried out by a complex system of enzymes

- DNA polymerase can only add nucleotides to the 3’ end of the primer

- Some regions of DNA do not code for proteins but have some other important function

Applications:

- Rosalind Franklin’s and Maurice Wilkins’ investigation of DNA structure by X-ray diffraction

- Tandem repeats are used in DNA profiling

- Use of nucleotides containing dideoxyribonucleic acid to stop DNA replication

Skills:

- Analysis of results of the Hershey and Chase experiment providing evidence that DNA is the genetic material

- Utilisation of molecular visualisation software to anaylyse the association between proteins and DNA within a nucleosome

Nature of science:

- Making careful observations: Rosalind Franklin’s X-ray diffraction provided crucial evidence that DNA is a double helix

Hershey-Chase experiments

Is DNA the genetic material?

Alfred Hershey and Martha Chase used radioisotopes to help prove this.

Radioisotopes

Forms of elements that decay over time at a predictable rate.

Particles given off in this decay allow detection of the specific isotope used

Hershey-Chase experiments

Grew bacteriophage viruses in two different types of cultures.

Radioactive phosphorous-32- Detectable phosphorous in DNARadioactive sulphur-32- Detectable sulphur found in outer coat of virus

Allowed to infect bacteria (E.coli)

Hershey-Chase experiments

As DNA contains phosphorous and not sulphur, they concluded that DNA was the

genetic material not protein.

Hershey-Chase experimentsDescribe the Hershey-Chase experiment:

DNA structure3 components:

– Pentose sugar (deoxyribose in DNA)

– Phosphate – Organic base (always contains nitrogen)

Phosphate

sugarbaseStay the same

ChangesContains nitrogen & carbon

Pentose sugar (5 Carbon atoms)

Single strand of DNA

Backbone is made of alternating phosphate and deoxyribose sugar

They are held together by a phosphodiester bond (just ester at SL!)

Condensation reaction – producing water

This produces a chain of DNA (as this links the single nucleotides together)

Phosphodiester bond

Where does the phosphodiester bonds occur?

Phosphate group reacts on the 5’ carbon

Hydroxyl group reacts on the 3’ carbon

Double strand of DNA

The strands of DNA run in opposite directions to each other

There is a 5’ carbon free to bond on this end…

…and a 3’ carbon free to bond on this end

There is a 3’ carbon free to bond on this end…

…and a 5’ carbon free to bond on this end

Double strand of DNA

Each end is either the 5-prime or 3-prime end5’

3’

3’

5’

Label the phosphodiester bond, hydrogen bonds, 3’ end and 5’ end for both strands, bases, ribose sugar, phosphate group, 3’ carbon and 5’

carbon in ribose.

DNA packaging

DNA molecules are paired with a protein called histone

Histones help to package DNA

Essential as DNA can be 4cm long, it must fit into a microscopic nucleus

DNA packagingUnfolded DNA looks like beads on a string

These beads are nucleosomes

8 histones (proteins) make up a nucleosome core

DNA wraps round these histones twice(slight negative charge on DNA attracts to

positive charge of histones)

Between the nucleosomes are strings of DNA

DNA sequencesFrom the Multinational Genome Project we have learnt

that less than 2% of human DNA codes for proteins.

What does the other 98% do?

- Regulate gene expression- Telomeres (protects DNA)

- Introns (interruptions in coding region)

Types of DNA sequencesFind out what the following are:

- Protein-coding genes- Highly repetitive sequences

- Structural DNA- Short tandem repeats

Protein-coding genes

Highly repetitive sequences

Structural DNA

Short-tandem repeats

Protein coding genesGenes that have coding functions

Provide base sequence to produce proteins

Genes will have interruptions of non-coding regions in between them then must be spliced out before proteins

are made.

Coding regions = ExonsNon-coding regions = Introns

Highly repetitive sequences3-500 base pairs long in a repetitive sequence

It could go up to 100,000 repeats of a single unit

If it is clustered together – satellite DNA

So far not discovered any coding function (not genes)

Transposable – can move from one location to another

AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT

Structural DNAHighly coiled DNA with no coding function.

Near centromere and telomeres

Could have lost their function due to mutations involving a base sequence change

Short Tandem RepeatsYour DNA is almost identical to the person next to you

Specific regions are varied – polymorphisms

Analyse these using DNA profiling

Usually look at short tandem repeats

Short repeating sequences of DNA – 2-5 base pairs

T T T C C C T C A T C A T C A T C A T C C C GA A A G G G A G T A G T A G T A G T A G G G C

DNA replicationReplication starts as a bubble

Helicase breaks the hydrogen bonds between nucleotides

Two strands separate

At each end of the bubble there is a replication fork – where DNA strands open.

Bubbles enlarge in both directions – bidirectional

Bubble eventually fuse with one another to produce two identical daughter DNA molecules

DNA replication

1. Primer (short piece of RNA) is produced at the replication fork by primase

2. Primers match exposed DNA bases, marks the start of replication

3. DNA polymerase III allows addition of nucleotides in 5’ to 3’ direction

4. DNA polymerase I removes the primers from the 5’ end

Energy to create the bondsEach nucleotide molecule is a deoxynucleoside triphosphate (dNTP) molecule

Contains - Deoxyribose - A base- Three phosphate groups

As the molecules are added together to form nucleotides, two phosphates are lost

This provides energy needed for the nucleotides to bind

DNA replication

When DNA is replicated – it assembles 5’ to 3’ due to the action of polymerase III.

5’

3’

3’

5’

DNA replicationThis means that there is a difference in the assembly of DNA from the

templates

Leading strand – continuous and fast (5’ to 3’)Lagging strand – slowly (3’ to 5’)

DNA replication1. The leading strand assembled continuously in 5’ to 3’ direction2. Lagging strand is assembled by fragments produced moving away

from the replication fork – in the 5’ to 3’ direction3. Fragments of the lagging strand called Okazaki fragments 4. Primer, primase and DNA polymerase III are needed to begin both

the leading and lagging strand 5. Once Okazaki fragments assembled, DNA ligase enzyme attaches

them together to make a single strand

Formation of the lagging strand

1. 2.

3.

4.

5.

6.

Proteins in replication

What do they do?

Protein Role

Helicase

Primase

DNA polymerase III

DNA polymerase I

DNA ligase

Proteins in replication

What do they do?

Protein Role

Helicase Uniwnds double helixBreaks H bonds

Primase Synthesises RNA primer

DNA polymerase III Synthesises new strand by adding nucleotides onto the primer (5’ to 3’

direction)DNA polymerase I Removes primer

DNA ligase Joins Okazaki fragments

Complete for your homework

DNA sequencing

- What is DNA sequencing?- What is the process?- What is it used for?

DNA profiling

- What is DNA profiling?- What is the process?- What is it used for?- How is it different to DNA sequencing?