dna stands for deoxyribose nucleic acid · • dna replication 5’->3’ • dna proof reading...

DNA stands for deoxyribose nucleic acid

This chemical substance is present in the nucleus

of all cells in all living organisms

DNA controls all the chemical changes which

take place in cells

DNA

DNA Structure

• Watson & Crick 1953

– Proposed the double helix shape of DNA and

received a Nobel Prize

• Maurice Wilkins and Rosalind Franklin

– Produced images of DNA using x-ray

diffraction

– Franklin passed before Nobel Prize was

awarded

Four requirements for DNA to be genetic material

Must carry information

– Cracking the genetic code

Must have the ability to be reproduced

– DNA replication

Must allow for information to change

– Mutation

Must govern the expression of the phenotype

– Gene function

DNA is a very large molecule made up of a long

chain of sub-units

The sub-units are called nucleotides

Each nucleotide is made up of

a sugar called deoxyribose

a phosphate group -PO4 and

an organic base

DNA molecule

Ribose is a sugar, like glucose, but with only five

carbon atoms in its molecule

Deoxyribose is almost the same but lacks one

oxygen atom

Both molecules may be represented by the symbol

Ribose & deoxyribose

The most common organic bases are

Adenine (A)

Thymine (T)

Cytosine (C)

Guanine (G)

The bases

The deoxyribose, the phosphate and one of the bases

adenine

deoxyribose

PO4

Combine to form a nucleotide

Nucleotides

A molecule of

DNA is formed

by millions of

nucleotides

joined together

in a long chain

PO4

PO4

PO4

PO4

sugar-phosphate

backbone+ bases

Joined nucleotides

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

2-stranded DNA

The bases always pair up in the same way

Adenine forms a bond with Thymine

and Cytosine bonds with Guanine

Bonding 1

Adenine Thymine

Cytosine Guanine

PO4

PO4

PO4

thymine

PO4

PO4

PO4

PO4

adenine

cytosine

PO4

guanine

Bonding 2

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

Pairing up

A

The paired strands are coiled into a spiral called

A DOUBLE HELIX

sugar-phosphate

chain

bases

THE DOUBLE

HELIX

Before a cell divides, the DNA strands unwind

and separate

Each strand makes a new partner by adding

the appropriate nucleotides

The result is that there are now two double-

stranded DNA molecules in the nucleus

So that when the cell divides, each nucleus

contains identical DNA

This process is called replication

Replication overview

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

The strands

separate

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

PO4

Each strand builds up its partner by adding

the appropriate nucleotides

Overview of Processes

• DNA replication 5’->3’

• DNA proof reading

• Lagging strand, back-stitching, Okazaki fragment

• Proteins involved:1. DNA polymerase,

primase

2. DNA helicase and single-strand DNA-binding protein (SSB)

3. DNA ligase, and enzyme to degrade RNA

4. DNA topoisomerases

5’ and 3’

ATP

dATP

Semi-Conservative DNA Replication

Step 1:

• Untwisting and Unzipping

• Helicase separates the strands by un-pairing

the bases

• How can you remember helicase?

• Replication Fork- Y-shaped region where

the parental strands of DNA are being

unwound

• Leading Strand- will be replicated

continuously

• Lagging Strand- will be replicated in

sections

• Template Strands- ?

Step 2

• Stabilization

• Single-Strand Binding Proteins- will bind to

unpaired DNA strands to keep them stable

Step 3

• Maintenance

• Topoisomerase- enzyme that breaks,

swivels and rejoins parental DNA strands

Step 4

• Initiation

• The enzymes that synthesize DNA cannot

start without a guide

• Primase- an enzyme that adds a strip of

RNA nucleotides to the parental strands

• This strip is approximately 5-10 nucleotides

long and is referred to as the Primer

• RNA Primers- point of initiation of DNA

replication

– Base pairs or nucleotides are similar with the

exception of Uracil.

– Uracil replaces Thymine and so, pairs with

Adenine

Uracil

Step 5

• DNA Synthesis

• DNA Polymerase III- starts at the primer,

adding bases in only one direction

• DNA Polymerase moves from the 5’ end

toward the 3’ end

Caveats of Elongation

• DNA has a defined 5’ and 3’ end which

gives it directionality

• DNA exists as antiparallel strands which

means the new strands must also be

antiparallel

• What’s the issue?

Leading Strand

• This template strand is called as such

because it is replicated continuously

• The DNA Polymerase binds to replication

fork at the primer and simply adds free

nucleotides to the leading strand until it has

completely synthesized a complimentary

strand.

Lagging Strand

• For the second template strand it is a bit

more complicated

• The DNA Polymerase must synthesize the

complimentary strand working away from

the replication fork

• **this must also be synthesized 5’ to 3’

• It is synthesized discontinuously

• Okazaki Fragments- the segment of the

lagging strand (100-200bp in eukaryotes)

Lagging Strand

• Thus, several primers are needed on the

lagging strand

• DNA Polymerase I- enzyme that will

replace the RNA primers

– Adds to the 3’ end of each Okazaki Fragment

– Exonuclease- detaches the RNA primers

• DNA Ligase- joins the sugar-phosphate

backbones of the strands together to make a

continuous strand

https://www.youtube.com/watch?

v=TNKWgcFPHqw

DNA Repair

• Errors occur during replication

• There is approximately 1 error in every 10

billion nucleotides after replication

• However, errors due occur during the

process and more frequently

– 1 in 100,000 bps

Proofreading

• While the base pairs are being added, DNA

Polymerase check to make sure they are

added correctly

• It is not a flawless proofread and correction

of these nucleotides is referred to as

mismatch repair

Proofreading

• Nucleotides can be incorrectly paired or

even altered after replication

• DNA has to frequently be repaired

• Chemical or physical agents can lead to

alterations of bps and warrant repair

• Reactive chemicals, radioactive emissions,

X-rays, UV rays and other molecules can

lead to nucleotides changes

– Often, these changes are repaired before

mutations can occur

Nuclease

• An enzyme called a nuclease will excise the

altered nucleotides

• Then other enzymes will replace those

nucleotides with the correct bps, and then

make the strand whole again

• This is done by using the unaffected strand

of DNA as a template

• This is an example of nucleotide excision

repair

Application

• UV light can lead to skin cancer

• UV light can catalyze the production of

thymine dimers

• This causes a covalent bond to form

between thymine nucleotides

Xeroderma Pigmentosum

• Xeroderma pigmentosum (XP) is a rare

autosomal recessive genetic disorder of

DNA repair in which the ability to repair

damage caused by ultraviolet (UV) light is

deficient

Problems with Replication

• Over the course of several replications, the

DNA faces some changes

• The Strands of DNA actually become

shorter

Telomeres

Prokaryote VS Eukaryote DNA Replication

Eukaryotes

• X-shaped Chromosome

• Semi-Conservative

• Replicates in Nucleus

• Many points of Origin

• Two templates

• Leading and Lagging

Strands

• At least 11 major

Polymerases

• 50 nucleotides/sec

Prokaryotes

• Ring-shaped Chromosome

• Semi-Conservative

• Replicates in Cytoplasm

• One point of origin

• One template

• Bidirectional and

Continuous

• 2 major polymerases

• 500 nucleotides/sec

Prokaryote DNA Replication

Prokaryote VS Eukaryote DNA Replication