DNA and Chromosomes

Outline the cell cycle diagram

What does interphase consist of?

Gap Phase 1 (G1) - cell grows bigger and replicates its organelles. A high amount of protein synthesis is taking place in order to build new organelles.

Synthesis Phase (S) - the cell replicates its DNA

Gap Phase 2 (G2) - the cell keeps growing until all of the organelles have duplicated.

When does interphase occur?

Most of the time

6 stages of M phase

1. Prophase

2. Prometaphase

3. Metaphase

4. Anaphase

5. Telophase

6. Cytokinesis

In interphase, DNA is —- long and

DNA: 30 nm fibre

46 balls of unravelled string in nucleus

Show what happenes in the S phase

What happens in Prophase?

1. Chromosomes condense

2. Mitotic spindles form

What happens in Prometaphase?

1. Nuclear membrane disintegrates

2. Spindles attach to kinetochores

What happens in metaphase?

1. Chromosomes align at equator

What happens in anaphase?

1. Sister chromatids separate

2. Pulled towards spindle poles

What happens in telophase?

1. Chromosomes arrive at poles

2. Nuclear envelope reform

What happens in cytokinesis?

1. Cytoplasm divides resulting in two genetically near-identical cells

~X% cancers have mutations in p53

~50% cancers have mutations in p53

Which phase do chemotherapy drugs target?

Chemotherapy drugs target S and M phases – kill rapidly replicating cells

What is the cell cycle controlled by?

Controlled by cyclins and protein kinases (Cdks): phosphorylation of cdk/cyclin complexes

DNA replication is conservative or semi-conservative

DNA replication is semi-conservative

• each strand acts as a template

• 2 identical copies will be made → any mutation will be passed down

Which end are nucleotides added to?

3’ end

Which direction is DNA synthesised in>

5’ to 3’

How is energy provided for DNA replication?

Energy provided from breakage of triphosphate bond

Lagging strand – discontinuous synthesis

Okazaki fragments

Leading strand

1. DNA Helicase: Unwinds double helix

2. DNA Primase: adds small RNA primer

3. DNA Polymerase: binds, adds nucleotides to 3’ end (of leading strand)

Lagging strand

4. Exposed lagging strand protected by single-strand DNA binding proteins

5. DNA Primase: adds small RNA primer

6. DNA Polymerase: adds nucleotides to 3’ end (of lagging strand)

7. DNA Nuclease: removes RNA primers, DNA polymerase fills in

8. DNA Ligase: joins together small gaps

What does DNA topoisomerases do?

DNA topoisomerases (including gyrases) – also help unwind

Multiple subtypes of each class of enzyme

Why are syndromes caused and why are they dangerous? 

Mutations to mitosis or DNA replication genes usually lethal

Range of syndromes result from loss of minor components

What is Werner syndrome?

Werner Syndrome (1/200,000 in USA)

Premature aging disorder

Mutation in a DNA Helicase (WRN)

Errors in DNA replication and DNA repair

Increase in risk of cataracts, atherosclerosis, osteoporosis and cancer

Model for the aging process

Preventing the accumulation of mutations

1. Proof-reading capacity of DNA polymerase during DNA replication

2. Excision repair systems act throughout cell life repairing DNA damage

These systems reduce error during DNA replication from:

1 mistake in 105 nt without proofreading or repair

to 1 mistake in 109 nt with both

What is the internal source of DNA damage? 

Products of normal cell function

What is the external source of DNA damage?

Mutagenic chemicals (benzene, cigarette smoke)

UV

Ionising radiation

Excision repair systems

Most mutations in DNA repair are 

lethal

What is Xeroderma pigmentosum?

XP: mutation in UV repair

Unable to remove thymine dimers

Autosomal recessive disorder

Symptoms of XP

Acute sun sensitivity

Hypo- and hyper-pigmentation

Multiple cancers at young age

Intellectual disability

Progressive degeneration

Define mutation

Any large/small change to DNA sequence (good or bad)

Assumes a deviation away from ‘normal’

Low frequency (<1% population)

Associated with disease (might refer to as gene variants when discussing with patients)

Define SNP

Single nucleotide polymorphism (SNP)

Single base change in DNA sequence

Normal genetic variation in population

Synonymous: no change in amino acid sequence

Non-synonymous: change to amino acid sequence

Characterising mutations

Impact individual or offspring (somatic or germ line)

Scale of mutation (chromosome or SNP)

Effect on normal function (loss or gain of function)

Somatic or germ-line

Give an example of substitution mutation

Sickle cell anaemia - loss of function

Give an example of a deletion mutation

Cystic fibrosis - loss of function

Give an example of an intersertion

Hunington’s disease - gain of function

What is sickle cell

Single nucleotide substitution in HBB gene (beta chain of haemoglobin)

Misshapen blood cells do not survive as long (can cause anaemia) and clog up capillaries

What is cystic fibrosis?

Abnormal lung mucus in lungs, results in lung infections and difficulty breathing and digesting food

11,000 cases in the UK

Impaired chloride transport (loss of function)

Misfolded CFTR protein, unstable or not transported

70% of patients share 3bp deletion in CFTR gene on chr 7

(cystic fibrosis transmembrane conductance regulator)

Hungington’s Disease

Neurodegenerative disease (starts to appear age 30-50)

uncontrollable muscular movements

loss of memory and depression

difficulties with speech and swallowing

Damage of the nerve cells in areas of the brain

~7,000 cases in the UK

Hungington’s disease mutation

Caused by increase in number of CAG trinucleotide repeats (encoding glutamine) in the Huntingtin (HTT) gene

Polyglutamine residues stick together creating a toxic product (gain of function) which causes neuron cell death through multiple mechanisms

How is Hungington’s disease treated?

Use microRNA to bind to huntingtin mRNA – reduce gene expression

Infused deep into the brain using real-time MRI scanning and a microcatheter: 12-18h neurosurgery

Results not published yet