MTM - DNA and the Genome (Lecture 04)

DNA

What is the ‘central dogma’ of molecular biology?

1. DNA replication occurs

2. DNA transcribed into RNA

3. RNA translated into protein

What is an exception to the ‘central dogma’ of molecular biology?

A retrovirus using reverse transcriptase turning RNA back into DNA

Give two properties of RNA.

1. single-stranded with intra-molecular base pairing

2. heterogeneous in size

Give two properties of DNA.

1. double-stranded

2. high molecular weight

How are nucleotides joined together?

A 3,5 phosphodiester linkage

What are the three main differences between RNA and DNA?

1. RNA has an -OH group at ribose C2, DNA does not

2. RNA uses uracil whereas DNA uses thymine

3. RNA is unstable whereas DNA is stable

What is a chromosome?

A single molecule of DNA

What is a gene?

A specific stretch of DNA where the sequence contains genetic instructions

How are genes arranged?

Genes are arranged one after the other along a chromosome, with stretches of non-coding DNA between them.

How are mitotic chromosomes formed?

They are formed from tightly packed chromatin.

Give seven properties of the organisation of the human genome.

1. Contains 3.2 × 10 to the power of 9 base pairs - around 2m long in length

2. Contains around 19,000 protein coding genes

3. Contains 5000 non-protein coding genes (inc. structural, catalytic and regulatory RNAs)

4. Protein coding genes vary in size and internal organisation

5. Genes unevenly distributed between and within chromosomes

6. Several mRNAs can be transcribed from some genes

7. Genes can overlap on different strands of DNA molecule; some genes within introns of other genes

How many DNA bases and different chromosomes are found in the nucleus?

3.2 billion DNA bases and 24 different chromosomes

How many DNA bases and genes are found in the mitochondria?

16,600 DNA bases and 37 genes

How is DNA distributed and packaged in human chromosomes?

• DNA distributed between 23 chromosome pairs - 22 somatic and 1 sex

• DNA is packaged into chromatin by histones and other chromosomal proteins

How can structural differences in DNA be found?

using Giemsa staining of mitotic chromosomes

How can each chromosome pair be revealed?

Specific probes of different colours can be used to ‘paint’ chromosomes

What does ‘splicing’ machinery do?

Removes introns from primary RNA

What are the differences between exons and introns?

• Exons - coding DNA

• Introns - non-coding DNA

What are ‘promoter’ and ‘enhancer’ elements?

• promoter - includes specific DNA motifs where transcription factors and their complexes can access

• enhancer - DNA regions that amplify transcription initiation

How many promoter and enhancer elements are there per gene?

Typically several per gene

What is the percentage of the genome that is made up of repeated sequences?

50%

How much of the DNA sequence is identical between humans?

99.9%

How do repeated sequences often occur?

They often occur in blocks of tandem repeats. Often called ‘Satellite DNA’

What are ‘mini satellites’?

• They are highly polymorphic up to 1000 copies in one block

• Common core sequence: GGGCAGGAXG

• They are also found at telomeres

What are ‘micro satellites’?

• Small arrays of simple sequence repeats

• Usually in intergenic/intronic (non-coding) DNA

What are telomeres?

They allow replication to tip of chromosome.

What happens to long tracts of repeats and why?

They can be unstable so deletions occur immediately below telomeres.

What can ‘mini satellites’ cause?

They can cause mispairing during cell division to give:

• large scale duplication/deletion between homologous chromosomes

• translocation of DNA between non-homologous chromosomes

What two molecules are involved in the translation of mRNA?

tRNA and rRNA

What does ‘microRNA’ do?

Binds to 3’UTR of mRNA and inhibit translation

Which RNA molecules modify other RNA molecules?

snRNA and snoRNA

How much DNA does each human cell contain?

2 metres of DNA in a nucleus with 5 micrometres of diameter

What does detergent treatment do to a nucleus?

Causes release of decondensed chromatin threads from non-dividing interphase cells

What does chromatin do?

plays a role in DNA packaging and condensation

What is a nucleosome?

The fundamental unit of chromatin - this is DNA wound around a histone core

What is the organisation of decondensed chromosomes in non-dividing cells?

• Attached to the nuclear skeleton

• They have a functional role such as clustering ribosomal genes in the nucleolus.

What is the nucleolus?

A large structure within the nucleus where ribosomal RNA is transcribed and ribosomal subunits are assembled

What is euchromatin?

• Prevalent in gene-rich areas.

• It allows access for proteins involved in transcription as it is less compact.

What is heterochromatin?

It is a highly condensed region of an interphase chromosome that is generally gene poor and transcriptionally inactive.

What are the two types of heterochromatin? Give an example of each.

• Facultative - X-chromosome inactivation (not always inactive and condensed)

• Constitutive - centromeres (always inactive and condensed)

When can chromatin structure be modified and why is this?

During gene regulation to allow access to RNA polymerase and regulatory proteins

Where are nucleotides added during DNA replication?

The 3’ end

What molecules are required for DNA replication?

dNTPs (deoxynucleotide triphosphates)

In DNA replication, what does the template strand determine?

The sequence of the newly-synthesised strand

How and where is DNA replication initiated?

It is initiated at replication origins and these are recognised by origin proteins.

What are the five properties of DNA polymerase when it comes to DNA replication?

1. requires dNTPs (deoxynucleotide triphosphates)

2. proceeds in a 5’ to 3’ direction

3. adds 1000 bases per second to the chain

4. must have a template and a RNA primer to work

5. has ‘proof reading’ (editing) activity

What sort of movement does DNA replication follow?

A fork movement

What are the five steps of joining an old Okazaki fragment to a new one?

1. new RNA primer synthesis occurs by DNA primase

2. DNA polymerase adds to new RNA primer to start new Okazaki fragment

3. DNA polymerase finishes new DNA fragment

4. old RNA primer erased and replaced by DNA

5. nick sealing by DNA ligase joins Okazaki fragment to growing chain

What is the frequency of mistakes that DNA polymerase makes and why is this the case?

1 mistake per 10,000,000 base pairs replicated (high fidelity)

Why:

• stability of base pairing

• proof reading by DNA polymerase

What is the frequency of mistakes after the mismatch repair system and why is this the case?

1 mistake per 1 billion base pairs replicated as mismatch repair system corrects 99% of the errors made previously

What is a mutation?

A permanent change in the sequence of DNA

What happens when there are DNA mismatches?

1. A DNA mismatch repair protein binds to DNA mismatches and removes sections of the newly synthesised DNA strand

2. The gap is repaired using DNA polymerase and DNA ligase

Describe the three chemical reactions that damage DNA.

1. Depurination - A hydrolysis reaction which removes the purine from a nucleotide

2. Deamination - A reaction which causes the loss of an amino group from a DNA base

3. Thymine dimers can be formed from UV radiation as the two thymine bases can react together