Lecture 2

Question 1

How many hydrogen bonds are there between A and T?

Answer 1

2

Question 2

How many hydrogen bonds are there between C and G?

Answer 2

3

Question 3

Which of the nitrogenous bases are purines?

Answer 3

Adenine and Guanine (double ringed structure)

Question 4

Which of the nitrogenous bases are pyrimidines?

Answer 4

Cytosine and Thymine also Uracil

Question 5

Where do the phosphodiester bonds in the DNA sugar-phosphate back bone form?

Answer 5

Between the phosphate group of one nucleotide and the carbon sugar of another nucleotide

Question 6

What are some of the reasons that DNA needs to be packaged into chromosomes?

Answer 6

• ‘Naked’ DNA molecules are unstable within cells, packaging helps to protect the molecule from damage
• Compaction allows for the DNA to actually fit into the cell
• Allows for efficient DNA replication and transfer to daughter cells
• The organisation of the genome allows for regulation of gene expression

Question 7

How does a piece of DNA get packaged into a chromosome?

Answer 7

Positively charged proteins called histones bind to the negatively charged DNA molecule. Groups of these histones for a nucleosome, these get connected to each other through linker DNA which forms coils. These coils end up super coiling and fitting into the chromosome

Question 8

Do prokaryotes have more or less DNA than eukaryotes?

Answer 8

They have less

Question 9

What is important to remember about the shape of prokaryotic DNA?

Answer 9

It can be either circular or linear

Question 10

What were the 3 models of DNA replication that were proposed and which model does experimental evidence support?

Answer 10

Conservative, Semi-conservative and Dispersive

The evidence supports the Semi-conservative replication

Question 11

Where is replication of DNA initiated?

Answer 11

It starts at the ‘Origin of replication’ (oriC)

Question 12

What is the Origin of replication?

Answer 12

A specific region of double stranded DNA, that is denatured and separated into 2 single strands ready for DNA helicase to bind and start building up the new DNA strand

Question 13

How does DNA replication happpen?

Answer 13

• Initiator protein (DNaA Gene Protein) binds to the replicator sequence (OriC) denaturing the A and T rich regions as the strands stick to the the DNaA proteins
• DNA helicase (DNaB protein) binds to the OriC using a helicase loader protein, this ‘unzips’ the DNA in both directions away from the OriC, breaking H bonds via hydrolysis of ATP forming the ‘replication fork’

Question 14

What is a replication fork?

Answer 14

A Y-shaped region of DNA that is created when DNA untwists, forming 2 single stranded templates

Question 15

What direction does the replication occur?

Answer 15

It occurs in the direction of the replication fork towards the yet untwisted DNA

Question 16

What happens when there are 2 replication forks and what does this cause?

Answer 16

When there are 2 replication forks this is called a ‘replication bubble’ and this causes bi-directional replication towards both ends of the replication forks

Question 17

What happens to the DNA at the end of the replication fork when the DNA at the other side is being unzipped?

Answer 17

It causes some of the DNA to coil tighter outside of the replication bubble/fork (supercoiling in the still double stranded part of the DNA)

Question 18

What happens when the supercoiling of the double stranded DNA is caused but the replication fork and what is used to resolve the problem?

Answer 18

The supercoiling causes tension in the DNA that needs to be relieve, this happens through the use of Topoisomerases

Question 19

There are 2 Topooisomerases present during DNA replication, what are they and what do they do to relieve the strain and tension of the DNA?

Answer 19

• Topoisomerase I - Nicks the DNA on one strand, relieving the tension
• Topoisomerase II (DNA Gyrase) - Cuts double stranded DNA then rejoins it to relieve the built up tension

Question 20

How does Double stranded circular DNA become replicated?

Answer 20

Circular DNA (Like in Bacteria) has only one OriC and when replication is happening it always happens bi-directionally in a replication bubble instead of a single replication fork

Question 21

What happens in linear (human) DNA that doesn’t happen in circular (bacterial) DNA?

Answer 21

Linear DNA makes multiple sometimes hundreds and thousands of replication bubbles during replication, circular DNA only has one replication bubble as it only has one OriC

Question 22

What are some of the enzymes and proteins that are used when synthesising new DNA?

Answer 22

• Single strand binding proteins - They stabilise the single stranded DNA and stop the strands from ‘sticking’ back together
• Primase - To prepare the DNA strand for DNA polymerase
• DNA Polymerase III - This synthesises the new strand of DNA

Question 23

What does the synthesis of a new DNA strand start with?

Answer 23

It starts with the RNA synthesis, Primases create primosomes that synthesise RNA primers which are short sequences of RNA which compliment the DNA template

Question 24

What does the RNA primer allow the the strand to do?

Answer 24

It allows for the addition of new nucleotides onto the parent strand of DNA

Question 25

What happens when the nucleotides bind and start forming the new DNA strand?

Answer 25

With every nucleotide that binds, 2 phosphates are lost as a pyrophosphate molecule. The Pyrophosphate is then hydrolysed into 2 separate molecules, this drives polymerisation

Question 26

Why is it important for there to be an RNA primer?

Answer 26

Because DNA polymerase III cannot act without it and so no DNA synthesis would be able to occur

Question 27

Why does it have to be an RNA primer that is used?

Answer 27

The RNA molecule contains ribose sugar, which has an extra hydroxyl group compared to deoxyribose. This - OH group is utilised by DNA polymerase III to bind new nucleotides and extend the strand

Question 28

What direction is DNA always synthesised in?

Answer 28

The 5’ to 3’ direction

Question 29

What is the leading strand in DNA replication?

Answer 29

The strand of DNA that is being synthesised using one RNA primer and is synthesised continuously with new nucleotides (5’ to 3’ direction)

Question 30

What is the lagging strand in DNA replication?

Answer 30

The strand of DNA made up of discontinuous replication of around 100bp each in eukaryotes as the DNA cannot be synthesised in the 3’ to 5’ direction , these discontinuous regions are called Okazaki fragments

Question 31

When the DNA polymerase III has synthesised the new strand of DNA what are you left with?

Answer 31

An RNA - DNA hybrid because of the RNA primer that was added at the start of the process

Question 32

How do we get rid of the RNA primer at the start of the DNA strand?

Answer 32

DNA polymerase I produces exonuclease activity that digests the RNA primer

Question 33

What does DNA polymerase I do?

Answer 33

DNA polymerase digests the RNA primer sequence and replaces it with DNA nucleotides, however this creates a gap between the already synthesised strand and the newly synthesised replacement for the RNA primer

Question 34

How are the gaps between the nucleotides and the Okazaki fragments bound together?

Answer 34

An enzyme named DNA ligase comes and forms phosphodiester bonds between the nucleotides, creating a fully formed newly synthesised strand of DNA