Lecture 2 Flashcards
How many hydrogen bonds are there between A and T?
2
How many hydrogen bonds are there between C and G?
3
Which of the nitrogenous bases are purines?
Adenine and Guanine (double ringed structure)
Which of the nitrogenous bases are pyrimidines?
Cytosine and Thymine also Uracil
Where do the phosphodiester bonds in the DNA sugar-phosphate back bone form?
Between the phosphate group of one nucleotide and the carbon sugar of another nucleotide
What are some of the reasons that DNA needs to be packaged into chromosomes?
- ‘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
How does a piece of DNA get packaged into a chromosome?
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
Do prokaryotes have more or less DNA than eukaryotes?
They have less
What is important to remember about the shape of prokaryotic DNA?
It can be either circular or linear
What were the 3 models of DNA replication that were proposed and which model does experimental evidence support?
Conservative, Semi-conservative and Dispersive
The evidence supports the Semi-conservative replication
Where is replication of DNA initiated?
It starts at the ‘Origin of replication’ (oriC)
What is the Origin of replication?
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
How does DNA replication happpen?
- 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’
What is a replication fork?
A Y-shaped region of DNA that is created when DNA untwists, forming 2 single stranded templates
What direction does the replication occur?
It occurs in the direction of the replication fork towards the yet untwisted DNA
What happens when there are 2 replication forks and what does this cause?
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
What happens to the DNA at the end of the replication fork when the DNA at the other side is being unzipped?
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)
What happens when the supercoiling of the double stranded DNA is caused but the replication fork and what is used to resolve the problem?
The supercoiling causes tension in the DNA that needs to be relieve, this happens through the use of Topoisomerases
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?
- 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
How does Double stranded circular DNA become replicated?
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
What happens in linear (human) DNA that doesn’t happen in circular (bacterial) DNA?
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
What are some of the enzymes and proteins that are used when synthesising new DNA?
- 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
What does the synthesis of a new DNA strand start with?
It starts with the RNA synthesis, Primases create primosomes that synthesise RNA primers which are short sequences of RNA which compliment the DNA template
What does the RNA primer allow the the strand to do?
It allows for the addition of new nucleotides onto the parent strand of DNA
What happens when the nucleotides bind and start forming the new DNA strand?
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
Why is it important for there to be an RNA primer?
Because DNA polymerase III cannot act without it and so no DNA synthesis would be able to occur
Why does it have to be an RNA primer that is used?
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
What direction is DNA always synthesised in?
The 5’ to 3’ direction
What is the leading strand in DNA replication?
The strand of DNA that is being synthesised using one RNA primer and is synthesised continuously with new nucleotides (5’ to 3’ direction)
What is the lagging strand in DNA replication?
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
When the DNA polymerase III has synthesised the new strand of DNA what are you left with?
An RNA - DNA hybrid because of the RNA primer that was added at the start of the process
How do we get rid of the RNA primer at the start of the DNA strand?
DNA polymerase I produces exonuclease activity that digests the RNA primer
What does DNA polymerase I do?
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
How are the gaps between the nucleotides and the Okazaki fragments bound together?
An enzyme named DNA ligase comes and forms phosphodiester bonds between the nucleotides, creating a fully formed newly synthesised strand of DNA
