Lecture 12 (DNA replication) Flashcards
Direction of DNA synthesis
DNA (or RNA) is always synthesised in the 5’ to 3’ direction (remember the 3rd carbon OH group). Thus the parental template strands are said to be/run in the 3’ to 5’ direction
What direction is the parental template strand said to be run in…?
The 3’ to 5’ direction
How many chromosomes in humans?
23 pairs therefore 46 chromosomes
Eukaryotic DNA replication
Multiple large linear chromosomes (23 pairs in humans)
Multiple origins of replication (replication doesn’t start at one end and go to the other, this would take too long, instead there are many start points along the chromosome)
Bidirectional (occurs in both directions)
Replication bubble
A replication bubble is an unwound and open region of a DNA helix where DNA replication occurs.
Origin of replication
Helicase unwinds only a small section of the DNA at a time in a place called the origin of replication. It is the place where the two strands are initially pulled apart (A-T rich region)
What is needed to make a DNA copy?
Progressive addition of new nucleotides (A,C,T or G)
A starting point for nucleotide addition
Unwinding of the helical double-stranded DNA (to give two parental templates)
Release of tension generated by unwinding the DNA helix
Prevention of unwound double-stranded helical DNA i.e. single-stranded DNA, from reforming and protecting it
Joining of ends of newly synthesised fragments together (lagging as well as leading strands)
DNA replication is …
Semi-discontinuous
Leading strand
The leading strand is a single DNA strand that, during DNA replication, is replicated in the 5’-3’ direction (same direction as the replication fork). DNA is added to the leading strand continuously, one complementary base at a time.
Lagging strand
A lagging strand is one of two strands of DNA found at the replication fork, or junction, in the double helix; the other strand is called the leading strand. A lagging strand requires a slight delay before undergoing replication, and it must undergo replication discontinuously in small fragments known as Okazaki fragments. The small fragments are synthesised in the 5’ to 3’ direction, when the fragments are added together there overall growth is the other way
Summary of leading and lagging strand
Leading strand - Continuously synthesised in its 5’ to 3’ direction
Lagging strand - Discontinuously synthesised in its 5’ to 3’ direction as Okazaki fragments
Okazaki fragments
Okazaki fragments are short sequences of DNA nucleotides which are synthesised discontinuously and later linked together by the enzyme DNA ligase to create the lagging strand during DNA replication.
Replication fork
The replication fork is the area where the replication of DNA will actually take place. There are two strands of DNA that are exposed once the double helix is opened. One strand is referred to as the leading strand, and the other strand is referred to as the lagging strand.
The more the replication bubble opens, the more…
The rest of the DNA twists/coils up
Primase
Primase is an enzyme that synthesizes short RNA sequences called primers. These primers serve as a starting point for DNA synthesis. Since primase produces RNA molecules, the enzyme is a type of RNA polymerase.
It does not make a primer of DNA nucleotides
The 3’ hydroxyl group is very important nucleotides together which will then react with the 5’ phosphate. At the very beginning, there is nothing to start from on the template strand so how do we get the initial 3’ hydroxyl group when there is nothing to start off with? This is when primate comes in - this enzyme has an internal 3’ hydroxyl group (inside the enzyme itself) and so it can start building nucleotides using its 3’ hydroxyl group.
Primase makes a primer that is a short stretch of RNA nucleotides which can act as a starting point for DNA or RNA synthesis by providing a 3’ hydroxyl group.
DNA polymerase III
Needs an OH group onto which the phosphate group of the incoming nucleotide can be attached
Only makes DNA in the 5’ to 3’ direction
Enzyme that synthesises a new DNA strand by adding nucleotides complementary to the parental template strands
Cannot bind to single stranded DNA and start copying it
Adds complimentary nucleotides, one by one. As it does this it knocks off the single-strand binding proteins
Topoisomerase
Topoisomerase also plays an important maintenance role during DNA replication. This enzyme prevents the DNA double helix ahead of the replication fork from getting too tightly wound as the DNA is opened up.
This is an enzyme that travels ahead of replication forks and cuts the tightly twisted DNA, allowing them to unwind and then stick them back together
Acts likes scissors and topoisomerase sticks it back together untwisted
Moves ahead of the replication fork and releases tension in the tightly wound DNA
Helicase
DNA helicase is the enzyme that unwinds the DNA double helix by breaking the hydrogen bonds down the centre of the strand. It begins at a site called the origin of replication, and it creates a replication fork by separating the two sides of the parental DNA.
Single-strand binding proteins
Single-stranded DNA-binding protein (SSB) binds to single-stranded regions of DNA.
During DNA replication, SSB molecules bind to the newly separated individual DNA strands, keeping the strands separated by holding them in place so that each strand can serve as a template for new DNA synthesis.
Prevents two parental DNA strands from coming back together before synthesis has finished because they are needed for template strands
The proteins also prevent the single strands of DNA from being degraded (enzymes in the cell might try ‘eat’ these single strands if the ssbp aren’t there)
DNA polymerase I
Removes RNA primers (RNase H) (recognises DNA/RNA hybrids and removes the RNA part and then uses the 3’ hydroxyl group from the other Okazaki fragment to fill the gap) and fills the gap with DNA nucleotides (DNA polymerase)
DNA ligase
Joins newly synthesised Okazaki fragments together (creates phosphdiester bonds), once the RNA primers have been removed and replaced by DNA nucleotides
Uses 3’ OH group and 5’ phosphate group to join newly synthesised fragments with strong phosphodiester bonds
Not only joins together the lagging strand (Okazaki) fragments together, but also the newly synthesised fragments from the multiple replication bubbles, including the leading strands
What are the two activities that DNA polymerase I carries out?
1 - RNase activity - RNase H is an endonuclease enzyme that recognises DNA:RNA hybrids and degrades the RNA part
2- DNA polymerase activity - synthesise DNA by adding nucleotides (complementary to the parental DNA template of the lagging strand)
DNA replication summary of steps
1- Helicase unwinds the parental double helix
2- Single-strand binding proteins stabilise the unwound parental DNA
3- The leading strand is synthesised continuously in the 5’ to 3’ direction by DNA polymerase
4- The lagging strand is synthesised discontinuously. Primase synthesises a short RNA primer, which is extended by DNA polymerase to form an Okazaki fragment
5- After the RNA primer is replaced by DNA (by another DNA polymerase), DNA Ligase joins the Okazaki fragment to the growing strand
How do you replicate a whole chromosome?
Replicaiton bubbles eventually grow into each other and thats how you replicate a whole chromosome with lots of replication bubbles
Progressive addition of new nucleotides (A,C,T,G)
DNA polymerase III
A starting point for nucleotide addition
Primase enzyme makes RNA primer
Unwinding of the helical double-stranded DNA to give two parental templates
Helicase
Release of tension generated by unwinding the DNA helix
Topoisomerase nicks and rejoins the DNA strands
Prevention of unwound double stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it from degradation
Single-stranded DNA binding protein
DNA polymerase I removes RNA primer (RNase H) and fills the gap with DNA nucleotides (DNA polymerase)
Joint of ends of newly synthesised fragments together (lagging as well as leading strands, within and between replication bubbles)
DNA ligase
When can DNA errors be repaired?
1 - DURING replication using an exonuclease (removes nucleotides from the end only)
2- AFTER replication using an endonuclease (can remove a chunk from within a DNA strand)
Exonuclease
an enzyme which removes successive nucleotides from the end of a polynucleotide molecule.
Endonuclease
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain so that it can remove chunks from within the DNA strand
Repair of DNA errors during DNA replication
DNA replication shows high accuracy (DNA polymerase III has a replication error rate of 1 in 10^8-10^10 base-pairs replicated)
DNA polymerase III has a proofreading mechanism - it checks the newly inserted nucleotide bases against the template (therefore this is occurring at the end of the strand)
These types of incorrect bases are removed by a 3’ to 5’ exonuclease activity of DNA polymerase III and then DNA synthesis can continue
Repair of DNA errors AFTER DNA replication
A variety of things can cause DNA damage or errors such as incorrectly inserted bases that are not corrected by DNA polymerase III, radiation damage (e.g. UV) and chemical modifications of bases (natural and chemical causes)
These types of incorrect or damaged nucleotide bases are removed by an endonuclease
1- Damaged/incorrect DNA
2- Damage, including some flanking regions, are removed by endonuclease (removes a large chunk as it does not have the fine motor skills to remove individual bases)
3 - A DNA polymerase makes a new DNA (uses 3’ hydroxyl group and extends from it)
4 - DNA ligase joins new DNA to existing DNA (creates phosphodiester bonds)
Importance of correcting DNA errors
If not corrected, the DNA error becomes part of the DNA template which leads to permanent DNA change i.e. DNA damage/mutation
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is a method widely used in molecular biology to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail.
In vitro DNA synthesis
In vitro method of making multiple DNA copies so that there is enough DNA material to work with
Only ‘targeted’ DNA region will be copied
Rapid exponential increase of DNA molecules
Method utilises cycles of heating and cooling
To analyse DNA, you need many DNA molecules therefore PCR technique is used
PCR applications
Medical applications
Forensic applications
Infectious disease detection and identifications
Molecular biology research applications
For example - COVID-19 is tested with a PCR swab. There are designed PCR primers which are specific for a particular region of the virus and if you are infected with the virus then the primers will bind to those specific regions of the virus only (nothing else in your body), and then you amplify the DNA from the virus and then you can measure and investigate it
How PCR works
Denaturation - temperature is increased to seperate DNA strands. Around 94-98 degrees and this is enough energy to break the hydrogen bonds that exist between the bases (the heat takes the role of helicase which would have been used in the body to break the hydrogen bonds)
Annealing - Temperature is decreased to allow remade primers to base pair to complimentary DNA template. Primers will bind when the temperature is lowered. Primers have the 3’ hydroxyl group from which the DNA polymerase can then extend from. Primers therefore define the region you are interested in
Extension - Polymerase extends primer to form nascent DNA strand
Repeat up to 35 times (exponential amplification occurs. As the process is repeated, and the region of interest is amplified exponentially)
Functions of PCR components used in ‘in vitro’ DNA replication
DNA template - DNA molecule to which complementary nucleotides can be matched to make identical copies via DNA synthesis
Primers- Provides a free 3’ OH group, the chemical group that is essential to initiate DNA synthesis. Defines the region of the DNA molecule that needs to be replicated
DNA polymerase - Enzyme which adds nucleotides, (complementary to the DNA template), and joins them together forming a phosphodiester bond
dNTPs - Free nucleotides (equal amount of A, G, C and T - the building blocks used by the DNA polymerase)
In vivo vs in vitro DNA replication - Primer
In vivo
RNA primer
Synthesised by primase
Removed and replaced by DNA nucleotides
In vitro
DNA primer
It is added completed
Part of newly synthesised DNA
In vivo vs in vitro DNA replication - DNA strands
In vivo
One continuous strand (leading), and discontinuous strand (lagging)
In vitro
Two continuous strands
In vivo vs in vitro DNA replication - Start
In vivo
At ‘origin of replication’ sites (there are multiple ori sites in eukaryotes)
In vitro
Two sites where DNA primers are designed to bind
In vivo vs in vitro DNA replication - Synthesis
In vivo
5’ to 3’ direction
All DNA (whole genome) is synthesised
In vitro
5’ to 3’ direction
Region between the primers is synthesised
In vivo vs in vitro DNA replication - Temperature
In vivo
One constant temp of 37 degrees
In vitro
Cycling of different temperatures between 48-98 degrees
In vivo vs in vitro DNA replication - Proteins and enzymes
In vivo
Several enzymes and proteins (DNA helicase, topoisomerase, single-stranded DNA-binding proteins, primase, DNA polymerase III, DNA polymerase I, DNA Ligase)
In vitro
One enzyme - heat stable DNA polymerase adding nucleotides (all other steps are regulated by temperatures)
