Lecture 12 (DNA replication)

Question 1

Direction of DNA synthesis

Answer 1

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

Question 2

What direction is the parental template strand said to be run in…?

Answer 2

The 3’ to 5’ direction

Question 3

How many chromosomes in humans?

Answer 3

23 pairs therefore 46 chromosomes

Question 4

Eukaryotic DNA replication

Answer 4

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)

Question 5

Replication bubble

Answer 5

A replication bubble is an unwound and open region of a DNA helix where DNA replication occurs.

Question 6

Origin of replication

Answer 6

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)

Question 7

What is needed to make a DNA copy?

Answer 7

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)

Question 8

DNA replication is …

Answer 8

Semi-discontinuous

Question 9

Leading strand

Answer 9

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.

Question 10

Lagging strand

Answer 10

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

Question 11

Summary of leading and lagging strand

Answer 11

Leading strand - Continuously synthesised in its 5’ to 3’ direction

Lagging strand - Discontinuously synthesised in its 5’ to 3’ direction as Okazaki fragments

Question 12

Okazaki fragments

Answer 12

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.

Question 13

Replication fork

Answer 13

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.

Question 14

The more the replication bubble opens, the more…

Answer 14

The rest of the DNA twists/coils up

Question 15

Primase

Answer 15

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.

Question 16

DNA polymerase III

Answer 16

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

Question 17

Topoisomerase

Answer 17

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

Question 18

Helicase

Answer 18

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.

Question 19

Single-strand binding proteins

Answer 19

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)

Question 20

DNA polymerase I

Answer 20

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)

Question 21

DNA ligase

Answer 21

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

Question 22

What are the two activities that DNA polymerase I carries out?

Answer 22

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)

Question 23

DNA replication summary of steps

Answer 23

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

Question 24

How do you replicate a whole chromosome?

Answer 24

Replicaiton bubbles eventually grow into each other and thats how you replicate a whole chromosome with lots of replication bubbles

Question 25

Progressive addition of new nucleotides (A,C,T,G)

Answer 25

DNA polymerase III

Question 26

A starting point for nucleotide addition

Answer 26

Primase enzyme makes RNA primer

Question 27

Unwinding of the helical double-stranded DNA to give two parental templates

Answer 27

Helicase

Question 28

Release of tension generated by unwinding the DNA helix

Answer 28

Topoisomerase nicks and rejoins the DNA strands

Question 29

Prevention of unwound double stranded helical DNA, i.e. single-stranded DNA, from reforming and to protect it from degradation

Answer 29

Single-stranded DNA binding protein

DNA polymerase I removes RNA primer (RNase H) and fills the gap with DNA nucleotides (DNA polymerase)

Question 30

Joint of ends of newly synthesised fragments together (lagging as well as leading strands, within and between replication bubbles)

Answer 30

DNA ligase

Question 31

When can DNA errors be repaired?

Answer 31

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)

Question 32

Exonuclease

Answer 32

an enzyme which removes successive nucleotides from the end of a polynucleotide molecule.

Question 33

Endonuclease

Answer 33

Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain so that it can remove chunks from within the DNA strand

Question 34

Repair of DNA errors during DNA replication

Answer 34

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

Question 35

Repair of DNA errors AFTER DNA replication

Answer 35

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)

Question 36

Importance of correcting DNA errors

Answer 36

If not corrected, the DNA error becomes part of the DNA template which leads to permanent DNA change i.e. DNA damage/mutation

Question 37

Polymerase chain reaction (PCR)

Answer 37

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

Question 38

PCR applications

Answer 38

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

Question 39

How PCR works

Answer 39

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)

Question 40

Functions of PCR components used in ‘in vitro’ DNA replication

Answer 40

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)

Question 41

In vivo vs in vitro DNA replication - Primer

Answer 41

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

Question 42

In vivo vs in vitro DNA replication - DNA strands

Answer 42

In vivo
One continuous strand (leading), and discontinuous strand (lagging)

In vitro
Two continuous strands

Question 43

In vivo vs in vitro DNA replication - Start

Answer 43

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

Question 44

In vivo vs in vitro DNA replication - Synthesis

Answer 44

In vivo
5’ to 3’ direction
All DNA (whole genome) is synthesised

In vitro
5’ to 3’ direction
Region between the primers is synthesised

Question 45

In vivo vs in vitro DNA replication - Temperature

Answer 45

In vivo
One constant temp of 37 degrees

In vitro
Cycling of different temperatures between 48-98 degrees

Question 46

In vivo vs in vitro DNA replication - Proteins and enzymes

Answer 46

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)