D1.1 DNA Replication Flashcards
What is DNA replication?
The production of exact copies of DNA with identical base sequences
Outline the purposes of DNA replication
Growth: involves the addition of new cells to make an organism larger, each new cell requiring a complete copy of the organisms DNA through DNA replication that must occur prior to cell division.
Reproduction
Repair: new cells need to be produced to replace the damaged/destroyed ones, DNA replication occurs before cell division. As they need to be exactly like the ones around them, they also need to be differentiated.
Describe what is meant by semi-conservative nature of DNA replication
1) As the double strands of DNA come apart, each strand acts as a template for DNA replication
2) The new stands are synthesised by adding nucleotides one by one and linking them together
3) When replication is complete each new double strand synthesised will contain an original stand of DNA
What is the role of complementary base pairing in DNA replication?
To maintain and conserve the correct sequence of bases in the molecule, ensuring newly synthesised strands are exact copies of the original (ensures genetic code remans intact between generations).
Complementary bases form H-bonds which stabilise the structure.
(A-T, C-G)
What experiment proved that DNA replication is semi-conservative?
Meselson and Stahl differentiated between different DNA theories by using a density-labelling method to track DNA replication
They found that each newly replicated DNA molecule consisted of one old and one new strand, thus confirming the semi-conservative model of DNA replication.
Why must DNA strands be separated prior to replication?
The two strands of the parent DNA molecule must separate so they can each serve as a template for the new DNA strands being built.
What is the role of helicase in DNA replication?
The enzyme helicase unwinds the DNA double helix and separates the two strands by breaking H-bonds between the bases.
ATP is used to expose these bases that are usually protected within the molecule.
What is the role of DNA polymerases in DNA replication?
Once strands are separated and bases exposed, the enzyme DNA polymerase moves along the separate DNA strands (towards the replication fork), using them as templates.
Then, it will begin building the new strand of DNA by placing and attaching fee nucleotides in a chain.
[Overall is responsible for synthesis of DNA molecules during DNA replication]
Are there seperate DNA polymerases for each strand of template DNA?
Yes
What is the polymerase chain reaction (PCR)?
A technique used to amplify and separate small fragments of DNA and copy these specific sections in a cell sample.
Outline the 3 step process of the PCR
In a thermal cycler temperature variations are used to control DNA replication:
- Denaturation— temperature is increased (98degreesC) to separate the DNA strands
- Annealing—temperature is decreased (55degreesC) allowing RNA primers (short RNA sequences) to serve as starting points for DNA polymerase, attaching to the 3’ ends of the target sequence
- Elongation— a heat resistant DNA polymerase (Taq) binds to the primer and copies the strand by adding free nucleotides (72degreesC/2mins)
What is Taq polymerase and why is it so important?
A heat-resistant DNA polymerase frequently used in the PCR.
It is used because the enzyme can withstand high temperatures without denaturing that are required in the PCR.
Outline the function of the 4 components needed to carry out the PCR: Primers, Taq polymerase, Nucleotides, Buffer/cofactors
DNA template—
Primers— short RNA sequences that serve as a starting point for DNA syntheses by Taq
Taq polymerase— heat-resistant enzyme that synthesises DNA
Nucleotides— many and free, used by Taq to synthesise new DNA strands
Buffer/cofactors—maintain pH balance of the mixture for optimal enzyme activity
Deduce the number and relative size of DNA fragments from the number of bands in an electrophoresis gel
What is gel electrophoresis?
A technique used to separate and analyse DNA fragments based on their size and charge using an electric field and gel matrix.
Outline the 3-step process of DNA electrophoresis
[Before GE is carried out a DNA sample must be amplified using PCR]
- DNA is then cut using restriction enzymes, which act like molecular scissors, cutting the DNA at specific points
- DNA samples are placed in a well on a gel acting as a molecular sieve/filter (gel consists of mesh filaments)
- The sugar phosphate backbone of DNA is negatively charged: once the gel is immersed in a connecting fluid, electricity is applied and DNA samples move towards positive electrode
- The movement of the samples and distance is restricted by molecular size of DNA —> the smaller the fragments, the furthest down the gel
How and why do DNA fragments separate during electrophoresis?
The gel is porous, and the DNA must travel through the spaces within the gel.
Smaller pieces can slip through the spaces more easily, allowing them to travel further along the gel in a given amount of time.
Usually, one or more of the wells is filled with a ‘DNA ladder’, which contains DNA fragments with a range of known lengths.
By using the DNA ladder, the length of sample fragments can be determined.
List the applications of PCR and electrophoresis
Viral infection testing— eg. Covid testing
DNA profiling—a technique that examines variable portions of DNA to create a profile or ‘fingerprint’ that is unique to the individual.
[Every cells contains our entire genome, and we shed cells continually, leaving them in the environment around us]
What are the advantages (3) and disadvantages (3) of the use of PCR to test for viral infection?
Advantages— highly specific, high sensitivity, rapid detection
Disadvantages—high cost, technical complexity, does not differentiate between active and inactive infections
Outline the process of DNA profiling
Most genomes have short, repeated DNA sequences called tandem repeats (they do not code for proteins: STRs&VNRs).
Number of repeats vary between individuals in a population, allowing for a specific fingerprint for every individual.
What are VNTRs and STTRs?
Sections of DNA that are repeated a number of times (AGGAGGAGGAGG)
Outline the process of DNA profiling with VNTRs
Each individual inherits 2 alleles from every gene (or VNTR) they receive from both parents, this allows for no one having the same VNTR combination.
The combination of alleles is the basis of DNA profiling as VNTR shows variations between individuals in terms of the number of times the sequence is repeated.
Outline the process of DNA profiling with STRs
STRs are specific sites in the DNA where highly variable numbers of a short, repeated sequence are found.
An individual inherits one copy of STR from each parent, giving either a heterozygous or homozygous genotype, depending on whether there are similar repeat sizes.
Typically used and compared to create the genetic fingerprint of an individual.
What are the 5 steps for making a DNA profile?
- DNA sample is collected (blood, semen, saliva), extracted and amplified using PCR
- Satellite DNA are cut with a specific restriction enzymes to generate fragments
- DNA fragments are separated using gel electrophoresis
- Fragments length will differ between individuals due to the variable length of STRs
- A pattern of bands of DNA is produced on the gel— this is the individuals DNA profile and can be compared with other samples using a DNA ladder
What is a phosphodiaster bond and when does it occur?
Nucleotides are held together by these bonds.
The ester bond that forms between the phosphate group of one nucleotide and the pentose sugar of the adjacent nucleotide.
Occurs when 2 of the OH groups in phosphoric acid react with the OH groups on other molecules to form 2 ester bond.
What is meant by the 5’ and 3’ ends of the DNA double strand?
The carbon atom with a free OH group on the pentose sugar (C-3) marks the 3’ end.
The carbon atom to which the phosphate group is attached (C-5) marks the 5’ end.
Why is the directionality of DNA polymerases 3’ to 5’?
When assembling a new strand of DNA, DNA polymerase III adds the 5ʹ end of a DNA nucleotide to the 3ʹ end of the previously added nucleotide.
Like other enzymes, DNA polymerase III has an active site that is complementary to only a very specific shape.
This is why it is only able to build new DNA strands in a 5ʹ to 3ʹ direction. If it worked in the other direction, the shape would be different.
Why is directionality important for the DNA polymerase?
DNA polymerases can only add the 5’ phosphate of a free nucleotide to the 3’ deoxyribose of the elongating strand.
The formation of a single strand of DNA involves repeated condensation reactions between nucleotides.
Why are nucleotides added only on the 3’ OH group of a nucleotide?
Once the DNA polymerase has recognised the 3’ end, the formation of a covalent bond occurs between the previous nucleotide and the free one.
DNA polymerase cannot initiate the process of DNA replication without the 3’ end of the free nucleotide can attach to.
Why is replication different on the leading and lagging strands of DNA?
Due to DNA’s antiparallel arrangements, and the directionality 5’ to 3’, replication occurs in two different directions from the origin and on both strands:
The strand that can be replicated in the same direction as helicase, towards the replication fork, moves is called the leading strand. This strand is orientated from 3ʹ to 5ʹ and can be replicated continuously.
The other strand must work in the opposite direction, this strand is known as the **lagging strand ** and the replication along this strand is discontinuous.
Compare the pace and direction of replication on the leading and lagging strands of DNA,
Leading: towards replication fork, continuous process, only use of DNA polymerase III
Lagging: away from replication fork, added in a series of Okazi fragments (discontinuous), 4-step process
Outline the 4-step process of lagging
- Primer
- DNA polymerase III
- DNA polymerase I
- DNA ligase
Outline the 4-step process of lagging
- Primer
- DNA polymerase III
- DNA polymerase I
- DNA ligase
What are Okazaki fragments?
Short DNA fragments that are synthesised on the lagging strand during DNA replication and later joined together using DNA ligase.
What is the function of the enzyme helicase?
Unwinds and unzips the DNA molecule by breaking the H-bonds that hold the complementary base pairs together
This forms the replication fork.
What is the function of RNA primase?
Attaches small RNA primers to the template strand, allowing DNA polymerase III to begin its function.
In the lagging strand, primers need to be placed at regular intervals (vs only once in leading).
What is the function of DNA polymerase III?
Synthesises the new strands of DNA by placing free nucleotides in the 5’ to 3’ direction.
What is the function of DNA polymerase I?
Removes the RNA nucleotides of the primers and replaces them with the correct DNA nucleotides.
What is the function of DNA ligase?
Catalyses the formation of the covalent (phosphodiester) bonds between the Okazaki fragments.
This makes the replicated strand built using the lagging strand into a single strand that can function normally.
Why are there are gaps between adjacent Okazaki fragments on the lagging strand?
DNA polymerase can only synthesize DNA in the 5’ to 3’ direction.
As a result, the lagging strand, which runs in the 3’ to 5’ direction, is synthesized discontinuously in short segments.
What is the function of DNA proofreading?
DNA polymerase III additionally proofreads the newley formed DNA strand as it is being built.
If a nucleotide is placed with a mismatched base, the incorrect nucleotide is removed and replaced with the correctly matching one.
This is a way to prevent potential mutations.
What is meant by the 5’ and 3’ ends of the double strands?
Carbon atoms in a nucleotide have a defined sequence and are numbered from 1-5.
The carbon atom to which the phosphate group is attached is called C-5 and marks 5’ end.
The carbon atom with a free OH group on the pentose sugar C-3 marks 3’ end.
What are 2 reasons for why directionality of DNA is important for the DNA polymerase?
New nucleotides can only form at the 3’ OH end.
The energy for the attachment of a new nucleoside triphosphate (the base) comes from the hydrolysis of the 2 phosphate groups that are located on the 5’ end of the new base.
What type of bonds are formed between the previous nucleotide and the free one?
Covalent (phosphodiaster) bonds
Why is replication different in the leading and the lagging strand?
Because DNA is antiparallel, replication occurs in two different directions from the origin (replication fork).
This causes replication to be continuous in one strand (leading) and discontinuous in the other (lagging).
Leading strand direction vs lagging strand direction
Leading— towards replication fork, synthesised continuously in the 5’ to 3’ direction as helicase unwinds and unzips the DNA.
As the replication fork opens up, DNA polymerase III attaches and begins synthesising the new strand in the same direction as the unwinding.
Lagging— away from replication fork, runs in the 5’ to 3’ direction, can’t be replicated in one piece like the leading strand. Instead, it’s replicated in segments known as Okazaki fragments.
How are Okazi fragments formed on the lagging strand?
Short DNA sequences synthesised discontinuously on the lagging strand.
Their formation begins when RNA primase synthesises a short RNA primer.
DNA polymerase III extends the primer, creating an Okazaki fragment.
After synthesis of Okazaki fragment, DNA polymerase I replaces the RNA primer with DNA, and DNA ligase connects the fragments, creating a continuous DNA strand.
How are Okazi fragments formed on the lagging strand?
Short DNA sequences synthesised discontinuously on the lagging strand.
Their formation begins when RNA primase synthesises a short RNA primer.
DNA polymerase III extends the primer, creating an Okazaki fragment.
After synthesis of Okazaki fragment, DNA polymerase I replaces the RNA primer with DNA, and DNA ligase connects the fragments, creating a continuous DNA strand.
What does antiparallel in DNA mean?
One strand runs in the 3’ to 5’ direction, while the other runs in the 5’ to 3’ direction.
What does antiparallel in DNA mean?
One strand runs in the 3’ to 5’ direction, while the other runs in the 5’ to 3’ direction.
DNA helicase
It catalyses the separation of the double helix structure of DNA by breaking the hydrogen bonds between the base pairs.
This action creates a ‘replication fork’ - a Y-shaped structure from where the replication begins.
DNA Polymerase
Responsible for creating new DNA strands by adding complementary nucleotides along the template strands.
It moves along the template strand, matching the base on the template with a complementary base and then linking the complementary bases together to form the new strand.
The polymerase chain reaction (PCR) technique, inspired by this natural process, is a powerful tool used in molecular biology to amplify DNA sequences.
Why are Osaka fragments formed on the lagging strand?
Because DNA polymerase can only synthesize DNA in a 5’ to 3’ direction.
As the replication fork progresses, it exposes sections of the template strand in a 3’ to 5’ direction.
Since DNA polymerase cannot synthesise in this direction, it starts at different points as new sections are revealed, creating separate fragments of DNA, known as Okazaki fragments.
Explain the need for RNA primers in DNA replication
(RNA primers are crucial for initiating DNA synthesis.
An enzyme called RNA primase synthesises these short RNA sequences complementary to the DNA template strand.
The RNA primer provides a free 3’ OH group, which is a prerequisite for the DNA polymerase to add nucleotides.)
RNA primers lay the foundation for DNA synthesis to commence. —> Once the primer is in place, DNA polymerase adds DNA nucleotides to it, and eventually, another enzyme replaces the RNA nucleotides of the primer with DNA nucleotides.
Compare the number of RNA primers on the leading and lagging strands.
Leading— many RNA primers
Lagging— 1 RNA primer
Outline the function of the enzyme DNA primase?
Begins the replication process
Outline the function of the enzyme DNA polymerase III
Assembles the components of cases in 5’ to 3’ direction
Outline the function of the enzyme DNA polymerase I
DNA polymerase I functions to fill DNA gaps that arise during DNA replication, repair, and recombination.
State the function of DNA proofreading
State the function of DNA proofreading
Outline the process of DNA proofreading by DNA polymerase III
Outline the process of DNA proofreading by DNA polymerase III
