DNA Replication Flashcards
How is DNA stored in cells. How does it work?
Stored in the cell as chromosomes.
How it works:
Nucleosomes: DNA wraps around histone proteins, forming nucleosomes. This packaging helps condense the DNA.
Chromatin: Nucleosomes further coil and fold to form chromatin, which can be in a loose (euchromatin) or tightly packed (heterochromatin) state.
Chromosomes: During cell division, chromatin condenses into distinct chromosomes, which ensures that DNA is accurately replicated and distributed to daughter cells.
Mitochondrial DNA: In addition to nuclear DNA, eukaryotic cells also contain DNA in mitochondria, which is inherited maternally and plays a role in energy production.
why is DNA stored in Chromosomes.
Structural Support: Chromosomes sturdy structure helps protect DNA from damage during cell division and other cellular processes.
Compact Shape: By coiling and folding, DNA can fit in the nucleus. This compact arrangement is crucial, especially in eukaryotic cells, where the amount of DNA can be quite large.
Gene Regulation: The way DNA is packaged affects gene expression. Tightly packed regions (heterochromatin) are less accessible for transcription (gene expression), while loosely packed regions (euchromatin) are
more accessible, allowing for more active gene expression.
where does DNA replication take place?
Happens in the S-phase of interphase.
What are the three stages of DNA Replication?
what is the main driver of DNA replication ?
Initiation
Elongation
Termination
MCM2-7 Helicase
DNA Replication - Initiataion
1- Origin Selection
- Replication origins are first selected by the Origin Recognition Complex (ORC).
- During S-phase, thousands of origins are selected throughout all chromosomes.
2- Loading of MCM2-7 Helicase:
- At each origin, the MCM2-7 helicase complex is loaded (complex is important for unwinding the double-stranded DNA)
- Two MCM2-7 helicases are loaded at each origin to facilitate bidirectional replication.
3- Activation of Helicases:
- The MCM2-7 helicases are activated by the binding of CDC45 and the GINS complex. This activation is crucial for helicase function to unwind DNA.
4- DNA Unwinding:
- Once the helicases are activated, they egin to unwind the DNA at the origin, creating a ‘replication bubble’. This bubble is where the DNA double strands separate into single strands, creating templates for new DNA synthesis (replication machinery).
5- DNA Synthesis:
- With the DNA unwound, the replication machinery, including DNA polymerases, is recruited to the replication forks. These enzymes synthesize new DNA strands by adding nucleotides complementary to the template strands.
The activation of origins is simultaneous. Explain why this coordination is important?
- allows rapid and efficient DNA replication.
- maintaining genomic integrity
- ensuring that each daughter cell receives an accurate copy of the genetic material.
DNA replication- Elongation
1) Formation of the Replication Fork:
- As MCM2-7 helicase unwinds DNA, a replication fork is formed. This fork consists of two single-stranded DNA templates available for replication.
2- Primase (an enzyme) Produces/synthesizes short RNA primers that are necessary for initiating DNA synthesis- they provide the starting point for DNA polymerases (DNA Synthesis).
3- DNA polymerases forms new DNA strands by adding nucleotides in a 5’ to 3’ direction-they can only add to the free 3’ hydroxyl (-OH) group of the growing strand.
4- DNA polymerase ε (epsilon) is responsible for creating the leading strand continuously as the DNA unwinds- moves along the template strand in the same direction as the replication fork.
5- DNA polymerase δ (delta) creates the lagging strand discontinuously. This strand is formed in short segments called Okazaki fragments, which are synthesized in the opposite direction to the replication fork movement.
6- Once the Okazaki fragments are formed, they are joined (ligate) together by DNA ligase. This enzyme seals the nicks between fragments, creating a continuous DNA strand on the lagging side.
what the role of Topoisomerase enzyme.
Topoisomerase enzyme works to relax supercoiling ahead of the fork.
As the DNA helix unwinds during replication, it can become supercoiled, which can hinder the progress of the replication machinery. Topoisomerases cut the DNA strands, allow them to unwind, and then rejoin them, thus preventing tangling and ensuring smooth replication.
There are two main types:
- Type I (topoisomerase I), which makes single-strand cuts.
- Type II (topoisomerase II), which makes double-strand cuts.
which enzyme is an RNA polymerase?
Primase- which creates the short RNA primers.
what are the two proteins involved in DNA replication?
Sliding clamp (PCNA)
Single-strand binding (SSB) protein - RPA
what is the role of PCNA?
Tethers (links) DNA polymerase to DNA.
By forming a ring around the DNA, it enhances DNA polymerases ability to synthesize long stretches of DNA without dissociating from the template strand.
The clamp is loaded onto DNA in an ATP-dependent reaction- facilitated by the complex called the clamp ladder which uses the energy from ATP hydrolysis.
RPA
RPA (replication protein A) binds to and stabilises unwound single-stranded DNA (ssDNA) during DNA replication and repair.
By coating the ssDNA, RPA protects it from degradation by nucleases and prevents the strands from re-annealing or forming secondary structures.
*Re-annealing refers to the process where two complementary single-stranded DNA molecules bind together to form a double-stranded structure. After being separated during processes like DNA replication or denaturation, the strands can spontaneously pair up again through hydrogen bonding, returning to their original double-helix form.
What is the name given to this DNA replication Machinery AND Summarise it .
The replisome.
- the complex of proteins and enzymes that orchestrates DNA replication at the replication fork. It includes several key components:
DNA Polymerases: These enzymes synthesize new DNA strands by adding nucleotides complementary to the template strands.
Helicase: This enzyme unwinds the double-stranded DNA, creating two single strands for replication.
Single-Strand Binding Proteins (like RPA): These stabilize the unwound single-stranded DNA to prevent it from degrading or re-annealing.
Sliding Clamp (PCNA): This protein enhances the processivity of DNA polymerases by tethering them to the DNA.
Clamp Loader: This complex loads the sliding clamp onto the DNA in an ATP-dependent manner.
Primase: This enzyme synthesizes short RNA primers needed for DNA polymerases to initiate synthesis.
DNA Replication - Termination
where does it happen?
DNA replication termination happens midway between two origins when two replication forks meet one another.
The replisome components (DNA polymerases, helicase…) are UNloaded from the DNA by proteins such as p97 or VCP (Valosin-Containing Protein), which help disassemble the replication complex.
Finally, DNA replication is complete, ending with two identical DNA molecules, each consisting of one old (template) strand and one new strand, reflecting the semi-conservative nature of DNA replication.
How are RNA primers laid down by Primase removed?
- eukaryotes
- prokaryotes
- replacement
- joining
In eukaryotic cells, the enzyme RNase H recognizes and removes the RNA primers
In prokaryotic cells, the enzyme DNA polymerase I has both polymerase and exonuclease activities. The exonuclease activity removes the RNA primers.
Once RNA primers are removed, the gaps are filled/replaced with DNA nucleotides by DNA polymerase.
The enzyme DNA ligase then seals the nicks in the sugar-phosphate backbone, to make sure the newly synthesised strands are continuous.
