FUN Quiz 2 Flashcards
What type of replication is DNA replication?
Semi-conservative
What are the requirements for DNA Replication?
DNA Replication has the following requirements:
- Single-Stranded Template
- Deoxyribonucleotide Triphosphates (dNTPs)
- Replisome: Nucleoprotein complex that co-ordinates the replication activities. Numerous enzymes and proteins
- A Primer with a free 3’ end hydroxyl group
True or False?
Eukaryotes have multiple points of replication
True
Where or when is the replisome assembled
Origin of Replication
DNA Helicase
Separates DNA strands in an ATP-dependent process
Single-Strand Binding Proteins (SSB)
Binds to prevent strands from reassociating
Topoisomerase
Regulates twisting of the DNA Supercoiling.
It has nuclease and Ligase Activity
Nuclease
Cut DNA
Ligase
Bind DNA (backbone)
Describe the process of initiation in DNA Replication
Initiation: Involves the separation of two complimentary strands and occurs at the origin of replication which are specific points where DNA replication begins. Eukaryotes have several points of replication. From the origin, two replication forks move outwards in opposite directions. The replisome is assembled at the origin.
- DNA Helicase: Separates the DNA strands in an ATP-dependent process
- Single-Strand Binding Proteins (SSB): Binds to prevent the strands from re-associating
- Topoisomerase: Regulate twisting of the DNA supercooling. It has nuclease (cut DNA) and ligase activity (binding
RNA Polymerase
Makes RNA Primer
What is the Significance of RNA Polymerase in DNA Replication
DNA polymerase can only extend a chain and cannot synthesize DNA from scratch. After replication, the RNA primer is removed and the gap is filled
What are the general functions and roles of DNA Polymerase in DNA Replication
• DNA Polymerase:
- Uses single strand DNA as template
- Reads its template 3’ to 5’
- Makes new DNA from 5’ to 3’
- Aligns and adds nucleotides along the template strand with the correct pairing
- Catalyzes the formation of the phosphodiester bond
- Has Proofreading activity prevent errors. DNA polymerase’s active site can bind all 4 dNTP types. Catalysis occurs only when the correct one is bound. It also has proofreading with 3’-5’ exonuclease activity which removes nucleotides at the 3’ end of the new strand that are mismatched
What is PCNA?
- PCNA is a protein that acts as a sliding clamp which encircles the DNA template and keeps DNA polymerase closely associated to template allowing it to speed up the process
Do both strands in DNA Replication have the same process of Elongation? Explain
DNA replication occurs in two directions but DNA Polymerase can only work in one direction. Because of this, there is a leading and a lagging strand. The leading strand is replicated continuously whereas the lagging strand is replicated discontinuously. In the lagging strand, DNA is replicated into pieces of 1000bp Okazaki Fragments. This means that there are several primers, and DNA nuclease removes the RNA primers replacing it with the correct nucleotides (DNA pol), DNA ligase connects the backbone
What are the different types of DNA Polymerase
Alpha, beta, epsilon, delta, and gamma
Function of Alpha DNA pol
- Alpha a-DNA Pol is involved in initiating replication and associates tightly with primase to make the complex. Replicates DNA by extending primer 5’-3’. It has no Exonuclease activity
Functions of epsilon and delta DNA polymerases. What are the differences between them
- Epsilon e-DNA pol and Delta d-DNA pol: Not associated with primase, replicates DNA, highly processive, and has 3’-5’ exonuclease activity. eDNA pol works with the leading strand synthesis and d-DNA pol works with the lagging strand synthesis
Function of Beta DNA polymerase
Involved in DNA Repair
Function of Gamma DNA polymerase
Replicates mitochondrial DNA
Function of RNAse H1
Removes most of the RNA leaving one 5’ ribonucleotide adjacent to the DNA
Does DNA pol A have endonuclease activity?
No lol
Flap Endonuclease (FEN1)
FEN performs the endonuclease activity removing the last 5’ primer and up to 15 BP from the DNA synthesized by Pol a
When is DNA replication normally terminated
When each fork collides with the fork from the adjacent replicon
Briefly list the steps of DNA replication
Full Overview of DNA Replication: Helicase => SSB => Topoisomerase => RNA Polymerase => DNA pol a => DNA pol e & d with PCNA => Proofreading with DNA pol => Ligase => RNAse H1 => FEN1 => DNA pol e & d fill in gaps
Explain the process of primer removal in DNA Replication
Primer Removal: Involves 2 enzymes: Removes 5’ ribonucleotide. Pol a lacks proofreading so it cannot detect the error in the RNA primer.
- RNAse H1: Removes most of the RNA leaving one 5’ ribonucleotide adjacent to the DNA.
- Flap Endonuclease 1 (FEN1): FEN1 performs this endonuclease activity removing the lat 5’ primer and up to 15 BP from the DNA synthesized by Pol a
- DNA Pol d/e fills gaps
LEARNING OUTCOME: Describe and Explain the full process of DNA replication (Initiation, Elongation, Primer Removal, and Termination)
Initiation: Involves the separation of two complimentary strands and occurs at the origin of replication which are specific points where DNA replication begins. Eukaryotes have several points of replication. From the origin, two replication forks move outwards in opposite directions. The replisome is assembled at the origin.
- DNA Helicase: Separates the DNA strands in an ATP-dependent process
- Single-Strand Binding Proteins (SSB): Binds to prevent the strands from re-associating
- Topoisomerase: Regulate twisting of the DNA supercooling. It has nuclease (cut DNA) and ligase activity (binding)
Elongation:
• RNA Polymerase makes an RNA primer. This happens because DNA polymerase can only extend a chain and cannot synthesize DNA from scratch. After Replication, the RNA primer is removed and the gap is filled
• DNA Polymerase:
1. Uses single strand DNA as template
2. Reads its template 3’ to 5’
3. Makes new DNA from 5’ to 3’
4. Aligns and adds nucleotides along the template strand with the correct pairing
5. Catalyzes the formation of the phosphodiester bond
6. Has Proofreading activity prevent errors. DNA polymerase’s active site can bind all 4 dNTP types. Catalysis occurs only when the correct one is bound. It also has proofreading with 3’-5’ exonuclease activity which removes nucleotides at the 3’ end of the new strand that are mismatched.
7. PCNA is a protein that acts as a sliding clamp which encircles the DNA template and keeps DNA polymerase closely associated to template allowing it to speed up the process.
DNA replication occurs in two directions but DNA Polymerase can only work in one direction. Because of this, there is a leading and a lagging strand. The leading strand is replicated continuously whereas the lagging strand is replicated discontinuously. In the lagging strand, DNA is replicated into pieces of 1000bp Okazaki Fragments. This means that there are several primers, and DNA nuclease removes the RNA primers replacing it with the correct nucleotides (DNA pol), DNA ligase connects the backbone.
There are 3 main enzymes involved in replication
1. Alpha a-DNA Pol is involved in initiating replication and associates tightly with primase to make the complex. Replicates DNA by extending primer 5’-3’. It has no Exonuclease activity.
2. Epsilon e-DNA pol and Delta d-DNA pol: Not associated with primase, replicates DNA, highly processive, and has 3’-5’ exonuclease activity. eDNA pol works with the leading strand synthesis and d-DNA pol works with the lagging strand synthesis
Primer Removal: Involves 2 enzymes: Removes 5’ ribonucleotide. Pol a lacks proofreading so it cannot detect the error in the RNA primer.
- RNAse H1: Removes most of the RNA leaving one 5’ ribonucleotide adjacent to the DNA.
- Flap Endonuclease 1 (FEN1): FEN1 performs this endonuclease activity removing the lat 5’ primer and up to 15 BP from the DNA synthesized by Pol a
- DNA Pol d/e fills gaps
Termination: DNA Replication proceeds until each replication fork collides with fork from adjacent replicon.
Telomeres
Telomeres are at the 3’ end of each chromosome and it is made up of 1000s of tandem repeats
Telomerase
Telomeres are synthesized and maintained by Telomerase
What are the components of telomerase
Ribonucleoprotein made up of RNA and Protein
LEARNING OUTCOME: Outline the Role of Telomerase:
No need to include everything below as long as you include the main ideas
Termination: DNA Replication proceeds until each replication fork collides with fork from adjacent replicon. Continuous synthesis on leading strand can proceed to very tip of template but the lagging strand will have some missing nucleotides at the beginning when the primer is removed. As this is the lagging strand, the primer is removed but no OH group is available for DNA polymerase to add nucleotides. In normal removal of the primer, DNA pol would use the preceding fragment to fill in the gap but at the beginning there are no preceding fragments. This causes DNA to be shorter after every replication. These ends are called Telomeres.
Telomeres: telomeres are at the 3’ end of each chromosome and it is made up of 1000s of tandem repeats (TTAGGG in humans). Telomeres DNA is synthesized and maintained by Telomerase (Ribonucleoprotein make up of RNA + Protein). Telomerase’s RNA acts as a template for synthesis of DNA adding tandem repeats to the 3’ end. Telomerase activity is normal in gamete production and germ line cells. During development, as cells divide and differentiate, Telomerase function declines
When the telomeres become too short, it causes the cell to enter G0 or go for apoptosis or can develop into a cancer where there is uncontrollable division and hence shortening affecting coding sequences
Does the proof-reading activity of all endonuclease activity eliminate errors that could arise?
No lmao
What factors may damage DNA?
• Radiation: UV light - Fuse adjacent pyrimidines
• High Energy Radiation: Double stranded breaks
• Chemicals: Nitrous Acid - Deaminates amines turning C into U binding with A and A into Hypoxanthine binding with C
Most of this damage is repaired by the cell
What are the 5 mechanisms of DNA repair? List the names
Mismatch Repair Base Excision Repair Nucleotide Excision Repair Non-Homologous End-Joining (NHEJ) Recombination or Homologous Repair
Describe the mechanism of Mismatch repair
Mismatch Repair (MMR): Occurs shortly after replication and replaces mismatched bases or loops. First MUT S detects which is the Parental and which is the daughter strand through methylation (parental strand is methylated), then MUT L and PCNA form the Tetrameric Complex and cut the strand recruiting Exonuclease 1 and DNA polymerase. Exonuclease removes bases around the mutation and DNA polymerase rebuilds the copy strand. DNA Ligase then joins the backbone. Defects in human MMR causes a high chance of cancer, specifically HNPC (Hereditary Non Polyposis Cancer). This cancer affects MLH1 and MLH2 genes which produce MutL proteins
Describe the mechanism of Base Excision Repair
Base Excision Repair: Replaces bases lost though chemical processes such as Depurination (purines) or Deamination (Pyrimidines). DNA Glycosylase identifies and removes damaged base leaving an Apurinic or Apyrimidinic Site where an OH is now present. AP Endonuclease identifies the site and cuts the backbone and Exonuclease removes the sugar and several adjacent bases. DNA pol and Ligase then repair the issue