Chapter 12 Flashcards
Conservative Model
Entire DNA strands remain or are used as a template to synthesize new strands
Semi-Conservative Model
Each nucleotide gets conserved, this means that equal base paring in each strand
Explain Meselson and Stahl’s Experiment
Meselson and Stahl grew a culture of E.coli in a medium that contained N15 isotope of Nitrogen
They took a sample of these bacteria, switched the rest of the bacteria to a medium that contained only N14 nitrogen
Meselson ada Stahl distinguished between the heavy and light isotope by using an equilibrium density gradient centrifugation
A single strand band was produced after one generation that was intermediate of the two isotopes – this went against the conservative model
In the second generation, a N14 band and a hybrid band was formed – this went for the semi-conservative model and again the dispersive model
Hence, DNA semi-conservatively replicates
Replicon
A segment of DNA that undergoes replication
Origin of Replication
Each replicon contains and origin of replication, replication starts at the origin and continues until the entire replicon has been replicated
Theta Replication
A replication that occurs in circular DNA common in bacteria, which has an intermediate stage that looks like the greek letter theta
Replication Bubble
The unwinding of the double helix generates a loop known as the replication bubble
Replication Fork
The point of unwinding where the two strands separate
Bidirectional Bubble
In theta replication, where the two strands separate from the double helix
Who discovered theta replication?
John Cairns – who used an electron microscope technique to detect radioactive DNA replication
Rolling Circle Replication
https://www.youtube.com/watch?v=ZDqsojQ8A5k
Plasmids, Viruses, Bacteria Chromosomes
1. Initiator Protein repA binds to the double-stranded DNA at the origin of replication – ORI
2. RepA breaks one strand of the DNA
3. The RepA protein holds on the 5’-PO4- group
4. The 3’ end hydroxyl group serves as a primer for a host DNA polymerase
5. The DNA polymerase gets to replicate the intact complementary strand
6. The RepA protein recruits Helicase to unwind the DNA
7. The inside strand replicates
8. The outside strand gets cleaved off to form a circular DNA
9. The circular DNA replicates itself
10. You have accomplished two new replicated strands…
***For more info –> watch the Link above
Circular DNA molecules that undergo theta or rolling circle replication have a single origin of replication
This cycle may repeat
Explain why Linear Eukaryotic Replication happens faster
They have multiple origins of replication
1. Each chromosome contains numerous origins
2. At the origin, the DNA unwinds, producing a replication bubble
3. DNA synthesis takes place on both strands at each end of the bubble as the replication forks proceed outwards
4. Eventually, the forks of adjacent bubbles run into each other and the segments of DNA fuse..
5. Producing two identical linear DNA molecules
Characteristic of ___ replication
1. Theta
2. Rolling Circle
3. Linear Eukaryotic
Theta (circular DNA template, No breakage of nucleotide, 1 replicon, uni/bidirectional, two circular molecules as products)
Rolling Circle (circular DNA template, yes breakage of nucleotides, 1 replicon, unidirectional, and products include one circular and one linear molecule)
Linear Eukaryotic(Linear template, No breakage of nucleotide, many replicons, bidirectional, and two linear molecules as products)
What are the requirements of replication?
- A template consisting of single-stranded DNA
- Raw material – substrates – to be assembled into a new nucleotide strand
- Enzymes and other proteins that read the template and assemble the substrates
substrates - dNTPS
Enzymes – DNA polymerases, RNA polymerases, DNA gyrase, etc.
Which end are dNTPs added?
“The 3’-OH end”
“The 3’-OH group of the last nucleotide attacks the 5’phosphate on the incoming dNTP”
What bond is created between DNA nucleotides?
Phosphodiester bonds
Leading Strand
The template strand for the leading strand runs in the 3’ –> 5’ direction, and so the new strand can be synthesized continuously towards the replication fork in the 5’ –> 3’ direction
Lagging Strand
The template strands runs in the 5’—> 3’ direction, so the new strand must be built discontinuously in the 5’–> 3’ direction away from the replication fork
Okazaki Fragments
The short lengths of DNA when produced discontinuously on the lagging strand
What are 4 stages of bacterial replication?
Initiation, Unwinding, Elongation, and Termination
Initiator Proteins
DNAa – bind to OriC and cause a short section of the DNA to unwind
DNA Helicase
Breaks the Hydrogen Bonds that exists between the bases of the DNA molecules
**Helicase binds to the lagging strand only, and it moves in the 5’–> 3’ direction
**Helicase needs the help of initiator proteins too separate
Single-stranded binding proteins
Protect single stranded nucleotides, prevent the formation of secondary structures such as hairpins
DNA Gyrase
It relives tension ahead of the replication fork
AKA: Topisomerase II
Topoisomerase I
Alter the supercoiling by making single-stranded breaks
Topoisomerase II
Creates double-stranded breaks using ATP
Place the order to which the enzymes used in Initiation: DNA gyrase, helicase, initiator proteins, Single-stranded binding proteins
Initiator Proteins, Helicase, Single-Stranded Binding proteins, and DNA Gyrase
Primase
Synthesizes short stretches of RNA nucleotides or Primers…which provide a 3’-OH group to which DNA polymerase can attach DNA nucleotides
***new primer is required in each Okazaki fragment
DNA Polymerase III
Synthesizes DNA by adding nucleotides to the 3’ end of a growing DNA strand
When does DNA polymerase move 3’–>5’?
Exonuclease activity –> allows it to remove incorrect nucleotides in the 3’–> 5’ direction (proofreading)
B - beta sliding clamp
The ring-shaped polypeptide encircles the DNA and allows polymerase to slide easily along the DNA template strand during replication
This polypeptide is part of DNA polymerase enzyme
DNA Polymerase I
5’–>3’ polymerase and 3’–>5’ exonuclease which allows to synthesize and to correct DNA
***DNA POL I also possess 5’–>3’ exonuclease activity which is used to remove primers and replace them with DNA nucleotides synthesized in the 5’–>3’ direction
What are the functions of DNA polymerases?
- Synthesize any sequence specified by the template strand
- Synthesize in the 5’–>3’ direction by adding nucleotides to a 3’-OH group
- Use dNTPs to synthesize new DNA
- Require a 3’-OH group to initiate synthesis
- Catalyzes the phosphodiester bonds, and cleaves two phosphates from the end
- Produce newly synthesized strands that are complementary and antiparallel to the template strand
- Are associated with a number of proteins
DNA Ligase
DNA Ligase seals the gap with a phosphodiester bond between the 5’-phosphate group of the initial nucleotide aded by DNA polymerase III and the 3’-OH group of the final nucleotide added by DNA polymerase I
How many units of DNA Pol III at the replication fork?
Two for each strand
How does replication termination occur in E.coli bacteria?
- Specific termination sequence – Ter – blocks further replication
- A termination protein TUS binds to the TER sequence creating TER-TUS complex
- This complex blocks the movement of helicase
- The complex stalls the replication fork and prevents further DNA replication
- The complex only blocks the replication fork moving in one direction not the other
Explain the steps of proofreading
- DNA polymerase inserts an incorrect nucleotide into the growing strand
- The 3’-OH group is not correctly position in the active site of DNA Polymerase Enzyme
- The incorrect positioning stalls the polymerization reaction
- The 3’–>5’ exonuclease activity of DNA polymerase removes the incorrectly paired nucleotide
- DNA polymerase than inserts the correct nucleotide
Explain the steps of Mismatch Repair
- Any incorrectly paired nucleotide remaining after replication produce a deformity in the secondary structure of DNA
- The deformity is recognized by enzymes – DNA POL
- Removes incorrect nucleotide
- Uses the original template strand to replace the incorrectly paired nucleotide
- DNA is fixed!
Things to know:
***Occurs after replication, methyl groups are added to the original parent strand to make it easy to fix the new strand of DNA
Why are Eukaryotic chromosomes more challenging than prokaryotic chromosomes?
A) Bigger Genome
B) Linear Chromosome
C) Histone Proteins
Origin Recognition Complex – ORC
Serves as the initiator by binding to defined organs than are actually used during DNA synthesis
Difference Between Bacteria and Eukaryotic Origin of Replication
—> In Bacteria, helicase is unable to bind to the double-stranded DNA…initiator protein first melts DNA
—> In Eukaryotes, Initiator ORC recruits and loads helicase onto double-stranded DNA at the origin…takes place in G1…where in S…helicase activates
What is the telomeric DNA?
5’-TTAGGG-3’
G-rich 3’overhang
The single stranded protruding end of the telomere DNA
Telomerase
An enzyme that build telomeres…and is composed of RNA and protein
Explain The Activity of Telomerase
- The telomere has a protruding end with a G-rich sequence – TTAGGG
- The RNA part of telomerase is complementary to the G-rich strand and pairs with it
- Nucleotides are added to the 3’ end of the sequence
- The telomerase is removed
- Synthesis of the complementary strand occurs during replication
How does Initiating start in Eukaryotic Organism DNA replication?
- In GAP 1 - G1 - Initiator protein called ORC binds to the DNA double helix
- The Initiator protein recruits licensing proteins and helicase
- The whole complex recruits MCM2-7 complex
- Then in S-phase…these complexes help activate helicase to start doing replication
Replication Licensing System
It ensures that the DNA is not replicated again until after the cell has passed through mitosis
Autonomously replicating sequences – ARS
They enable DNA that were attached to them to replicate
What are the different DNA polymerases and their functions in Eukaryotic organism?
- DNA polymerase alpha – Primate activity and initiates nuclear DNA synthesis by synthesizing RNA primers
- DNA polymerase delta – completes replication of the lagging strand
- DNA polymerase epsilon – completes replication of the leading strand