14.4: Prokaryotic Replication Flashcards
How does Prokaryotic Replication start?
- It starts at a single place= Origin
- The 2 separate replisomes are loaded onto the origin & start synthesis in the opposite directions on the chromosome & continue until they reach termination site
What is the Replicon?
-It controls the DNA at the Origin
What consists of Single Replicon?
-Chromosome + Origin (theres more in eukaryotic rep)
What are the 3 different polymerases that help in E.Coli replication?
- DNA Polymerase I
- DNA Polymerase II
- DNA Polyermase III
What are the key characteristics of all 3 of the DNA Polymerases?
- They all synthesize polynucleotide strands ONLY in the 5’-3’ direction & requires chi-che from a primer
- They also have 3’-5’ Exonuclease activity= proofreading for enzymes to back-up & remove mispaired base
What else can some Polymerases do?
-They can remove nucleotide=Nuclease
What are the 2 classifications for enzymes that can act as Nucleases?
- Endonucleases
- Exonucleases
What is Endonucleases?
-They are a class of enzymes that act as nucleases that cut out DNA internally
What is Exonucleases?
-They are a class of enzymes that act as nucleases that removes nucleotides from the end of the DNA
What is the role of DNA Polymerase III?
-It is the main replication enzyme & responsible for the bulk of DNA synthesis
What is the role of DNA Polymerase I?
-It acts on the lagging strand to remove primers & replace them w/ DNA
What is the role of DNA Polymerase II?
-This one is more involved in the DNA repair processes instead of replication
True or False: all polymerases are active in DNA replication
-False bc DNA Polymerase II is not since its more involved w/ DNA repair
What is the most efficient way to replicate DNA?
-If the helix is unwound ahead of the polymerase
What is a Helicase?
-It is the enzyme that unwinds the double helix
What does unwinding of the DNA require? what does it cause?
- DNA unwinding requires ATP
- It also causes torsional strain
Why would single strands after the unwound DNA be unstable? How would cells solve this problem?
- The hydrophobic bases are exposed to water
- Cells would solve this problem via Single-Strand-Binding protein (SSB)= coats the exposed strands
What is Supercoiling?
-The coiling in space of double-stranded DNA molecules due to torsional strain= occurs when helix is unwound
How do we describe the coiling of the double helix?
-Topological state of DNA
What are Topoisomerases?
-They are enzymes that act to relieve the torisional strain that is caused by unwinding & prevent this supercoiling from happening
What is DNA Gyrase
-It is the topoisomerase involved in DNA replication
What is a key characteristic of Replication?
-It is semi discontinuous
Why is DNA replication semi-discontinuous?
-Bc the polymerases can only synthesize in 5’-3’ direction= the leading strand can ONLY be synthesized continuously while the lagging strand must be made in pieces each w/ its own primer
What are the DNA fragments that are synthesized on the Lagging strand called?
-Okazaki fragments
What are the purpose of the Okazaki Fragments?
-They introduce a need for more enzymic activity on the lagging strand
What is it called when there is a partial opening of a DNA helix to form 2 single strands?
-Replication Fork
Where do all of the enzymatic activities occur?
-The Replication Fork
What is the role of DNA Ligase?
-It joins the ends of DNA segments & repairs DNA
What enzyme are the primers required by DNA polymerase during replication synthesized by?
-DNA Primase
What is the role of DNA Primase?
-It is an RNA polymerase that synthesizes short stretches of RNA 10-20 base pairs= function are primers for DNA polymerase
How is the leading strand synthesized?
- A single priming event is required
- Then the strand can be extended indefinitely by DNA Polymerase III
What is Processivity?
-It is the ability of a polymerase to remain attached to the template
What enzyme has high Processivity?
-Polymerase III
Why does Polymerase III have high Processivity?
-Because it consists of multisubunit & the high processivity is due to the “B subunit”
What does the B subunit of Polymerase III do?
- It forms a ring that encircles DNA= forms sliding clamp that keeps the polymerase attached to the template
- This mech can also be found in Eukaryotes
How does replication occur in the Lagging Strands?
- Primase (polymerase III) synthesizes the primers for each Okazaki fragments
- Then the RNA primers are removed & replaced w/ DNA by DNA Polymerase I
- Finally the fragments are stitched together by DNA Ligase
Where does Termination occur?
-It occurs at a specific site that is opposite of the oriC on the circular chromosome
What do the last 2 stages of replication produce?
-They produce 2 daughter molecules that are intertwined like 2 rings in a chain
What are the intertwined daughter molecules unlinked by?
-DNA gyrase= same enzyme that relieves torsional strain at replication fork
What do all the enzymes involved in DNA replication form?
-A macromolecular assembly= Replisome=forms “replication organelle”
What are the 2 subcomponents of the Replisome?
- Primosome (primase & helicase w/ other proteins)
- A complex of 2 DNA Polymerase III enzymes on each strand
True or False: The DNA Polymerase III enzyme are active on both strand simultaneously
- True!
- Because there is a loop formed on the lagging strand = allows the polymerases to move in the same direction
What is the Replisome complex held together by?
-By a number of proteins including clamp loader
What is the role of the Clamp Loader in the Polymerases III?
-It loads a B subunit onto the next fragment & transfers the Polymerase III to this new B subunit
Does the DNA replication complex move?
-No bc it is stationary= DNA moving through it like thread in a showing machine
What are the benefits of having a stationary Replication Complex?
-It pushes newly synthesized DNA outward= aids in chromosomes segregation
