10.2 DNA Replication (Prokaryotes) Flashcards
T/F: The main mechanisms of DNA replication are shared in all living organisms
True
What is the main difference between DNA replication in bacteria and eukaryotes?
- Because bacteria contains circular genomes, they need mechaisms to create closed circles of DNA.
- Eukaryotes contain linear chromosomes so they do not have to do this
What are the three phases of DNA replication (in order)?
- Initiation
- Elongation
- Termination
What are the steps/goal/end product of the initiation phase of DNA replication?
- Steps: The packaging of DNA in chromosomes is unwound, a starting place for replication is found, and the replication fork is built.
- Goal: create single stranded template DNA so that replication can begin
- End product: replication forks
What is the goal/end product of the elongation phase of DNA replication?
- Goal: To replicate the template DNA
- End product: linear dsDNA
What is the goal/end product of the termination phase of DNA replication?
- Goal: stop replicating DNA
- End product (prokaryotes): circular DNAs
vocab (definition)
Topoisomerases
enzymes that participate in the over/underwinding of DNA
initiation
vocab (definition)
Gyrase
A topoisomerase that removes positive supercoiling in a process requiring ATP
initiation
vocab (definition)
quinolones
Class of antibiotics that prevent bacterial replication by targeting the ATP binding sites of gyrase
initiation
In bacteria, how is DNA compressed?
through supercoiling
Initiation
The first step of initiation is to unpack the DNA. What is required for this process? How is this step completed?
- Gyrase/ATP/template DNA
- Gyrase removes positive supercoiling. First, gyrase binds to the supercoiled loop of DNA, then cuts through the dsDNA and unwinds one of the loops. This shifts the DNA from one domain of the enzyme to the other. Then, gyrase glues the ends back together
Initiation
Why is the first step of DNA replication to unpack the chromatin?
So that replication machinery can find and access ori sequences
Initiation
What can happen once the DNA is unwound in initiation phase?
proteins involved in DNA replication can access the DNA
Initiation
How many ori sites on circular (prokaryotic) chromosomes
one
Initiation
What do ori sites contain?
- AT-rich regions (3 replicated regions, proceeds the DnaA box)
- DnaA recognition sequence/box (9 bp sequence)
- GATC sequences (interspersed throughout the ori sequence, Cs are methylated to help proteins find the ori sequence)
Initiation
What is DnaA and what does it do?
- the DNA binding protein that scans the genome looking for Dna boxes where it can bind to
- When DnaA binds to dnaA box, it causes the dsDNA to bend. Several DnaA proteins bind which causes significant bending and physical stress (tension to the “B” structure of DNA)
Initiation
What happens as a result of DnaA proteins creating tension to the “B” structure of DNA?
- H bonds are broken between the AT base pairings in the AT rich region of the ori site
- This separates the dsDNA, so now ssDNA is exposed to act as templates for replication
vocab (definition)
Helicase (DnaB)
- enzyme (motor protein) that unwinds dsDNA, separating the dsDNA to expose more ssDNA for its replication
- moves in 5 prime to 3 prime direction (so each are going in opposite directions)
- multimeric complex shaped like a donut
initiation
Initiation
What loads helicase onto the single strands of Dna?
Dna helicase loader, dnaC
Initiation
How is helicase donut ring put around the ssDNA?
- DnaC binds to DnaA and to helicase
- Then, DnaC separates the helicase ring, wraps it around the ssDNA, and reforms the donut ring
- A helicase is loaded onto each of the single strands of DNA
Initiation
What disloges DnaA and DnaC from the ori site?
- The movement of helicase (they are no longer needed once th helicase has been loaded)
Initiation
What structure is created by helicase (DnaB)?
- The replication bubble
vocab (definition)
Replication bubble
- consists of two replication forks
- formed at the end of initiation
initiation
vocab (definition)
Replication forks
the sites where DNA is being replicated
initiation
Initiation
What do ss binding proteins do?
- ssDNA is unstable and will try to form dsDNA
- To prevent dsDNA from reforming in the replication bubble, ss binding proteins bind non speciically to ssDNA to prevent the H-bonds from reformign between the two strands of ssDNA
Initiation
Explain the entire initiation process
- Gyrase unwinds supercoiled dsDNA to expose the ori sequence
- DnaA binds to DnaA boxes within the ori sequences
- DnaA proteins bend the dsDNA, creating stress/tension on the structure
- To relievve the tension, H bonds holding the A-T pairs within the AT-rich region are broken, creating the initiation bubble
- Helicase (DnaB) is loaed onto the single stranged DNA by helicase loader (DnaC)
- Helicase moves from 5’ to 3’ to unwind dsDNA
- Initiation is complete once 2 replication forks have been established
Initiation
What is needed for initiation phase?
- gyrase
- ori sequence (A-T rich region, DnaA boxes, GATC sequences)
- DnaA
- helicase (dnaB) and helicase loader (dnaC)
- ss binding proteins
- ATP
- template dsDNA
vocab (definition)
DNA polymerase
- enzyme responsible for DNA replication
- binds loosely to ssDNA, wrapping around the DNA
- Main function is to add complementary (free) nucleotides to the 3’ hydroxyl end of a polynucleotide chain, in a 5’ to 3’ direction
- Can only add free nucleotides to an existing chain, cannot begin replication from scratch
- Helps reshap the dsDNA helix by aiding n H-bond formation between the bases in the old and new DNA strand
- proof reads and corrects mistakes
elongation
vocab (definition)
primer
a short RNA chain (oligonucleotide sequence) that provides a starting point for DNA polymerase to begin synthesizing a new DNA strand
elongation
vocab (definition)
processivity
the ability of an enzyme to catalyze consecutive reactions without releasing its substrate
elongation
vocab (definition)
Primosome
helicase and primase complex
elongation
vocab (definition)
primase
- an RNA polymerase that builds a primer from a specific recognition sequence
- builds rna chains from 5’ to 3’ direction
- can create a new nucleotide chain from scratch
elongation
What are the four steps of elongation?
- Build an RNA primer
2.Create the replisome
3.New strand synthesis by DNA polymerase - Closing the gaps on Okazi fragments
Elongation
How is an RNA primer built?
- primase binds to the DNA helicase and is carried along the replication bubble until it finds a specific target sequence on the ssDNA.
- Once it finds the target sequence on the ssDNA, it uses this as a template for RNA primer synthesis.
- The helicase and primase complex is called the primosome
elongation
Why is the first step of elongation the synthesis of a primer?
Because DNA pol can not begin replicating DNA without a free 3’ end of a nucleotide chain to build from
Elongation
How is the replisome created?
- Once the primer has been formed Dna pols are recruited, and held onto the template dnas by a complex of the sliding clamp and the sliding clamp loader.
- This complex simultaneously tethers 2 different dna polys, one on each strand (in part to make sure replication is occuring on each strand simultaneously)
- Dna replication occurs at the replisome
vocab (definition)
Replisome
- Complex of helicase, primase, dna poly, sliding clamp, and sliding clamp loader
- Where dna replication occurs
elongation
vocab (definition)
Sliding clamp loader
assembles sliding clamp/DNA pol complex and tethers DNApol to the helicase
elongation
Elongation
Describe the process of new strand synthesis by DNA polymerase
- Once the replisome is formed, dna polys create the new strands of DNA, adding free nucleotides to the 3’ end of the RNA primer in a 5’ to 3’ direction.
- Polys also help to reshape the dsDNA helix by aiding in H-bond formation between the bases in the old template strand of DNA and the newly synthesized dna strand
- Poly will also proofread and correct the mistakes that it makes
vocab (definition)
leading strand
- newly synthesized dna strand headed in the same direction as the helicase
- Dna synthesis occurs continuously
elongation
vocab (definition)
lagging strand
- dna synthesis occurs in opposite direction from the replisome
- DNA replication on this strand occurs until DNA poly bumps into an RNA primer
- discontinuous DNA synthesis
elongation
vocab (definition)
Okazaki fragments
- the fragments formed from DNA synthesis on the lagging strand (due to synthesis being discontinous)
elongation
elongation
What does the lagging strand contain after DNA synthesis?
- little bits of DNA/RNA hybrids
- breaks in the phosphate backbone where the 5’ and 3’ ends of the phosphate bone are not covalently bonded
Elongation
How does poly fix mistakes?
Elongation
What rate does poly work at?
750 bp/second
Elongation
How often do mistakes happen w poly before and after fixing mistakes?
- Before: 1bp/10,000bp
- After: 1bp/100,000,000bp
This high accuracy is called high fidelity
elongation
How does dna poly fix mistakes?
- The poly recognizes mispaired bases, halts activity, and relaxes the dsDNA (supercoiling) that is being formed to expose the new template to a new domain of the enzyme
- Then uses exonuclease activity to remove a few bases from the end of a nascent strand, and then restarts polymerase activity
Elongation
What is the process of closing hte gaps on okazaki fragments?
vocab (definition)
ligase
connects broken phosphate backbones, creating a smooth continuous DNA strand
elongation
Termination
What problem does the Tus/Ter complex solve?
- Ensures that the replisome stops when it reaches halfway around the circle of dna to ensure DNA replication does not continue forever
Elongation
Full breakdown of elongation
- Primase binds to helicase (DnaB), and is carried along as helicase unzips the dsDNA.
- When primase finds a recognition sequence on the ssDNA, primase builds a complementary RNA chain in the 5’ to 3’ direction (using the 3’ to 5’ DNA strand as a template)
- The sliding clamp loader tethers DNA poly to the helicase.
- Sliding clamp loader also loads the sliding clamp onto the DNA. Sliding clamp helps DNA poly remain attached to the template DNA. Each helicase can bind to 2 DNA poly. Replisome is now formed.
- DNA poly finds the RNA primers and begins adding free DNA nucleotides to the RNA primer in a 5’ to 3’ direction.
- One DNA polymerase builds its newly synthesized DNA chain in the same direction that the helicase is moving (leading strand). The other moves in the opposite direction (lagging strand).
- The lagging strand synthesis will be blocked whenever the polymerase bumps into a new RNA primer. This discontinuous strand synthesis creates Okazaki fragments
- A different poly identifies RNA/DNA complexes, removes the RAN component using exonuclease activity (3’ to 5’) and fills in the gap with DNA.
- Ligase seals the gap left in the phosphate backbone
Elongation
What is needed for elongation phase?
- template ssDNA
- primase
- DNA polymerase
- sliding clamp and sliding clamp loader
- dATP, dGTP, dTTP, dCTP
- ligase
- ATP (energy)
Termination
When does DNA replication end?
When both replication forks meet at the termination of replication. It is critical that each replication fork meets at the terination sequence at the same time.
Vocab (definition)
Ter 1 and Ter 2
- Around 20 bp palindromic sequences (sequences that are the same but on opposite sides of DNA) found on the DNA in the termination sequence.
- Binding domains for tus
Termination
Where does tus bind to ter1 and ter2?
-Ter 1 on top of dna strand
-Ter 2 on bottoms of DNA strand
-This directionality can help guide termination
Termination
What is the overall process of termination?
- On the opposite side of the ori sequence is a termination sequence, with two domains (Ter 1 and 2)
- Tus binds to these domains directionally
- The replication fork will approach the tus/ter complex fro the top or bottom. When helicase approaches the complex on the same strand where tus is binding, this is non-permissive. Helicase is blocked and the replisome sits and waits. When helicase approaches from the opposite side, the helicase can disloge tus from the ter complex and continue replication (permissive).
- Eventually the 2 replication forks will meet at 1 tus/ter complex.
- When the 2 replication forks meet, tus/ter help finish DNA replication, releasing 2 circles of DNA
Termination
How are the DNA circles completed?
- Once both the replication forks are blocked, the helicases are removed and polymerases and ligases, with ATP energy, complete the DNA circles.
Termination
What is needed for termination?
- Termination sequences (Ter 1/Ter2)
- Tus
