DNA replication Flashcards
DNA structure
- Strands run in opposite polarity
- New nucleotides added in the 5’ –> 3’ direction (added onto the 3’ end)
- Complementary base pairing (held together by hydrogen bonds)
Requirements for DNA replication
- template DNA to be copied
- raw materials (nucleotides)
- enzymes
DNA replication must be . . .
- accurate
- fast
(bacteria is 1000 bp/sec)
(humans are 33 bp/sec)
Who is credited for discovering DNA replication?
Watson and Crick publish DNA has a double helix in 1953
DNA replication potential models
- Conservative
- Semi- conservative
- Dispersive
Conservative potential model
Both strands serve as template (parental and new)
Semi conservative potential model
One strand serves as template
Dispersive
Parental DNA is “Dispersed” randomly
When did Matt Meselson and Franklin Stahl meet?
Met in summer 1954
Meselson & Stahl Experiment
- they grew E. coli in a medium containing 15N- incorporated into DNA bases, which made DNA “Heavy”
- grew long enough so both strands are labeled
- Parent DNA is Heavy:Heavy (H:H)
- Transferred the DNA to medium containing 14N and grew several generations
- “Light” nucleotides are incorporated into newly made DNA
- Pattern reveals model of replication
- Labeled DNA analyzed on CESIUM CHLORIDE gradients
Conservative
L:L and H:H
Semi-conservative
H:L and H:L
Dispersive
H:L and H:L
What does DNA migrate to in the experiment?
its buoyant density: heavy labeled DNA runs further
- H:H DNA runs near bottom of tube
- H:L DNA (15 N & 14N) runs in between
- L:L DNA runs near top of tube
When were the different models ruled out and which model won?
Conservative was ruled out first because bands did not match up. Dispersive was ruled out after second round because two different bands. Therefore, Semi-conservative model won!
DNA replication is bi-directional
DNA synthesis initiates at an origin of replication and proceeds outwards in opposite directions
Prokaryotic DNA replication
- Millions of base pairs
- 1000 bp/sec
- ONE origin of replication
Eukaryotic DNA replication
- 100s of millions of base pairs (billions
- 33 bp/sec
- MULTIPLE origins of replication (solves slow rate of elongation)
Replicon model
Proposed in E. coli
- Contains:
~ Origin of replication (replicator)
~ Initiator (DnaA)
~ Replication forks
Origin of replication (replicator)
sequence where DNA replication starts
Initiator (DnaA)
protein(s) that bind to the origin and initiate replication
Replication forks
move away from the origin in a bidirectional fashion
DNA replication in eukaryotes
- multiple origins of replication
- opens multiple replication bubbles that fuse together to complete replication
First step in DNA Replication
1.) Find the origins of replication
- Short stretches of DNA having a specific nucleotide sequence
- DnaA (the initiator) binds to OriC (the origin)
- Looping causes stress to form in nearby AT-rich sequence= short stretch unwinds
- Unwound section provides access for DNA replication enzymes
Second step in DNA Replication
2.) Two DNA helicase attach and unwind the DNA molecule by breaking hydrogen bonds between nucleotides
- Move away from each other and separate the two parental strands, making them available as template strands
- Creates replication forks: Y-shaped region where DNA is unwound
Third step in DNA Replication
3.) Untwisting of the double helix causes strain ahead of the replication fork
- Topisomerase keeps the tension down
Fourth step in DNA Replication
4.) Single-Stranded Binding Proteins (SSB) bind to unpaired DNA strands, keeping them from repairing
- keeps unraveled DNA single-stranded/stabilizes DNA
Fifth step in DNA Replication
5.) Unwound sections of parental DNA are now able to serve as templates for synthesis of new complementary DNA strands
One problem with DNA replication (5th step)
Enzymes that synthesize DNA cannot initiate synthesis of a polynucleotide
- Can only add nucleotides to the end of an existing chain that is base-paired with the template strand
Solution to problem with DNA replication (5th step)
PRIMASE
- can synthesize a short stretch of RNA called the RNA primer
- new DNA strand can start from the 3’ end of the RNA primer
Sixth step of DNA Replication
6.) DNA polymerase III catalyzes the synthesis of new DNA
- Adds complementary (to the parental DNA strand) nucleotides to the 3’ -OH end of the RNA primer
- Only adds in the 5’ –> 3’ direction!
- each nucleotide added comes from triphosphate nucleotide –> when added, releases two phosphates as pyrophosphate (broken down by pyrophosphatases)
Seventh step of DNA Replication
7.) DNA added to both strands in different ways depending on if leading strand (continuous) or lagging strand (not continuous)
- helicases continue to unwind parental DNA
- LEADING strand: synthesizes continuously
- LAGGING strand: once enough of the parental DNA is unwound, primase lays down another RNA primer (DNA polymerase III extends unit it hits the previous primer)
Leading strand
Template strand with 5’ end at replication fork
- Continuous synthesis in the 5’ –> 3’ direction
- Grows towards the replication fork
- Only ONE primer needed and DNA polymerase III remains attached to this strand
Lagging strand
Template strand with 3’ end at replication fork
- Discontinuous synthesis in the 5’ –> 3’ direction
- Grows away from the replication fork
- MULTIPLE primers needed to be laid down by primase
- DNA polymerase III extends until it hits the previous primer = creates Okazaki fragments
Which strand creates Okazaki fragments?
Lagging strand
Eighth step of DNA Replication
8.) RNA primers are removed and Okazaki fragments are ligated
DNA polymerase 1
removes the RNA primer and fills gap with DNA nucleotides
DNA ligase
covalently links the Okazaki fragments together
When does termination occur?
When replication bubbles merge
Issues with the 5’ end of lagging strand
- RNA primer removed from lagging strand, but DNA polymerase III cannot add to it because can only add to 3’ end
- Therefore, next round of replication: shorter leading strand and shortened/staggered lagging strand
Do prokaryotes have an issue with 5’ end of lagging strand?
No issue due to circular chromosome
What is the solution for the issue with the 5’ end of the lagging strand in Eukaryotes?
Telomeres!
- Multiple repetitions of one short nucleotide sequence (no genes!)
- (ex. humans is TTAGGG)
Issues with proofreading
- DNA polymerase III works from 5’ to 3’
- It proofreads (3’ to 5’) so each nucleotide against its template as soon as it is added to the growing strand (opposite direction)
