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DNA Replication
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DNA Replication
process by which a cell doubles its DNA before divison
What were the 3 proposed models for DNA?
conservative, dispersive, semi-conservative
Semi-conservative model of DNA Replication
2 nucleotide strands of DNA separate, and each strand serves as a template for a new strand
1 original strand, 1 new strand
Conservative model of DNA replication
entire double-stranded DNA molecule serves as template for a whole new molecule
original double helix and new double helix
Dispersive model for DNA replication
both nucleotide strands disperse into fragments = templates for new synthesis of new DNA fragments → then reassemble into new DNA molecules
original and new DNA fragments mixed, no original strands preserved
What did the Meselson and Stahl centrifugation experiment prove and how?
Grew bacteria in medium of 15N, 15N then one round in 14N, and in just 14N.
15N = heavier isotope
DNA of bacteria incorporated their respective nitrogen isotope into their nitrogenous bases
Centrifugation revealed:
heavy DNA (15N) appeared as single band
DNA allowed one round in 14N = single band at intermediate weight
2 rounds in 14N = 2 bands, one light and one intermediate in weight
Several rounds of replication = progressively lighter, and 2 bands 1 intermediate, 1 very light.
Proved semi-conservative model
conservative cannot produce intermediate weight
dispersive wouldn’t produce 2 bands, all would be intermediate
How many bands of DNA would be expected in Meselson and Stahl’s experiment after 2 rounds of conservative replication in 14N medium?
Two bands
one heavy and one light
How can semiconservative replication differ between organisms?
differ in whether template DNA is linear or circular
Replicon
a segment of DNA that undergoes replication
origin of replication to end of replication on either side of origin
Origin of replication
site/DNA sequence where DNA replication is initiated
Bacterial mode of replication
circular chromosome
single origin of replication (ori)
Eukaryotic mode of replication
Linear chromosomes
multiple origins of replication
ends of linear molecules require special replication
In what kind of DNA does Theta replication occur?
circular DNA (bacteria)
What initiates Theta replication and how does it progress?
initiated by unwinding of the two nucleotide strands = replication bubble
unwinding continuously at one or both ends of bubble = making it larger
DNA repliation on both template strands simultaneous with unwinding until 2 replication forks meet
How did Theta replication get its name?
generates an intermediate structure that resembles Greek letter theta θ.
In which direction does DNA replication occur?
DNA replication is bidirectional, forming a replication bubble and 2 replication forks
Replication fork
point at which the 2 strands of DNA are separated to allow replication of each strand
Who provided the first visible evidence of theta replication and how?
John Cairns
grow bacteria in presence of radioactive nucleotides
after replication = 1 radioactive strand and 1 nonradioactive
Rolling-Circle Replication
viruses
replication of circular DNA
initiated by break in one of the nucleotide strands = double-stranded circular DNA + single-stranded circular DNA
single-stranded serves as template for synthesis of complementary strand
Why do Eukaryotic DNA molecules have more than one origin of replication, whereas circular DNA only has a single one?
Linear chromosomes of eukaryotic DNA are too large to be replicated quickly enough from a single origin
Which modes DNA replication are unidirectional or bidirectional, or both?
Unidirectional = Rolling-circle
Unidirectional or Bidirectional = Theta
Bidirectional = Linear Eukaryotic
Which type of replication requires a break in the nucleotide strand to get started?
Rolling circle replication
Describe Eukaryotic DNA replication and what it produces:
Replication forks of adjacent replicons run into each other and fuse to yield 2 identical linear molecules
How does the rate of eukaryotic replication compare to prokaryotes?
20x slower
What are the 3 main requirements of replication?
A template consisting of single stranded DNA
Raw materials (substrates) to be assembled into a new nucleotide strand
Enzymes and other proteins that “read” the template and assemble the substrates into a DNA molecule
What are the raw materials from which new DNA molecules are synthesized?
deoxyribonucleoside triphosphates (dNTPs)
deoxyribose sugar + nucleoside base + 3 phosphate groups
Where and how are nucleotides added during DNA synthesis?
DNA polymerase adds nucleotides to the free 3’-OH group on the growing strand
In which direction is DNA synthesized?
5’ to 3’ direction
How does the phosphodiester bond between the last nucleotide on a strand and the new nucleotide form?
3’-OH group of last nucleotide attacks 5’-phosphate group of incoming dNTP
2 phosphate groups are cleaved from the incoming dNTP
phosphodiester bond created between the 2 nucleotides
Does DNA synthesis occur spontaneously?
No, requires host of enzymes and proteins that function in a coordinated manner
Why is there a leading strand and laggig strand
because the template strands are anti-parallel but DNA can only be synthesized in the 5’ to 3’ direction
Leading strand
new strand synthesized in same direction as the movement of the replication fork (direction of unwinding)
unwinding → & synthesis →
continuous replication
Lagging strand
new strand synthesized in the opposite direction as the movement of the replication fork (direction of unwinding)
unwinding → & ← synthesis
discontinuous replication
Discontinous replication
runs out of template as it proceeds away from replication fork
synthesis starts again further up template as fork unwinds it
short fragments of DNA produced = okazaki fragments
Lagging strand
Okazaki fragments
short length of newly synthesized DNA produced by discontinuous replication on lagging strand
eventually joined together by DNA ligase
Discontinous replication is a result of which property of DNA?
antiparallel nucleotide strands
Which enzyme breaks hydrogen bonds at each replication fork?
helicase
Unzips
What is the function of single-stranded binding proteins?
prevent the single stranded DNA template from reforming base pairs after helicase unzipped them
Which enzyme relieves tension (supercoils) in the helix ahead of the replication forks?
Topoisomerase or Gyrase
What is the function of DNA polymerase?
adds new nucleotide to 3’ end of growing daughter strand
Which enzyme joins the okazaki fragments?
DNA ligase
What is the function of Primase?
Forms RNA primers to initiate replication
What are the 4 stages in which replication takes place?
Initiation
Unwinding
Elongation
Termination
What are the key players in the Initiation stage of replication?
Initiator proteins
Helicase
Single-Stranded Binding proteins
Describe the Initiation stage of replication:
Initiator proteins (DnaA) bind to the oriC (origin of replication)
= short stretch of DNA unwinds
Unwinding allows helicase and single-stranded binding proteins to attach to the single stranded DNA
What are the key players in the Unwinding stage of replication?
Helicase
single-stranded binding proteins
DNA gyrase
Describe the Unwinding stage of replication:
DNA helicase binds to lagging strand template at each replication fork and moves 5’ to 3’, breaking hydrogen bonds and moving R. fork
Single-stranded binding proteins stabilize the exposed single -stranded DNA
DNA gyrase relieves strain ahead of replication fork
How does DNA synthesis begin if DNA polymerases require an existing 3’-OH group to add a new nucleotide to?
Primase synthesizes short stretches of RNA nucleotides (primers), providing a 3’-OH group
What are the key components of the Elongation stage of replication?
Primase
DNA polymerase III
DNA polymerase I
DNA Ligase
Describe the Elongation stage of replication:
Primase make short stretched of RNA nucleotides.
= Provides free 3’-OH group for DNA polymerase III to add DNA nucleotides to.
DNA polymerase I replaces RNA nucleotides of primer with DNA nucleotides.
Ligase forms phosphodiester bond between 3’-OH and 5’-phosphate groups without adding another nucleotide = joins Okazaki fragments and seals breaks in sugar-phosphate backbone
Where are primers added during Elongation?
1 primer at 5’ end of newly synthesized leading strand
At beginning of each Okazaki fragment on lagging strand
Why is a nick/break left in the sugar-phosphate backbone after all the nucleotides of new strand are added?
The 3’-OH group of the last nucleotide replaced by DNA polymerase I is not attached to the 5’ phosphate group of the 1st nucleotide added by DNA polymerase III
When does DNA know when to Terminate (end) replication?
some terminate when 2 replication forks meet
some require specific termination sequences (Ter sites) to block further replication
Tus-Ter complex
Ter site and termination protein complex that blocks movement of helicase = stalling replication fork
blocks replication fork from moving in one direction but not the other
What is the error rate of DNA replication?
less than 1 mistake per billion nucleotides
How are errors in DNA polymerase III base-pairing corrected?
corrected immediately using DNA polymerase’s 3’ to 5’ exonuclease activity and repair
backwards removal and repair = proofreading
How are any mistakes that are present after DNA replication is completed rectified? How are the old and new strands identified?
Mismatch repair enzymes correct any error that remains after DNA replication is completed
excise incorrectly paired nucleotides from new strand and use original strand as template to replace them
In bacteria, original DNA is methylated, so repair enzymes know which strand to fix
Why is gene sequence erosion not an issue in the circular DNA of bacteria?
there is always a 3’-OH end, no need for the RNA primers to be removed and replaced
Why does gene sequence erosion occur in linear chromosomes?
end of chromosome has no 3’-OH group preceding the primer
terminal primer removed = cannot be replaced by DNA nucleotide
results in gap at end of chromosome
= chromosome progressively shorter with each round of replication
= gene sequence eventually eroded and cell dies
How do telomeres relate to gene erosion?
heterochromatic regions of many short repeated sequences at ends of linear chromosomes
postpone erosion of vital gene sequences
What would be the result if an organism’s telomerase were mutated and nonfunctional?
chromosomes would shorten with each new generation
How does telomere shortening relate to aging?
telomere shortening may contribute to aging
most somatic cells have a pre-programmed life span with limits on how many times they can divide before death (apoptosis)
How do germ line (produce gametes) cells, single cell organisms, proliferative cell types prevent telomere shortening?
using telomerase
enzyme that restores telomere length
Telomerase
enzyme that restores telomere length
composed of protein and RNA components complementary to G-rich strand that serves as template for DNA synthesis
Describe how telomerase restores telomere length:
Telomere has protruding end with a G-rich repeated sequence
RNA part of telomerase complementary to G-rich and pairs w it = template for synthesis of copies of the repeats
Nucleotides are added to 3’ end of G-rich strand