Download DNA Replication

Warm-up
1. Which parts of the nucleotides are
connected to make one strand (half) of
DNA?
2. Which parts of the nucleotides connect
the two strands (halves) of DNA?
3. What type of bond connects the two
strands together?
4. If a DNA molecule is 30%Adenine how
much Cytosine is there?
What does DNA look like?
• We can describe its molecular structure
• Next week we will see what DNA looks like
after isolation.
When a cell divides where
Does the extra DNA come
from?
DNA replication
DNA Replication
Whenever a cell divides into two new cells, it
needs to make an exact copy of the DNA.
What would happen if a cell divided without DNA
making a copy of itself?
The process of copying DNA is called DNA
replication.
DNA Replication 1.
• Replication could be:
– Conservative: Old strands stay together and
act as a template.
– Semi conservative: Strands split and act as a
template.
– Dispersive: Strands separate AND break
apart and act as templates.
Possible DNA Replication Models
The Meselson-Stahl Experiment
•
•
•
•
•
•
E. coli bacteria were cultured for several generations in heavy nitrogen (15N
- normal nitrogen is 14N) (Why is 15N heavier than 14N?).
The bacteria incorporated 15N into their nucleotides and thus, their DNA
Meselson and Stahl then transferred the bacteria to a medium containing
14N,
So…. any DNA that the bacteria synthesized would be lighter than the "old"
DNA made with the heavier 15N medium
The DNA was extracted from the cells and centrifuged in a cesium chloride
density gradient for 20 hours at 40,000rpm.
The DNA migrated to a point that was equivalent to their density
• http://www.dnaftb.org/dnaftb/20/concept/index.html
Possible outcomes
Experimental Results
･Results from the parental generation contained only a
single high density band - all DNA molecules contained
the "heavy" nitrogen.
･DNA taken from the two generations after the switch
contained an intermediate-density band - DNA contained
a "heavy" DNA strand from the parent and a
complementary "light" DNA strand.
･Density results from generation 3, displayed two bands.
They included an intermediate density band, composed
of one parental "heavy" strand and a new light band,
composed of only DNA strands with "light" nitrogen.
Actual Outcome
So Replication is Semi Conservative
DNA Replication 2
• DNA could be extended in a 3’ -> 5’
direction.
• DNA could be extended in a 5’ -> 3’
direction.
• DNA could be extended in a both 3’ -> 5’
and 5’ -> 3’ directions simultaneously.
DNA synthesis uses the energy released from the
triphosphate bond to “power” DNA synthesis…
Nucleotides add as 5’ NTPs…
Specificity determined by base pairing…
C
DNA polymerase
“Primer” strand
5’
C
A
T
T
G
G
T
A
A
C
G
C
T
A
G
T
G
T
C
A
Template strand
Incorrect nucleotide cannot base pair…
… and is removed by 3’→5’ exonuclease activity of DNA polymerase…
Correct base inserted and 5’→3’ replication continues…
Each nucleotide brings the energy needed for its own addition…
5’
If synthesis needs triphosphate bonds can 3’→5’
NO!
synthesis be self-correcting?
Consider a hypothetical scheme in which DNA synthesis occurs 3’ to 5’…
Triphosphates for addition of next nucleotide at 5’ end…
5’ triphosphate
T5’-3’ exonuclease
No high energy
phosphates
G
T
5’
3’
“Primer” strand
C
T
G
C
C
A
G
T
A
A
C
G
G
T
C
A
Template strand
Incorrect nucleotide cannot base pair…
Removed by 5’→3’ exonuclease activity…
No high energy phosphates left for insertion of next nucleotide…
Replication unable to proceed…
3’→5’ polymerase cannot be self correcting…
5’
Error: Some comparisons
Therefore:
DNA Replication proceeds in a
5’->3’ direction only.
DNA polymerase can only add to 3’ end of base-paired nucleotide…
SO..short lengths of RNA act as “primers” for DNA synthesis…
3’
5’
“Primase” uses DNA template to synthesize a
short ( ~10 b) stretch of RNA…
Leading strand is only primed at initiation…
5’
RNA primer of
previous Okazaki
fragment
5’
RNA primer of next
Okazaki fragment
Each Okazaki fragment of lagging strand
begins with new RNA primer…
5’
3’
Fork migration
3’
5’
DNA polymerase then elongates the RNA
primer by adding dNTPs to its 3’ end…
RNA primer removed by “repair exonuclease”…
Gap filled by “repair polymerase”…
5’
3’
“Ligase” seals nicks to complete strand…
DNA Replication 3.
• Does DNA Replication occur from one
point only?
OR
• Does DNA Replication occur from from
many origins?
Replication in Action
• 1 human chromosome is an average of 80
million base pairs long (3 billion/46).
• If a cell replicates in 10 hours how many bases
per second does replication proceed at?
• About 2200bp/sec!
• E. coli chromosome is 5 million bp, if it replicates
every 20 mins how many bases per second does
replication proceed at?
• About 4200bp/sec
The Speed of DNA Replication
• Radioactive nucleotide incorporation has
been measured at about 1000bp/sec in
prokaryotes and about 50bp/sec in
eukaryotes
• So DNA replication must occur
simultaneously at multiple places along a
DNA strand.
Replication origins: DNA replication begins at specific sites…
Initiator proteins (incl helicases)
“melt” double helix at origin…
ss DNA serves as template for
DNA polymerase…
“Replication origin”
Single strand DNA
5’
3’
3’
5’
“Replication bubble”
“Replication forks” then move
outward…
Prokaryotes generally
use just one origin…
Yellow = new DNA
Eukaryotes use
multiple origins…
Origin
Replication forks
Replication forks
Replication forks
Replication forks
Origins
Origins
Taking a closer look at replication forks…
Leading strand
template for L fork…
Lagging strand
template for R fork…
5’ 3’
Left fork
Right fork
Fork migration
5’
3’
3’
Most recently synthesized DNA
5’
Lagging strand
template for L fork…
Fork migration
5’
3’
5’
3’
3’ 5’
Leading strand
template for R fork…
The leading strand template for the right fork is the lagging strand template
for the left fork...
…and vice versa.
DNA Replication 4.
• At the replication fork there are MANY
enzymes that help in the synthesis of new
DNA
Enzymes in DNA replication
• Enzymes are proteins that act as machines
in the cell to do work.
• Many enzymes come together to form a
multi-functional replication machine called a
Replisome at the replication fork.
DNA replication: a caste of characters…
Replication:
Helicase: ATP-dependent separation (“melting”) of DNA strands…
Single strand binding protein (SSB): binds and stabilizes ss DNA…
Primase: synthesizes RNA primers (may be a component of DNA polymerase)…
DNA polymerase: synthesizes and “proof-reads” new DNA strand…
Repair Exonuclease: removes RNA primer…
Repair polymerase: fills “gap” after primer is removed…
Ligase: closes “nicks” in DNA
Topoisomerase I: creates ss nicks ahead of replication to allow unwinding…
Topoisomerase II: creates ds nicks to pass one ds DNA through another…
Proof-reading and repair:
DNA polymerase: 3’→5’ exonuclease/5’→3’ polymerase…
DNA mismatch repair enzymes: exonuclease, polymerase, ligase…
DNA Polymerases :
5 known in prokaryotes
At least 20 known in eukaryotes.
Complicated picture of replication…
The Whole Enchilada!
http://www.mcb.harvard.edu/Losick/images/TromboneFINALd.swf
OR
http://www.dnai.org/a/index.html
Go to: 1. Copying the code
2. Putting it together
3. Replication