DNA Replication
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Transcript of DNA Replication
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2007-2008
DNA Replication
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Watson and Crick1953 article in Nature
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Double helix structure of DNA
“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.”Watson & Crick
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Directionality of DNA• You need to
number the carbons!– it matters!
OH
CH2
O
4
5
3 2
1
PO4
N base
ribose
nucleotide
This will beIMPORTANT!!
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The DNA backbone• Putting the DNA backbone
together– refer to the 3 and 5 ends
of the DNA• the last trailing carbon
OH
O
3
PO4
base
CH2
O
base
OPO
C
O–O
CH2
1
2
4
5
1
2
3
3
4
5
5
Sounds trivial, but…this will beIMPORTANT!!
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Anti-parallel strands• Nucleotides in DNA backbone
are bonded from phosphate to sugar between 3 & 5 carbons– DNA molecule has “direction”– complementary strand runs in
opposite direction
3
5
5
3
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Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
3
5
3
5
covalentphosphodiesterbonds
hydrogenbonds
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Base pairing in DNA• Purines– adenine (A)– guanine (G)
• Pyrimidines– thymine (T)– cytosine (C)
• Pairing– A : T • 2 bonds
– C : G• 3 bonds
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Copying DNA• Replication of DNA– base pairing allows each
strand to serve as a template for a new strand
– new strand is 1/2 parent template & 1/2 new DNA• semi-conservative
copy process
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DNA Replication • Large team of enzymes coordinates replication
Let’s meetthe team…
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Replication: 1st step• Unwind DNA– helicase enzyme• unwinds part of DNA helix• stabilized by single-stranded binding proteins
single-stranded binding proteins replication fork
helicase
I’d love to behelicase & unzipyour genes…
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DNAPolymerase III
Replication: 2nd step
But…We’re missing something!What?
Where’s theENERGYfor the bonding!
Build daughter DNA strand add new complementary
bases DNA polymerase III
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energy
ATPGTPTTPCTP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPCMPmodified nucleotide
energy
We comewith our ownenergy!
And weleave behind anucleotide!
YourememberATP!Are there other waysto get energyout of it?
Are thereother energynucleotides?You bet!
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Energy of Replication• The nucleotides arrive as nucleosides– DNA bases with P–P–P
• P-P-P = energy for bonding– DNA bases arrive with their own energy source for
bonding– bonded by enzyme: DNA polymerase III
ATP GTP TTP CTP
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• Adding bases – can only add nucleotides
to 3 end of a growing DNA strand• need a “starter” nucleotide
to bond to
– strand only grows 53
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
DNAPolymerase III
energy
energy
energy
Replicationenergy
3
3
5B.Y.O. ENERGY!The energy rulesthe process
5
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energy
35
5
5
3
need “primer” bases to add on to
energy
energy
energy
3
no energy to bond
energy
energy
energy
ligase
3 5
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Limits of DNA polymerase III can only build onto 3 end of an existing DNA
strand
Leading & Lagging strands
5
5
5
5
3
3
3
53
53 3
Leading strand
Lagging strand
Okazaki fragments
ligase
Okazaki
Leading strand continuous synthesis
Lagging strand Okazaki fragments joined by ligase
“spot welder” enzyme
DNA polymerase III
3
5
growing replication fork
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DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading strand
lagging strandleading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing replication fork
growing replication fork
5
5
5
5
53
3
5
5lagging strand
5 3
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DNA polymerase III
RNA primer built by primase serves as starter sequence for DNA
polymerase III
Limits of DNA polymerase III can only build onto 3 end of an existing DNA
strand
Starting DNA synthesis: RNA primers
5
5
5
3
3
3
5
3 53 5 3
growing replication fork primase
RNA
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DNA polymerase I removes sections of RNA primer and
replaces with DNA nucleotides
But DNA polymerase I still can only build onto 3 end of an existing DNA strand
Replacing RNA primers with DNA
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
ligase
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Loss of bases at 5 ends in every replication
chromosomes get shorter with each replication limit to number of cell divisions?
DNA polymerase III
All DNA polymerases can only add to 3 end of an existing DNA strand
Chromosome erosion
5
5
5
5
3
3
3
3
growing replication fork
DNA polymerase I
RNA
Houston, we have a problem!
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Repeating, non-coding sequences at the end of chromosomes = protective cap
limit to ~50 cell divisions
Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells
high in stem cells & cancers -- Why?
telomerase
Telomeres
5
5
5
5
3
3
3
3
growing replication fork
TTAAGGGTTAAGGGTTAAGGG
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Replication fork
3’
5’3’
5’
5’
3’3’ 5’
helicase
direction of replication
SSB = single-stranded binding proteins
primase
DNA polymerase III
DNA polymerase III
DNA polymerase I
ligase
Okazaki fragments
leading strand
lagging strand
SSB
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DNA polymerases• DNA polymerase III– 1000 bases/second!– main DNA builder
• DNA polymerase I– 20 bases/second– editing, repair & primer removal
DNA polymerase III enzyme
Arthur Kornberg1959
Thomas Kornberg??
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• In 1953, Kornberg was appointed head of the Department of Microbiology in the Washington University School of Medicine in St. Louis. It was here that he isolated DNA polymerase I and showed that life (DNA) can be made in a test tube. In 1959, Kornberg shared the Nobel Prize for Physiology or Medicine with Severo Ochoa — Kornberg for the enzymatic synthesis of DNA, Ochoa for the enzymatic synthesis of RNA.
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Editing & proofreading DNA• 1000 bases/second =
lots of typos!
• DNA polymerase I – proofreads & corrects typos – repairs mismatched bases– removes abnormal bases
• repairs damage throughout life
– reduces error rate from 1 in 10,000 to 1 in 100 million bases
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Fast & accurate!• It takes E. coli <1 hour to copy
5 million base pairs in its single chromosome – divide to form 2 identical daughter cells
• Human cell copies its 6 billion bases & divide into daughter cells in only few hours– remarkably accurate– only ~1 error per 100 million bases– ~30 errors per cell cycle
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3
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What does it really look like?
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2007-2008
Any Questions??