AP Biology 2007-2008 DNA Replication
AP Biology 2007-2008
DNA Replication
AP Biology
Watson and Crick 1953 1953 article in Nature
AP Biology
Directionality of DNA § You need to
number the carbons! u it matters! u 3’ refers to
the 3 carbon on the sugar
u 5’ refers to the 5 carbon on the sugar.
OH
CH2 O
4ʹ
5ʹ
3ʹ 2ʹ
1ʹ
PO4
N base
ribose
nucleotide
AP Biology
The DNA backbone § Putting the DNA
backbone together u refer to sugar bonded
to the phosphate groups.
u The BASES are not the backbone.
OH
O
3ʹ
PO4
base
CH2 O
base
O P O
C
O –O
CH2
1ʹ
2ʹ
4ʹ
5ʹ
1ʹ
2ʹ
3ʹ
3ʹ
4ʹ
5ʹ
5ʹ
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Anti-parallel strands § Nucleotides in DNA backbone
are bonded from phosphate to sugar between 3ʹ & 5ʹ carbons u DNA molecule has
“direction” u One strand is 5’-3’ while the
other is 3’ to 5’ u Complementary strand
runs in opposite direction u Strands held together with
HYDROGEN BONDS. 3ʹ
5ʹ
5ʹ
3ʹ
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Bonding in DNA
….strong or weak bonds? How do the bonds fit the mechanism for copying DNA?
3ʹ
5ʹ 3ʹ
5ʹ
covalent phosphodiester
bonds
hydrogen bonds
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Base pairing in DNA § Purines (2 rings)
u adenine (A) u guanine (G)
§ Pyrimidines (1 ring) u thymine (T) u cytosine (C)
§ Pairing u A : T
§ 2 bonds u C : G
§ 3 bonds
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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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Copying DNA § Replication of DNA
u base pairing allows each strand to serve as a template for a new strand
u new strand is 1/2 parent template & 1/2 new DNA
u This is called SEMI-CONSERVATIVE
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DNA Replication § Large team of enzymes coordinates replication § Speeds up using multiple origins
Let’s meet the team…
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Slight differences in Prokaryotes
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Replication: 1st step § Unwind DNA with TOPOISOMERASE
u Helicase enzyme § splits apart the DNA helix (break H-bonds) § Stabilized each piece using single-stranded
binding proteins
single-stranded binding proteins replication fork
helicase
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DNA Polymerase III
Replication: 2nd step
But… We’re missing something!
What?
Where’s the ENERGY
for the bonding!
§ Build daughter DNA strand u add new
complementary bases using.. DNA polymerase III
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energy
ATP GTP TTP CTP
Energy of Replication Where does energy for bonding usually come from?
ADP AMP GMP TMP CMP modified nucleotide
energy
We come with our own
energy!
And we leave behind a nucleotide!
You remember
ATP! Are there other ways
to get energy out of it?
Are there other energy nucleotides? You bet!
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Energy of Replication § The nucleotides arrive as nucleosides
u DNA bases with P–P–P § P-P-P = energy for bonding
u DNA bases arrive with their own energy source for bonding
u bonded by enzyme: DNA polymerase III
ATP GTP TTP CTP
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§ Adding bases u can only add
nucleotides to 3ʹ end of a growing DNA strand § need a “starter”
nucleotide to bond to
u strand only grows 5ʹ→3ʹ
DNA Polymerase III
DNA Polymerase III
DNA Polymerase III
DNA Polymerase III
energy
energy
energy
Replication energy
3ʹ
3ʹ
5ʹ B.Y.O. ENERGY! The energy rules
the process
5ʹ
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energy
3ʹ 5ʹ
5ʹ
5ʹ
3ʹ
need “primer” bases to add on to
energy
energy
energy
3ʹ
no energy to bond
energy
energy
energy
ligase
3ʹ 5ʹ
û
AP Biology
Limits of DNA polymerase III u can only build onto 3ʹ end of
an existing DNA strand
Leading & Lagging strands
5ʹ
5ʹ
5ʹ
5ʹ
3ʹ
3ʹ
3ʹ
5ʹ 3ʹ
5ʹ 3ʹ 3ʹ
Leading strand
Lagging strand
Okazaki fragments
ligase
Okazaki
Leading strand u continuous synthesis
Lagging strand u Okazaki fragments u joined by ligase
§ “spot welder” enzyme
DNA polymerase III
!
û
3ʹ
5ʹ
growing replication fork
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DNA polymerase III
Replication fork / Replication bubble
5ʹ
3ʹ 5ʹ
3ʹ
leading strand
lagging strand
leading strand
lagging strand leading strand
5ʹ
3ʹ
3ʹ
5ʹ
5ʹ
3ʹ 5ʹ
3ʹ
5ʹ
3ʹ 5ʹ
3ʹ
growing replication fork
growing replication fork
5ʹ
5ʹ
5ʹ
5ʹ
5ʹ 3ʹ
3ʹ
5ʹ
5ʹ lagging strand
5ʹ 3ʹ
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DNA polymerase III
RNA primer u built by primase u serves as starter sequence
for DNA polymerase III
Limits of DNA polymerase III u can only build onto 3ʹ end of
an existing DNA strand
Starting DNA synthesis: RNA primers
5ʹ
5ʹ
5ʹ
3ʹ
3ʹ
3ʹ
5ʹ
3ʹ 5ʹ 3ʹ 5ʹ 3ʹ
growing replication fork primase
RNA
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DNA polymerase I u removes sections of RNA
primer and replaces with DNA nucleotides
But DNA polymerase I still can only build onto 3ʹ end of an existing DNA strand
Replacing RNA primers with DNA
5ʹ
5ʹ
5ʹ
5ʹ
3ʹ
3ʹ
3ʹ
3ʹ
growing replication fork
DNA polymerase I
RNA
ligase
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Loss of bases at 5ʹ ends in every replication u chromosomes get shorter with each replication u limit to number of cell divisions?
DNA polymerase III
All DNA polymerases can only add to 3ʹ end of an existing DNA strand
Chromosome erosion
5ʹ
5ʹ
5ʹ
5ʹ
3ʹ
3ʹ
3ʹ
3ʹ
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 u limit to ~50 cell divisions
Telomerase u enzyme extends telomeres u can add DNA bases at 5ʹ end u different level of activity in different cells
§ high in stem cells & cancers -- Why?
telomerase
Telomeres
5ʹ
5ʹ
5ʹ
5ʹ
3ʹ
3ʹ
3ʹ
3ʹ
growing replication fork
TTAAGGG TTAAGGG TTAAGGG
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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
u 1000 bases/second! u main DNA builder
§ DNA polymerase I u 20 bases/second u editing, repair & primer removal
DNA polymerase III enzyme
Arthur Kornberg 1959
Roger Kornberg 2006
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Editing & proofreading DNA § 1000 bases/second =
lots of typos!
§ DNA polymerase I u proofreads & corrects
typos u repairs mismatched bases u removes abnormal bases
§ repairs damage throughout life
u 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 u divide to form 2 identical daughter cells
§ Human cell copies its 6 billion bases & divide into daughter cells in only few hours u remarkably accurate u only ~1 error per 100 million bases u ~30 errors per cell cycle
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3 4
What does it really look like?
AP Biology 2007-2008
Any Questions??
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