1. DNA Replication
AP Biology 2007-2008

2. 1953 article in Nature
Watson and Crick
AP Biology

3. 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
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material.” Watson & Crick

4. Directionality of DNA
 You need to PO nucleotide
4
number the
carbons!
 it matters! N base
5′ CH2
This will be O
IMPORTANT!!
4′ ribose 1′
3′ 2′
AP Biology
OH

5. 5′
The DNA backbone PO4
 Putting the DNA
backbone together base
5′ CH2
 refer to the 3′ and 5′ O
4′ 1′
ends of the DNA C
3′ 2′
 the last trailing carbon O
–
O P O
Sounds trivial, but…
O base
this will be 5′ CH2
IMPORTANT!! O
4′ 1′
3′ 2′
OH
AP Biology 3′

6. Anti-parallel strands
 Nucleotides in DNA
backbone are bonded from
phosphate to sugar
between 3′ & 5′ carbons 5′ 3′
 DNA molecule has
“direction”
 complementary strand runs
in opposite direction
AP Biology 3′ 5′

7. Bonding in DNA
hydrogen
bonds
5′ 3′
covalent
phosphodiester
bonds
3′
5′
….strong or weak bonds?
AP Biology the bonds fit the mechanism for copying DNA?
How do

8. 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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9. 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
AP Biology

10. Let’s meet
the team…
DNA Replication
 Large team of enzymes coordinates replication
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11. I’d love to be
helicase & unzip
Replication: 1st step your genes…
 Unwind DNA
 helicase enzyme
 unwinds part of DNA helix
 stabilized by single-stranded binding proteins
helicase
single-stranded binding proteins
AP Biology replication fork

12. Replication: 2nd step
 Build daughter DNA
strand
 add new
complementary bases
 DNA polymerase III
But…
Where’s the
We’re missing
ENERGY
DNA something!
for the bonding!
Polymerase III What?
AP Biology

13. Energy of Replication
Where does energy for bonding usually come from?
We come
with our own
energy!
You energy
remember energy
ATP!
Are there
other ways
other energy
to get energy
nucleotides?
out of it?
You bet!
And we
leave behind a
CTP
GTP
TTP
ATP nucleotide! CMP
TMP
GMP
AMP
ADP
AP Biology modified nucleotide

14. 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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15. 5′ 3′
Replication energy
DNA
 Adding bases Polymerase III
 can only add energy
nucleotides to DNA
3′ end of a growing Polymerase III
DNA strand energy
 need a “starter”
DNA
Polymerase III
nucleotide to
bond to
DNA
energy
 strand only grows Polymerase III
5′→3′
B.Y.O. ENERGY!
The energy rules 3′ 5′
the process
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16. 5′ 3′ 5′ need “primer” bases to add on to 3′
energy
no energy

to bond
energy
energy
energy
energy
ligase
energy
energy
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3′ 5′ 3′ 5′

17. Okazaki
Leading & Lagging strands
Limits of DNA polymerase III
 can only build onto 3′ end of
an existing DNA strand 5′
ents
3′
Okaza
5′
ki fragm
3′
5′
3′ 5′ 5′
3′
Lagging strand
ligase
growing 3′
replication fork
5′
Leading strand
Lagging strand

3′ 5′
3′
DNA polymerase III
 Okazaki fragments
 joined by ligase Leading strand
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 “spot welder” enzyme  continuous synthesis

18. Replication fork / Replication bubble
3′ 5′
5′ 3′
DNA polymerase III
leading strand
5′
3′ 3′ 5′
5′ 5′
5′ 3′ 3′
lagging strand
3′ 5′
5′
3′ lagging strand leading strand
5′ growing
3′ replication fork 5′
5′ growing
replication fork 5′
leading strand 3′
lagging strand
3′
5′
5′ 5′
AP Biology

19. Starting DNA synthesis: RNA primers
Limits of DNA polymerase III
 can only build onto 3′ end of
an existing DNA strand 5′
3′ 5′ 3′
5′
3′
3′ 5′
growing 3′ primase
replication fork DNA polymerase III
5′
RNA 5′
RNA primer 3′
 built by primase
 serves as starter sequence
AP for DNA polymerase III
Biology

20. Replacing RNA primers with DNA
DNA polymerase I
 removes sections of RNA DNA polymerase I
primer and replaces with 5′
DNA nucleotides 3′
3′
5′ ligase
growing 3′
replication fork
5′
RNA 5′
3′
But DNA polymerase I still
can only build onto 3′ end of
an Biology
AP existing DNA strand

21. Houston, we
have a problem!
Chromosome erosion
All DNA polymerases can
only add to 3′ end of an DNA polymerase I
existing DNA strand 5′
3′
3′
5′
growing 3′
replication fork DNA polymerase III
5′
RNA 5′
Loss of bases at 5′ ends 3′
in every replication
 chromosomes get shorter with each replication
AP limit to number of cell divisions?
 Biology

22. Telomeres
Repeating, non-coding sequences at the end
of chromosomes = protective cap
5′
 limit to ~50 cell divisions
3′
3′
5′
growing 3′ telomerase
replication fork
5′
5′
Telomerase
TTAAGGG TTAAGGG TTAAGGG
 enzyme extends telomeres 3′
 can add DNA bases at 5′ end
 different level of activity in different cells
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 high in stem cells & cancers -- Why?

23. Replication fork
DNA
polymerase III lagging strand
DNA
polymerase I
3’
Okazaki primase
fragments 5’
5’ ligase
SSB
3’ 5’
3’ helicase
DNA
polymerase III
5’ leading strand
3’
direction of replication
AP Biology
SSB = single-stranded binding proteins

24. DNA polymerases
 DNA polymerase III
 1000 bases/second! Thomas Kornberg
??
 main DNA builder
 DNA polymerase I
 20 bases/second
 editing, repair & primer removal
DNA polymerase III Arthur Kornberg
enzyme 1959
AP Biology

25. 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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26. 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
AP Biology

27. What does it really look like?
1
2
3
4
AP Biology

28. Any Questions??
AP Biology 2007-2008