DNA Replication

AP Biology 2007-2008
 1953 article in Nature
Watson and Crick

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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
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material. Watson & Crick
 Directionality of DNA
You need to PO4 nucleotide
number the
carbons!
it matters! N base

5 CH2
This will be O
IMPORTANT!!
4 ribose 1

3 2
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OH
 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
 Anti-parallel strands
Nucleotides in DNA
backbone are bonded from
phosphate to sugar
5 3
between 3 & 5 carbons
DNA molecule has
direction
complementary strand runs

in opposite direction

AP Biology 3 5
 Bonding in DNA
hydrogen
bonds
5 3

covalent
phosphodiester
bonds

3
5

.strong or weak bonds?

How
AP do the bonds fit the mechanism for copying DNA?
Biology
 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

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 Lets meet
the team
DNA Replication
Large team of enzymes coordinates replication

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 Replication: 1st step
Unwind DNA
helicase enzyme
unwinds part of DNA helix
stabilized by single-stranded binding proteins
helicase

single-stranded binding proteins

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 Replication: 2nd step
Build daughter DNA
strand
add new
complementary bases
DNA polymerase III

But
Wheres the
Were missing
ENERGY
DNA something!
for the bonding!
Polymerase III What?

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 Energy of Replication
Where does energy for bonding usually come from?
We come
with our own
energy!
You energy
remember energy
ATP!
Are there
otherenergy
other ways
to get energy
nucleotides?
out
You of it?
bet!

And we
leave behind a
ATP
GTP
CTP
TTP nucleotide! CMP
TMP
GMP
AMP
ADP
AP Biology modified nucleotide
 Energy of Replication
The nucleotides arrive as nucleosides
DNA bases with PPP
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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 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
DNA
need a starter
Polymerase III
nucleotide to
bond to
DNA
energy
strand only grows Polymerase III
53
B.Y.O. ENERGY!
The energy rules 3 5
the process
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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
 Okazaki

Leading & Lagging strands

Limits of DNA polymerase III
can only build onto 3 end of
5
an existing DNA strand

3 5
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
 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

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 Starting DNA synthesis: RNA primers
Limits of DNA polymerase III
can only build onto 3 end of
5
an existing DNA strand
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
 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

But DNA polymerase I still

can only build onto 3 end of
an existing DNA strand
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 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
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 Biologyto number of cell divisions?
limit
 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 3
enzyme extends telomeres
can add DNA bases at 5 end
different level of activity in different cells
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high in stem cells & cancers -- Why?
 Replication fork
DNA
polymerase III lagging strand
DNA
polymerase I
3
Okazaki primase
fragments 5
5 ligase
3 5 SSB

3 helicase

DNA
polymerase III
5 leading strand
3
direction of replication
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SSB = single-stranded binding proteins
 DNA polymerases
DNA polymerase III
1000 bases/second! Roger Kornberg
2006
main DNA builder

DNA polymerase I
20 bases/second
editing, repair & primer removal

DNA polymerase III Arthur Kornberg

enzyme 1959

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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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 What does it really look like?

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 Any Questions??

AP Biology 2007-2008