1 AP Biology 2007-2008 DNA Replication AP Biology Watson and Crick 1953 article in Nature 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 AP Biology Directionality of DNA You need to number the carbons! it matters! OH CH 2 O 4 5 3 2 1 PO 4 N base ribose nucleotide This will be IMPORTANT!! AP Biology 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 PO 4 base CH 2 O base O P O C O – O CH 2 1 2 4 5 1 2 3 3 4 5 5 Sounds trivial, but… this will be IMPORTANT!! 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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AP Biology 2007-2008
DNA Replication
AP Biology
Watson and Crick 1953 article in Nature
AP Biology
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 AP Biology
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 be
IMPORTANT!!
AP Biology
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
O
P
O
C
O –O
CH2
1
2
4
5
1
2
3
3
4
5
5
Sounds trivial, but…
this will be IMPORTANT!!
AP Biology
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
2
AP Biology
Bonding in DNA
….strong or weak bonds?
How do the bonds fit the mechanism for copying DNA?
3
5 3
5
covalent
phosphodiester
bonds
hydrogen
bonds
AP Biology
Base pairing in DNA
Purines
adenine (A)
guanine (G)
Pyrimidines
thymine (T)
cytosine (C)
Pairing
A : T
2 bonds
C : G 3 bonds
AP Biology
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
DNA Replication Large team of enzymes coordinates replication
Let’s meet the team…
AP Biology
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 be helicase & unzip
your genes…
AP Biology
DNA
Polymerase III
Replication: 2nd step
But… We’re missing
something! What?
Where’s the ENERGY
for the bonding!
Build daughter DNA
strand
add new
complementary bases
DNA polymerase III
3
AP Biology
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!
AP Biology
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
AP Biology
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
DNA
Polymerase III
DNA
Polymerase III
DNA
Polymerase III
DNA
Polymerase III
energy
energy
energy
Replication energy
3
3
5 B.Y.O. ENERGY! The energy rules
the process
5
AP Biology
energy
3 5
5
5
3
need “primer” bases to add on to
energy
energy
energy
3
no energy
to bond
energy
energy
energy
ligase
3 5
AP Biology
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
5
3 5
3 3
Leading strand
Lagging strand ligase
Okazaki
Leading strand
continuous synthesis
Lagging strand
Okazaki fragments
joined by ligase
“spot welder” enzyme
DNA polymerase III
3
5
growing replication fork
AP Biology
DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading strand
lagging strand leading strand
5
3
3
5
5
3
5
3
5
3 5
3
growing replication fork
growing replication fork
5
5
5
5
5
3
3
5
5 lagging strand
5 3
4
AP Biology
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 5
3 5 3
growing replication fork
primase
RNA
AP Biology
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
AP Biology
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!
AP Biology
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
TTAAGGG TTAAGGG
AP Biology
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
AP Biology
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 Kornberg 1959
Thomas Kornberg ??
5
AP Biology
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
AP Biology
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
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What does it really look like?
AP Biology 2007-2008
Any Questions??
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