2007-2008 AP Biology DNA Replication
May 11, 2015
2007-2008 AP Biology
DNA Replication
Watson and Crick1953 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
Directionality of DNA You need to
number the carbons! it matters!
OH
CH2
O
4
5
3 2
1
PO4
N base
ribose
nucleotide
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
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
Bonding in DNA
….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?
3
5 3
5
covalentphosphodiester
bonds
hydrogenbonds
Base pairing in DNA Purines
adenine (A) guanine (G)
Pyrimidines thymine (T) cytosine (C)
Pairing A : T
2 bonds C : G
3 bonds
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
DNA Replication Large team of enzymes coordinates replication
DNA-pol of eukaryotes
DNA-pol : elongation DNA-pol III
DNA-pol : initiate replication and synthesize primers
DnaG, primase
DNA-pol : replication with low fidelity
DNA-pol : polymerization in mitochondria
DNA-pol : proofreading and filling gap
DNA-pol I
repairing
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
DNAPolymerase III
Replication: 2nd step Build daughter DNA
strand add new
complementary bases DNA polymerase III
energy
ATPGTPTTPCTP
Energy of ReplicationWhere does energy for bonding usually come from?
ADPAMPGMPTMPCMPmodified nucleotide
energy
We comewith our own
energy!
And weleave behind a
nucleotide!
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
§2.2 Primase
• Also called DnaG
• Primase is able to synthesize primers using free NTPs as the substrate and the ssDNA as the template.
• Primers are short RNA fragments of a several decades of nucleotides long.
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
DNA polymerase III
Replication fork / Replication bubble
5
3 5
3
leading strand
lagging strand
leading 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
53
3
5
5lagging strand
5 3
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
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
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
Roger Kornberg2006
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
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
1
2
3
4
What does it really look like?
Characteristics of replication
Semi-conservative replication
Bidirectional replication
Semi-continuous replication
High fidelity
§1.1 Semi-Conservative Replication
Semiconservative replication
Half of the parental DNA molecule is conserved in each new double helix, paired with a newly synthesized complementary strand. This is called semiconservative replication
Semiconservative replication
Experiment of DNA semiconservative replication
"Heavy" DNA(15N)
grow in 14N medium
The first generation
grow in 14N medium
The second generation
Significance
The genetic information is ensured to be transferred from one generation to the next generation with a high fidelity.
Bidirectional Replication
• Replication starts from unwinding the dsDNA at a particular point (called origin), followed by the synthesis on each strand.
• The parental dsDNA and two newly formed dsDNA form a Y-shape structure called replication fork.
3'
5'
5'
3'
5'
3'
5'3'
direction of replication
Replication fork
Bidirectional replication
• Once the dsDNA is opened at the origin, two replication forks are formed spontaneously.
• These two replication forks move in opposite directions as the syntheses continue.
Bidirectional replication
Replication of prokaryotes
The replication process starts from the origin, and proceeds in two opposite directions. It is named replication.
Replication of eukaryotes
• Chromosomes of eukaryotes have multiple origins.
• The space between two adjacent origins is called the replicon, a functional unit of replication.
origins of DNA replication (every ~150 kb)
Semi-continuous Replication
The daughter strands on two template strands are synthesized differently since the replication process obeys the principle that DNA is synthesized from the 5´ end to the 3´end.
5'
3'
3'
5'
5'
direction of unwinding3'
On the template having the 3´- end, the daughter strand is synthesized continuously in the 5’-3’ direction. This strand is referred to as the leading strand.
Leading strand
Semi-continuous replication
3'
5'
5'3'
replication direction
Okazaki fragment
3'
5'
leading strand
3'
5'
3'
5'replication fork
• Many DNA fragments are synthesized sequentially on the DNA template strand having the 5´- end. These DNA fragments are called Okazaki fragments. They are 1000 – 2000 nt long for prokaryotes and 100-150 nt long for eukaryotes.
• The daughter strand consisting of Okazaki fragments is called the lagging strand.
Okazaki fragments
Continuous synthesis of the leading strand and discontinuous synthesis of the lagging strand represent a unique feature of DNA replication. It is referred to as the semi-continuous replication.
Semi-continuous replication
2007-2008 AP Biology
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