DNA Replication
Lecture 11
Fall 2008
• Read pgs. 305-312
1
Nucleic Acid Structure
• Deoxyribonucleic Acid
(DNA)
– Double strand
• Ribonucleic Acid (RNA)
– Single strand
• Nucleic Acid: long chain of
nucleotides
3 components of nucleotides
• 5 carbon sugar
• Phosphate group (PO4-)
• Nitrogenous bases
– Nucleoside = sugar + base
(no phosphate group)
Fig. 5.27
2
Nucleic Acid Structure
Nitrogenous bases
• Pyrimidines
• 6-membered ring of
carbon and nitrogen
– Cytosine (C)
– Thymine (T)
– Uracil (U) replaces T in
RNA
• Purines
• 6-membered ring fused
to a 5-membered ring
– Adenine (A)
– Guanine (G)
Fig. 5.27
3
Nucleic Acid Structure
• 5 carbon sugar
– Ribose in RNA
– Deoxyribose in DNA
• Missing oxygen at 2’
• Carbons numbered 1 to 5 – prime’
DNA Structure
4
• Long chain of nucleotides
– Allows for unique
arrangement of 4 bases
• Sugar-phosphate
backbone
– Phosphodiester linkage
• Covalent bond between sugar
group of one nucleotide and
phosphate group of another
nucleotide
Fig. 5.27
• 5’ end with phosphate
group
• 3’ end with hydroxyl group
(OH)
5
DNA Structure
DNA molecule
• Double helix
– Two strands
• Antiparallel
– Strands oriented
in opposite
directions
• Complementary base
pairing
– T+A
– C+G
Fig. 16.27
• Hydrogen bonds between
the base pairs
• Van der Waals interactions
between stacked bases
6
DNA Replication
See Fig. 5.28
• Cell division requires the
duplication of genetic
material
• DNA is a template
– Two strands separate
– Free nucleotides bond to
template and form
“daughter” DNA strand
7
DNA Replication
• Origins of replications
– Short stretches of DNA with
specific nucleotide
sequence
– DNA separates, forming
replication bubble
– Replication continues in
both directions until
completed
– Prokaryotes
• One origin of replication
See Fig. 16.12
8
DNA Replication
• Origins of replications
– Eukaryotes
• Many origins (100s to 1000s)
• Replication bubbles eventually fuse
– Replication fork
• Y-shaped region where parental DNA strand is unwound
into 2 single strands
Fig. 16.12
DNA Replication
How does DNA separate?
• Helicases
– Unwinds and separates DNA strands
– Catalyzes breaking of hydrogen bonds between nucleotides
• Single-strand binding proteins
– Stabilizes separated strands
• Topoisomerase
– Releases strain on
unwinding DNA
– Cuts, twists and rejoins
DNA downstream of
replication fork
Fig. 16.13
9
10
DNA Replication
• How is DNA synthesis initiated?
– Primase
• Adds a primer - short section of RNA
– 5-10 nucleotides long
• Necessary because DNA polymerases can only
add nucleotides to an existing chain
Fig. 16.13
11
DNA Replication
• DNA polymerases
– Catalyze synthesis of new DNA by adding
nucleotides to preexisting chain
– Prokaryotes
• DNA polymerase III & DNA polymerase I
– Eukaryotes
• ~ 11 DNA polymerases identified
DNA Replication
• Nucleoside triphosphate
– Sugar, base + 3 phosphate groups
– Removal of 2 phosphates catalyzed by DNA polymerase III
• Nucleotides can only be added at 3’ end
• Elongation in 5’ to 3’ direction
Fig. 16.14
12
Leading and Lagging Strands
• Leading strand
– The new complementary DNA strand synthesized
continuously along the template strand toward the
replication fork
– DNA polymerase III and sliding clamp
Fig. 16.15
13
Leading and Lagging Strands
Lagging strand
• A discontinuously synthesized
DNA strand that elongated by
means of Okazaki fragments
– A short segment of DNA
synthesized away from the
replication fork
• 100-200 nucleotides (eukaryotes)
• Requires multiple primers
Fig. 16.16
14
Leading and Lagging Strands
• DNA polymerase I
– Replaces RNA nucleotides
of primer with DNA
nucleotides
• DNA ligase
– Joins the Okazaki
fragments
Fig. 16.16
15
16
DNA Repair
• Errors in completed DNA molecule
– 1 in 10 billion nucleotides
• Initial pairing errors in DNA replication
– 1 in 100,000 nucleotides
• Corrections during replication
– DNA polymerase proofreads
• If error in match, nucleotide removed and
replaced
– Mismatch repair
• Repair by other enzymes if DNA
polymerase missed the error
DNA Repair
• Corrections after replication
– ~100 repair enzymes identified in in
E. coli
– ~130 repair enzymes identified in
humans
• Nucleotide excision repair
– E.g., repair of thymine dimers
• Covalent linking of adjacent thymine
bases
• Causes DNA to buckle
• Caused by UV radiation
– Nuclease cuts damaged DNA at two
points
– DNA polymerase adds nucleotides
– DNA ligase joins nucleotides
Fig. 16.18
17
DNA Repair
Replicating ends of linear DNA
molecules
• Nucleotides can only be added
at 3’ end of existing strand
• No way to replace the primer
on the 5’ end
• Linear DNA molecules grow
shorter with each replication
– In somatic cells
Fig. 16.19
18
19
DNA Repair
• Telomeres
– Repeating sequence of nucleotides at ends of
linear chromosomes
• TTAGGG in humans
• Repeated 100 to 1000 times
– Do not contain genes
– Chromosomes continue to shorten
– Cell eventually dies
DNA Repair
Preserving DNA ends in
meiosis
• Telomerase
– Catalyzes lengthening
of telomeres in germ
cells
– Preserves length of
chromosomes in
gametes
– Not active in most
somatic cells
20
21
DNA Repair
• Telomerase and cancer
– Chromosomes of somatic cells gradually
shorten
• Telomere loss signals cells to enter non-dividing
stage
– If telomerase activated in somatic cells, cell
may continue to divide
• May become cancerous