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DNA ReplicationLecture 11Fall 2008
• Read pgs. 305-312
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)
1
Fig. 5.27
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)
2
Fig. 5.27
Nucleic Acid Structure
• 5 carbon sugar– Ribose in RNA– Deoxyribose in DNA
• Missing oxygen at 2’
• Carbons numbered 1 to 5 – prime’
3
DNA Structure
• 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
• 5’ end with phosphate group
• 3’ end with hydroxyl group (OH)
Fig. 5.27
4
DNA StructureDNA molecule• Double helix
– Two strands
• Antiparallel– Strands oriented
in opposite directions
• Complementary base pairing– T + A– C + G
• Hydrogen bonds between the base pairs
• Van der Waals interactions between stacked bases
Fig. 16.27
5
DNA Replication
• 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
6
See Fig. 5.28
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
7
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
8
DNA ReplicationHow 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
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
10
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
11
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
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
16
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
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
19
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
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
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