Download - Chapter 20
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Chapter 20
DNA Replication and Repair
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Watson and Crick Predicted Semi-conservative Replication of
DNA • Watson and Crick: "It has not
escaped our notice that the specific (base) pairing we have postulated immediately suggests a possible copying mechanism for the genetic material."
• The mechanism: Strand separation, followed by copying of each strand.
• Each separated strand acts as a template for the synthesis of a new complementary strand.
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The Semiconservative Model
• Matthew Meselson and Franklin Stahl tested semi-conservative model
• Template DNA labeled with 15N –nucleotides. (more dense than normal DNA)
• Fed 14N –nucleotides. (newly synthesized DNA was less dense than template)
• Isolated DNA at different times and fractionated DNA on a density gradient
• denser/heavier DNA found lower in the gradient.• Less dense/lighter DNA found higher in
gradient.
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Replication is bidirectional
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• E. coli genome size = 4.6 X 106 bp
• Bacteria have circular chromosome with single origin of replication.
• Replication rate is ~1000 base pairs per second.
• Duplicate chromosome in 38 minutes.
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• Eukaryotes have larger genomes 3 X 109 bps• Rate of Eukaryote chromosome replication is
slower• But because eukaryote chromosomes have
multiple origins of replication, it takes about the same amount of time to replicate complete genome.
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DNA Replication is Semidiscontinuous
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Okazaki Fragments
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The Enzymology of DNA Replication
• If Watson and Crick were right, then there should be an enzyme that makes DNA copies from a DNA template
• In 1957, Arthur Kornberg and colleagues demonstrated the existence of a DNA polymerase -
• Three DNA polymerases in E. coli- DNA polymerase I – DNA repair and
participates in synthesis of lagging strand- DNA polymerase II – DNA repair- DNA polymerase III – major polymerase
involved in DNA replication.
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DNA Polymerase III is a Multisubunit Enzyme
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DNA Polymerase III Subunit Organization
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DNA Replication is a Processive Process.
• DNA Polymerase remains bound to the replication fork.
• Dimer of -subunit forms ring structure around the growing DNA chains.
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DNA Polymerase also has proof reading
function• The polymerization reactions have an error rate of 1 mistake for every 100,000 base pairs incorporated (1 X 10-5 errors per base)
• DNA polymerase has 3’ to 5’ exonuclease function (epsilon-subunit) that recognizes base pair mismatches and removes them.
• Therefore proof reading function helps eliminate errors which could lead to detrimental mutations.
• However proof reading exonuclease has error rate of 1 mistake for every 100 base pairs (1 X 10-2 errors per base)
• Overall error rate is 1 X 10-7 errors per base.
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Stages of DNA Replication
• Initiation•Elongation •Termination
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Initiation of Replication in E. coli
• The replisome consists of: DNA-unwinding proteins, the priming complex (primosome) and two equivalents of DNA
• polymerase III holoenzyme • Initiation: DnaA protein binds to
repeats in ori, initiating strand separation and DnaB, a helicase delivered by DnaC, further unwinds. Primase then binds and constructs the RNA primer
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Elongation Stage of Replication
• Elongation involves DnaB helicase unwinding, SSB binding to keep strands separated.
• Primase Complex Synthesizes short RNA primers.
• DNA polymerase grinding away on both strands
• Topoisomerase II (DNA gyrase) relieves supercoiling that remains
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DNA Polymerase I/ Ligase Required to Join Okazaki
Fragments • DNA polymerase I has 5’ to 3’
exonuclease activity that removes RNA primer.
• Also has 5’ to 3’ DNA polymerase activity to fill in the gap. (proofreading 3’-5’ exonuclease activity)
• Ligase connects loose ends. Used NAD+ in phosphoryltransfer reaction, not a redox reaction (Page 643)
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Termination of Replication
• Termination occurs at ter region of E. coli chromosome.
• ter region rich in Gs and Ts, signals the end of replication.
• Terminator utilization substance (Tus) binds to ter region.
• Tus prevents replication fork from passing by inhibiting helicase activity.
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DNA Replication in Eukaryotes
• Occurs similarly to what occurs in prokaryotes.
• Multiple origins of replication• Replication is slower than in
prokaryotes.• 5 different DNA polymerases in
Eukaryotes.
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Eukaryotic DNA Polymerases
• Alpha – Primer synthesis and DNA repair
• Beta – DNA repair• Gamma – Mitochondrial DNA
replication• Delta – Leading and lagging strand
synthesis, and DNA repair• Epsilon – Repair and gap filling on
lagging strand.
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PCNA analogous to E. coli b-subunit of E. coli DNA
polymerase
• Proliferating cell nuclear antigen
• Trimeric protein • Sliding clamp
structure binds to newly synthesized DNA strand
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DNA Repair • A fundamental difference from RNA,
protein, lipid, etc. • All these others can be replaced, but
DNA must be preserved • Cells require a means for repair of
missing, altered or incorrect bases, bulges due to insertion or deletion, UV-induced pyrimidine dimers, strand breaks or cross-links
• Two principal mechanisms: methods for reversing chemical damage and excision repair.
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Repair of UV Induced Thymine
Dimers
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General excision-repair
pathway
•Excision-repair systems scan DNA duplexes for mismatched bases, excise the mispaired region and replace it
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Repair of damage resulting from the
deamination of cytosine
• Deamination of cytosine to uracil is one of most common forms of DNA damage
• DNA glycosylases cleave bases at N-glycosidic linkages. Leaving sugar-phosphate backbone.
• Endonuclease identifies abscent base and sugar phosphate.
• Gap then filled in by DNA polymerase and ligase.