Replikasyon -

DNA Replication and Repair
Watson and Crick Predicted Semiconservative 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
• The mechanism: Strand separation,
followed by copying of each strand.
• Each separated strand acts as a template
for the synthesis of a new complementary
The Semiconservative Model
• Matthew Meselson and Franklin Stahl tested semiconservative 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.
Replication is bidirectional
• E. coli genome size =
4.6 X 106 bp
• Bacteria have circular
chromosome with
single origin of
• Replication rate is
~1000 base pairs per
• Duplicate chromosome
in 38 minutes.
• Eukaryotes have larger genomes 3 X 109 bps
• Rate of Eukaryote chromosome replication is
• But because eukaryote chromosomes have
multiple origins of replication, it takes about
the same amount of time to replicate complete
DNA Replication is
Okazaki Fragments
The Enzymology of DNA
• If Watson and Crick were right, then there should
be an enzyme that makes DNA copies from a DNA
• 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.
DNA Polymerase II is a
Multisubunit Enzyme
DNA Polymerase II Subunit
DNA Replication is a
Processive Process.
• DNA Polymerase
remains bound to
the replication
• Dimer of b-subunit
forms ring
structure around
the growing DNA
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
• 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.
Stages of DNA Replication
• Initiation
• Elongation
• Termination
Initiation of Replication
in E. coli
• The replisome consists of: DNAunwinding proteins, the priming complex
(primosome) and two equivalents of
• 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
Elongation Stage of
• Elongation involves DnaB helicase unwinding,
SSB binding to keep strands separated.
• Primase Complex Synthesizes short RNA
• DNA polymerase grinding away on both
• Topoisomerase II (DNA gyrase) relieves
supercoiling that remains
DNA Polymerase I/ Ligase
Required to Join Okazaki
• DNA polymerase I has 5’ to 3’
exonuclease activity that removes RNA
• 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)
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.
DNA Replication in Eukaryotes
• Occurs similarly to what occurs in
• Multiple origins of replication
• Replication is slower than in
• 5 different DNA polymerases in
Eukaryotic DNA
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
PCNA analogous to E. coli bsubunit of E. coli DNA
• Proliferating cell nuclear
• Trimeric protein
• Sliding clamp structure
binds to newly
synthesized DNA strand
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 of UV
Induced Thymine
General excisionrepair pathway
•Excision-repair systems
scan DNA duplexes for
mismatched bases, excise
the mispaired region and
replace it
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.