Replikasyon - mustafaaltinisik.org.uk

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
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.
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.
• Replication rate is
~1000 base pairs per
second.
• Duplicate chromosome
in 38 minutes.
• 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.
DNA Replication is
Semidiscontinuous
Okazaki Fragments
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.
DNA Polymerase II is a
Multisubunit Enzyme
DNA Polymerase II Subunit
Organization
DNA Replication is a
Processive Process.
• DNA Polymerase
remains bound to
the replication
fork.
• Dimer of b-subunit
forms ring
structure around
the growing DNA
chains.
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.
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
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
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
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)
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
prokaryotes.
• Multiple origins of replication
• Replication is slower than in
prokaryotes.
• 5 different DNA polymerases in
Eukaryotes.
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.
PCNA analogous to E. coli bsubunit of E. coli DNA
polymerase
• Proliferating cell nuclear
antigen
• 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.
Repair of UV
Induced Thymine
Dimers
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.