DNA replication
• DNA replication
• Process of Replication
in E. coli
• Origin of replication
• Role of Primase: RNA
Primer
• Elongation
• Lagging strand
synthesis: Okazaki
fragments
• Error rate of DNA
synthesis
• Eukaryotic Replication
Types of replication
Meselson-Stahl Experiment:
Semi-conservative replication
Eukaryotic chromosomes with base
Analog 5-Bromodeoxyuridine with
staining
Process of Replication in E. coli
Polymerases
• DNA Polymerases
• I,II, III
• No initiation of
replication
• Primase: RNA
polymerase
• Lays down RNA
nucleotides (primer)
Origin of replication
• Origin: 245 bp, containing repeats
• Proteins involved, DNA A (initial denaturing), DNA B and
C (further opening/destabilize helix)
• unwinding of the helix: helicases (DNA B/C)
• stabilization of the helix: single stranded binding proteins
• role of topoisomerases, DNA gyrases
Initiation
Elongation
• Anti-parallel strands
• DNA Polymerase III
• Leading strand
synthesis
DNA polymerase
Replisome
Lagging strand synthesis
•
•
•
•
Role of DNA Polymerase I
removal of primer
exonuclease activity
DNA ligase
DNA Ligase
Proofreading
• Error rate of DNA
synthesis
• Proofreading
• Base Pairing rules
Eukayotic Replication
• Multiple origins
• Polymerases
• Linear chromosomes
Multiple origins
Eukaryotic DNA Replication
•
•
•
•
•
DNA helicase promotes unwinding
at the replication fork,
DNA pol δ with RFC and PCNA
synthesizes DNA on the leading
strand.
DNA pol α initiates synthesis on
the lagging strand by generating an
RNA primer (red segment) followed
by a short segment of DNA. Then,
RFC and PCNA load a second DNA
polymerase (δ or ε ) to continue
synthesis of the Okazaki fragment.
B, as DNA pol δ approaches the
downstream Okazaki fragment,
Cleavage by RNase H1 removes the
initiator RNA primer leaving a single
5 -ribonucleotide. Then, FEN1/RTH1
removes the 5 -ribonucleotide. The
resulting nick is sealed by DNA
ligase.
Volume 272, Number 8, Issue of February 21, 1997 pp. 4647-4650
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
Linear chromosomes
Telomerase= ribonucleoprotein with
RNA, template for DNA synthesis