dna replication - western oregon universityguralnl/311dna replication.pdf · semi-conservative...
TRANSCRIPT
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
Meselson-Stahl Experiment: Semi-conservative replication
Eukaryotic chromosomes with baseAnalog 5-Bromodeoxyuridine withstaining
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
Lagging strand synthesis
• Role of DNA Polymerase I • removal of primer • exonuclease activity • DNA ligase
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.